text:
Most Popular "Dunce Cap" Titles
- Movies or TV
- Genres
- Keywords
- IMDb Rating
- Instant Watch Options
- In Theaters
- On TV
- Release Year
1. Iron Man 3 (2013)
PG-13 | 130 min | Action, Adventure, Sci-Fi
When Tony Stark's world is torn apart by a formidable terrorist called the Mandarin, he starts an odyssey of rebuilding and retribution.
Writers: Drew Pearce, Shane Black, Stan Lee, Don Heck, Larry Lieber, Jack Kirby, Warren Ellis, Adi Granov | Director: Shane Black | Stars: Robert Downey Jr., Guy Pearce, Gwyneth Paltrow, Don Cheadle
2. House of 1000 Corpses (2003)
R | 89 min | Horror
Two teenage couples traveling across the backwoods of Texas searching for urban legends of murder end up as prisoners of a bizarre and sadistic backwater family of serial killers.
Writer: Rob Zombie | Director: Rob Zombie | Stars: Sid Haig, Karen Black, Bill Moseley, Sheri Moon Zombie
3. 8½ (1963)
Not Rated | 138 min | Drama, Fantasy
A harried movie director retreats into his memories and fantasies.
Writers: Federico Fellini, Ennio Flaiano, Tullio Pinelli, Brunello Rondi | Director: Federico Fellini | Stars: Marcello Mastroianni, Anouk Aimée, Claudia Cardinale, Sandra Milo
4. Pete's Dragon (1977)
G | 128 min | Adventure, Family, Fantasy
An orphan boy and his magical dragon come to town with his abusive adoptive parents in pursuit.
Writers: Malcolm Marmorstein, Seton I. Miller, S.S. Field | Director: Don Chaffey | Stars: Sean Marshall, Helen Reddy, Jim Dale, Mickey Rooney
5. Goya's Ghosts (2006)
R | 113 min | Biography, Drama, History
Painter Francisco Goya faces a scandal involving his muse, who is labeled a heretic by a monk.
Writers: Milos Forman, Jean-Claude Carrière | Director: Milos Forman | Stars: Javier Bardem, Natalie Portman, Stellan Skarsgård, Randy Quaid
6. Toot Whistle Plunk and Boom (1953)
Approved | 10 min | Animation, Short, Comedy
A crash course on the history of Western musical instruments.
Writer: Dick Huemer | Directors: Ward Kimball, Charles A. Nichols | Stars: The Mellomen, Loulie Jean Norman, Charlie Parlato, Bill Thompson
7. Street Smarts (2000–2005)
TV-PG | Comedy, Game-Show
In this test of general knowledge and human nature, contestants try to predict who answered trivia questions a certain way based on three separate man-on-the-street interviews.
Stars: Frank Nicotero, Michael Gambino, Kate Linder, Taylor Masamitsu
8. A Symposium on Popular Songs (1962)
Passed | 19 min | Animation, Comedy, Family
Professor Ludwig von Drake plays a variety of popular music, all of which he wrote. First, ragtime: the Rutabaga Rag, with vegetables dancing in stop-motion. Next, the Charleston, with ... See full summary »
Writer: Xavier Atencio | Director: Bill Justice | Stars: Paul Frees, Gloria Wood, Billy Storm, Skip Farrell
9. Katnip Kollege (1938)
Approved | 7 min | Family, Musical, Animation
At the Katnip Kollege, we see a roomful of cats taking a course in Swingology. Everyone swings except Johnny, who can't cut it and has to sit in the dunce chair. Miss Kitty Bright tells him... See full summary »
Writer: Dave Monahan | Directors: Cal Dalton, Cal Howard | Stars: Mel Blanc, Johnnie Davis, George MacFarland, Poley McClintock
10. Melody (1953)
Approved | 10 min | Animation, Short, Family
An owl teaches his class full of birds about melody. It's all around in nature. Only birds and man can sing; man "sings" even when he speaks. We see a quick survey of the stages of life, as... See full summary »
Writer: Dick Huemer | Directors: Ward Kimball, Charles A. Nichols | Stars: Loulie Jean Norman, Harry Stanton, Bill Thompson, Gloria Wood
11. Yankee Doodle Bugs (1954)
7 min | Animation, Short, Comedy
Bugs lectures his nephew Clyde about early America, inserting himself into events wherever possible.
Writer: Warren Foster | Director: Friz Freleng | Stars: Mel Blanc, Bea Benaderet
12. Goofy Goofy Gander (1950)
Approved | 7 min | Animation, Short
Little Audrey likes school, but doesn't like Mother Goose rhymes. She gets so involved in the crooks in her comic books that she doesn't hear when asked by the teacher to recite a rhyme, ... See full summary »
Writer: Isadore Klein | Director: Bill Tytla | Stars: Jack Mercer, Mae Questel, Sid Raymond
13. Infernum (2010)
7 min | Short, Drama, History
A high-stakes drama of redemption and death, set during the chaos and brutality of the medieval Inquisition.
Writer: Ted Marcus | Director: Ted Marcus | Stars: David Dayan Fisher, Charlie Hunter, Vera R. Taylor, Denis O'Mahoney
14. Bored of Education (1946)
Approved | Animation, Short
Lulu answers wrong on a history quiz after cheating off of Tubby. While sitting in the corner and wearing a dunce cap Lulu falls asleep and dreams of chasing Tubby through history to get back at him.
Writers: Marge, Isadore Klein, George Hill | Director: Bill Tytla | Stars: Jackson Beck, Cecil Roy, Arnold Stang
15. A Bout with a Trout (1947)
Approved | 8 min | Animation, Short, Family
Little Lulu, torn between playing hookey and going fishing, or going to school, chooses to go fishing...where she encounters the Fish from Cartoon Hades.
Writers: Isadore Klein, Marge, Jack Ward | Director: Izzy Sparber | Star: Cecil Roy
16. This Land (2004)
2 min | Animation, Musical, Comedy
The cut-out heads of George W. Bush and John Kerry alternate singing verses and choruses of a partisan political version of Woody Guthrie's "This Land Is Your Land." Bush and Kerry trade ... See full summary »
Writers: Evan Spiridellis, Gregg Spiridellis | Directors: Evan Spiridellis, Gregg Spiridellis | Star: Jim Meskimen
17. Insultin' the Sultan (1934)
7 min | Animation, Short
Willie Whopper tells a tall tale of his girlfriend being carried off by a Sultan and his efforts to rescue her.
Director: Ub Iwerks
The Shadow of the Tower (1972– )
Do the Sheep Sin?
Biography, Drama, History
Heavy-handed tax policies lead the peasants of Cornwall to march peacefully in protest on London, but an ambitions nobleman wants to use the pilgrimage to his own advantage.
Writers: Anthea Browne-Wilkinson, John Gould | Director: Prudence Fitzgerald | Stars: James Maxwell, John Castle, John Woodvine, Richard Warwick
Archie's Funhouse (1970–1971)
Ice Block/Circus Act
Animation, Comedy, Family
The Archies perform "Love Vibrations," "Falling in Love Is Fun" and "I'm Just a Puppet on a String." Big Ethel chases Jughead on the ice. Reggie and Moose perform a trapeze act.
Writer: Bob Ogle | Director: Hal Sutherland | Stars: John Erwin, Dal McKennon, Howard Morris, Jane Webb
Bonne nuit les petits (1962–1966)
Les oursons font leurs devoirs
5 min | Animation, Short, Family
Writer: Claude Laydu | Director: Michel Manini | Stars: Claude Laydu, Jean Martinelli, Martine Merri, Monique Messine
Once a Thief (1996–1998)
The Big Bang Theory
R | Action, Comedy, Crime
Creators: Glenn Davis, William Laurin | Director: David Wu | Stars: Ivan Sergei, Sandrine Holt, Nicholas Lea, Jennifer Dale
Milton the Monster (1965–1967)
The Bomb's Rush/There Auto Be a Law/Ghoul School
Animation, Comedy, Family
Goo Fee pretends to be dying in order to lure Fearless Fly into his trap. Penny Penguin causes trouble between her father and a mechanic. Aunt Hagatha visits to teach the monsters some manners.
Director: Hal Seeger | Stars: Bob McFadden, Dayton Allen, Beverly Arnold, Zel DeCyr
Milton the Monster (1965–1967)
The Goofy Doctor Goo Fee/From Riches to Rags/The Pot Thickens
Animation, Comedy, Family
Gung Ho captures Horsey, mistaking him for Fearless Fly. Muggy-Do, working as a bellboy, makes a dishonest buck off a movie-star guest. Professor Weirdo tries to prevent his Aunt Hagatha from seeing Milton.
Creator: Beverly Arnold | Director: Hal Seeger | Stars: Bob McFadden, Herb Duncan, Beverly Arnold
Alfred Hitchcock Presents (1955–1962)
The Schartz-Metterklume Method
30 min | Crime, Drama, Mystery
Charlotte Hope arrives in the Wellington household to act as a governess to four children. She is a somewhat brash, opinionated person who boldly announces that she uses the ... See full summary »
Writers: Marian B. Cockrell, Hector Hugh Munro | Director: Richard Dunlap | Stars: Alfred Hitchcock, Hermione Gingold, Elspeth March, Doris Lloyd


text:
'If it is not good for us, it is not good for the Jews in the Negev'
Haredi parties intent on entering coalition by start of Passover
Ya'alon: You don't have to be in intelligence to know that Iran is lying
Negotiating parties split on Likud push to increase number of ministers
First-ever effort between hospital and taxi company to ensure newborns are taken home safely
Jerusalem Municipality shuts down 'filthy, rodent-infested' butcher shop in Mahane Yehuda market
*New Worlds: The brain's 3D compass*
*IQ is not enough – develop your EQ, too*
Eco Wave Power to establish its first Chinese power station
Watch: Technion students recreate Passover story with robots
Noble Energy walks away from Ruth C exploration license
Cyber Chief: 8% of ministry budgets should go to cyber security
Play it once again, Sam
Anti-Semitic fans pressure Hungarian rock band to cancel Israel concert
Concert review: IPO
Eytan's Fox's 'Cupcakes' opening in New York
Bruised yellow-and-blue looks ahead after Real blow
Israeli figure skater Bychenko places in world championships
Soccer: Israel trounced by Wales in key qualifier
Scuffling Maccabi must dig deep on road at group leader Real
Legislators, environmentalists gather for launch of Knesset solar rooftop field
Court to render verdict in Talansky retrial today
Israelis part of int'l observers monitoring Uzbekistan election
Meet the new MK: Zouheir Bahloul hopes to promote better relations between Jews, Arabs
Top Hamas engineer, tactician convicted in Beersheba court
Exclusive: In exchange for freed tax funds, PA won't pursue Israel over settlements at ICC
Egyptian court convicts man and woman of spying for Israel
Abbas: Israeli-Palestinian relationship must change
US 'disappointed UN rights council continues to single out Israel'
Jewish Agency, IFCJ spar over Ukrainian immigration
Why are these South Africans wearing kippot?
Will or won't 'Borat' play Freddie Mercury in Queen biopic?
Limmud FSU marks Auschwitz liberation with emotional exhibition
Toulouse rabbi told to remove kippah at voting station
US says talks with Iran will go down to the wire
Egypt lists as terrorists top Muslim Brotherhood leader, 17 others from group
Officials: Iran indicating willingness to accept fewer centrifuges, ship out enriched uranium
Watch: Armed Houthi women threaten war against US, Saudi Arabia, Israel
Risk vs. reward: Saudis could become regional heavyweight, or serve to embolden Iran
Reverse course
Fundamentally Freund: Happy Land of Israel Day!
Detoxifying US-Israel relations
Letters to the editor
Right or wrong: Anti-Semitism or emasculation?
The blood libel Haggada
The turning point
Travel tech start-ups
On wings of carbon
Veterans: On target
Fusing modern and traditional in the kitchen
Book Review: Perceptive Haggada
Hot off the Arab press
Let's cooperate
Shabbat screenings
Passover perspective
On the field and in court
Happily Eva After: Times, they are a-interestin'
Music and the mamaloshen
What's the catch?
Kayaking for gold on the Kinneret
Why Tunisia?
Palestine – words matter but their meaning matters more
A nuclear deal with Iran – would it stick?
When words matter and when they don't
'מְלָגוֹת 'צוּק אֵיתָן
עוֹלִים חֲדָשִים
כּוּלָם רוֹצִים לִחְיוֹת
יִשְׂרָאֵל בְּמַבָּט פָּנוֹרָמִי
Getting a young start
A culinary festival starring the women next door
Global girl power
Apartheid South Africa: Israel edition
Sa'ar says national unity government is 'still on the table'
Israeli parties prepare legislative demands for Netanyahu's new coalition
With full term possible, Netanyahu may outlast Ben-Gurion (and Obama)
Arab sector turnout for recent elections reached 63.5%, polling data shows
Joubran: Polls have to include parties that don't pass threshold
Pope Francis pays tribute to Christians persecuted by ISIS during Palm Sunday mass
PHOTOS: Exercising the 'Right to Movement' at the Palestine Marathon
It's hard to be a Christian in Israel, but not because of prejudice
Unconvincing ISIS video shows Assyrian captive converting to Islam
'Those who hate Jews hate Christians': A closer look at US Presidential hopeful Ted Cruz
Green Leadership Conference in Hamalachim-Shahariya Forest
Scenic Lookout in Memory of Lt-Gen. Amnon Lipkin Shahak
Following in the Footsteps of Meir Har-Zion
Celebrating the 100th Birthday Anniversary of Sculptor Ahiam Shoshany
French Students Get to Know the Real Israel
Serbian PM Plants a Tree in the Grove of Nations
Sharing and Learning - German Forestry Students Intern with KKL-JNF
Israel and Finland Connect through the Environment
New Bio-filters Purify Surface Runoff Water for Reuse
Future Tour Guides Discover the Negev with KKL-JNF
The Shamir Drilling Project
Graduation Ceremony for Rosh HaAyin's Volunteer Firefighters
From Sunny Israel to Cold Canada
Walking for Water with KKL France in the Mountains of Eilat
JNF Ontario Community Mission Tours the Western Negev
Celebrating 50 Years of German-Israeli Diplomatic Relations
The Negev Conference on Agricultural Research and Development
Arava Open Day: Food Security for Israel and the Entire World
Living Green in Israel: the Contest and the Winners
Addressing Climate Change Conference
From Think Tank to Environmental Consultant
Award Winning Designer Launches Career as Wedding Dressmaker
Outdoor Fitness Israel: An Innovative Fitness Program Developed By UK Olim
The Burstein Brothers: 3 Oleh Siblings in the IDF
5 Tips to Land a Job in Israel
Starting Your Own Business in Israel: Real Life Examples
5 Strategies for Finding Your Professional Place in Israel
PetBNB:An Olah's Unusual Canine Startup
36 Hours in Southern Israel
Food Shopping In Israel
How Women Can Have it All in Israel
The Eisenberg's Long Journey Home
Why Retirees are Thriving in Israel
Thanks to Aliyah video
6 Frequently Asked Questions about Israeli Healthcare
Starting Over in Israel
VIDEO: Are you ready for the South?
Sovev Tel Aviv, Israel's Largest Biking Event
Running Through Color
Formula 1 Racecars Zip Through Jerusalem
Make yourself at home… in Israel
NBN Announces Bonei Zion Prize Recipients for 2015
NefeshB'Nefesh Provides Services to Bnot Sherut
How Doctors Are Impacting Israel's South
Over 1,000 IDF 'lone soldiers' attend personal errand day in Tel Aviv
03.30.2015 | 10 Nisan, 5775
Hillary Clinton: US, Israel must preserve 'special' ties
At Swiss palace, intrigue and press questions as Iran deal nears
Germanwings pilot pleads: 'Open the damn door' in desperate attempt to re-enter cockpit
Putin to Arab League: Palestinians have right to state with capital in east Jerusalem
Drugs to treat mental illness reportedly found at home of Germanwings co-pilot
Iran, world powers, close to written agreement outlining future long-term nuclear settlement
Said ex-lover of Andreas Lubitz: Crash co-pilot was psychiatric patient, planned big gesture
Lena Dunham stirs pot with controversial 'dog or Jewish boyfriend' magazine piece
Analysis: Iran is seeking hegemony via a nuclear deal
Deterring Hezbollah: The ex-IDF deputy chief of staff shares his vision for Israeli security
Politics: Netanyahu's coalition vision - From slavery to freedom?
10/10/2010 03:36
gaza flotilla raid 311.
(photo credit:IDF)
LONDON – A British news weekly has published an advertisement, signed by
activists accusing the BBC of a pro-Israel bias, that other publications refused
to publish.The New Statesman published the ad, a letter signed by 232
people berating the broadcaster for "a consistently pro-Israel bias" for its
coverage of the Gaza flotilla incident and the conflict in general across its
entire network."The BBC's decision to broadcast uncritically Israeli
film of the attack reflects a consistently pro-Israeli bias in its coverage of
the Israel-Palestinian conflict," the ad stated.It was signed by a host
of pro-Palestinian and anti-Israel activists including Liberal Democrat peer
Jenny Tonge, Green Party MP Caroline Lucas, Methodist Church minister Stephen
Leah, Palestinian academic Nur Masalha and Israel-born academics Avi Shlaim,
Moshe Machover and Haim Bresheeth along with many involved in the boycott,
divestment and sanctions campaign against Israel.Citing legal reasons,
The Independent and The Guardian, publications with a critical stance toward
Israel, rejected the advertisement, organized by a Brighton-based group calling
itself the "Campaign to end BBC bias on Palestine" but asking that checks be
made out to a radical group called "Jews for Boycotting Israeli
Goods."According to an activist who set up a Facebook protest page in
August, the Independent said it would not publish the ad as it was "based on
opinion and not facts.""The advertising department there does not seem
to appreciate the virtues of a free press, because they kept telling me it was
based on opinion not facts," Cheryl "Filasteen" Abdul from Newcastle said on the
Facebook page.The Independent said the ad would make it vulnerable to a
libel suit and raise questions from the Advertising Standards Authority, the
UK's advertising regulator.The Guardian also refused to publish the
letter without changes being made to the text."Legal have just got back
to me and said that we'll only be able to run your ad if you can make a few
changes to the copy," Jennifer Melmore from the Guardian's advertising
department told the activists in an email last month."The Guardian was
approached about running the advert, but the advertisers chose not to proceed,"
a Guardian spokesman said on Friday.Changes had been made to the
advertisement published in the New Statesman, compared to the original draft
seen by The Jerusalem Post.Taken out was the accusation that the "the
BBC's Orwellian production portrayed the victims of Israel's premeditated attack
on the ships as the ones guilty of violence," as was an accusation that Israeli
government spokesman Mark Regev "went unchallenged as he justified the murders
and vilified the victims."A blanket accusation that "throughout its
coverage" BBC News 24 broadcasted Israel's own film evidence was taken out to
read: "BBC News 24 broadcast uncritically Israel's own film 'evidence' of their
commandos being attacked, when it was fully aware that this had edited out the
initial lethal attack."The words, "By the time the flotilla survivors
were free to tell their side of the story, the BBC news agenda had conveniently
moved on" were completely removed, as was a statement in the original draft to
the effect that the Israeli commandos "were disarmed" on the Mavi Marmara,
leaving, "They [viewers] were also not shown the photographs smuggled past
Israel's pirates, showing activists giving medical treatment to Israeli
commandos."The advertisement asked why the BBC's Panorama show
documentary on the incident did not "investigate the attack from the standpoint
of those who were killed and injured instead of allowing itself to be bought off
with 'unique access' to the killers?" "Whilst the rest of the world expressed
incredulity at Israel's claim that its commandos had acted in self-defense,
BBC's radio and TV news broadcasted, without comment, Benjamin Netanyahu's
absurd claim that 'Israel did all it could to avoid violence,'" the signatories
claimed.The ad also described the Panorama documentary, titled Death on
the Med, as "a half-hour justification of the murder of 9 aid workers," and said
that Israeli troops deliberately fired a tear-gas canister directly at a
protester in the West Bank.
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subset: commoncrawl


text:
I have been victim of robbery on 11/19/2005 they stole my car and my wallet with everything in, i called 911 and my case # was : 200*-443071, one 1 year later i was arrest for steeling and cashing a check, i spent almost 3 days in jail for doing nothing, i went to courthouse and got release, after all that was the same person i have been victim before using my driver licence. Is it too late for me to be sue the Orlando Department. Anuel JEAN,
Business Attorney
It's probably too late. And it's difficult to win such cases against law enforcement anyway.
My answer to your question does not create an attorney-client relationship.
Post a question and get free legal advice from attorneys.Ask a Lawyer
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text:
12 of Kelly Rowland's Sweetest Family Moments view
The Ladies of 'Orange Is The New Black' view
Child Stars: Where Are They Now? view
Kim Kardashian threw her BFF Ciara a surprise birthday bash at NYC's Greenhouse....
Kim Kardashian threw her BFF Ciara a surprise birthday bash at NYC's Greenhouse. She looks surprised! Good job, Kim.
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text:
Books, Crime, Intellectual Property, Non-Sequiturs, Rank Stupidity, Richard Posner, Technology
* Additional thoughts, from Professor Josh Blackman, on Judge Richard Posner's awesome streak of book reviews. [Josh Blackman]
* Meanwhile, Professor Kyle Graham wonders: How would Judge Posner review Moby Dick, Fifty Shades of Grey, and other classic literature? Incredibly, that's how. [noncuratlex]
* Apple responded to Samsung's blame-the-jury appeal with knives out and guns blazing. [Ars Technica]
* This attempt at using a disguise to commit ID theft was so pathetic, I almost feel bad for the guy. And yes, there is a photo. [Lowering the Bar]
* A longtime Arby's employee fled when a knife-wielding robber broke into the restaurant in the middle the night. And then Arby's fired her. At least unemployment > dying alone in an Arby's. [Consumerist]
* Models, runway shows, and confidentiality agreements, oh my! [Fashionista]
Lawsuit of the Day, Nauseating Things
Lawsuit of the Day: The Urinal From Hell
Here's a whole new thing to be terrified about when using a public bathroom…
What Do Lawyers And Celebrities Have In Common?
Besides their good looks and fame, they're also increasing their focus on data security. In the wake of "Celebgate," the Sony Pictures hack, and nearly daily data breaches targeting massive corporations to individuals, law firms are finally recognizing the importance of bringing their cybersecurity policies up to speed.


text:
3 Backyards -- Film Review
The Bottom LineEmpty
PARK CITY -- A slice-of-life story cut too thin, "3 Backyards" is a minimalist glimpse into the lives of a burg of uninteresting people. It's likely exhibition neighborhood will be video-on-demand where cineastes will take a chance on recognizable cast names - Edie Falco, Elias Koteas, Embeth Davidtz.
Set in a bucolic New York coastal enclave, "3 Backyards' intercuts between the residents of three dwellings. Unfortunately, filmmaker Eric Mendelsohn has created such a humdrum assortment of folks that it's hard to get interested in any of their comings-and-goings. In one abode, the businessman husband (Koteas) and his hausfrau wife (Kathryn Erbe) don't communicate, in essence, they don't talk to each other - the marriage has gotten that bad. Unfortunately, they are so thinly drawn that we never care whether they get back together or not.
The second sub-story centers on a woman (Edie Falco) who dabbles away her days as a backyard artist. She also babbles, which she is more skilled at than painting. She's obsessed with the emotionally stricken mini-celebrity (Embeth Davidtz) who has sublet around the bend.
Then, there's this little girl (Rachel Resheff) who innocently wanders into some potentially deadly backyard depravity. There's also a hideous, poofed-up white, designer -dog that ambles throughout the story, which must be some attempt at highbrow symbolism.
The only character we care about is an African woman whose job quest intersects the wanderings of businessman John. In fact, she's the bright light of the whole opus, thanks to actress Danai Gurira's gleaming aura.
Smartly observed and precisely visualized, "3 Backyards" is nonetheless a bore: We never care for any of the characters and their lives of "quiet desperation."
Under Mendelsohn's capable hand, the technical contributions are astute and amplify the story. Special praise to composer Michael Nicholas for the drained sounds; a cacophony of agitated woodwinds continuously underscores the characters' frazzled mindsets.
Venue: Sundance Film Festival
Section: U.S. Dramatic Competition
Production: Caruso-Mendelsohn Prods. In association with Fred Berner Films
Cast: Embeth Davidtz, Edie Falco, Elias Koteas, Rachel Resheff, Kathryn Erbe, Danai Gurira
Director/Screenwriter: Eric Mendelsohn
Producers: Rocco Caruso, Amy Durning
Executive producer: Fred Berner
Director of photography: Kasper Andersen
Production designer: Markus Kirschner
Music: Michael Nicholas
Editors: Morgan Faust, Jeffrey K. Miller
Costume designer: Suzanne McCabe
No rating, 85 minutes
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text:
The Should I Date You Test
Their result for The Should I Date You Test ...
How you doin?
You scored 52 Tinability!
Their Analysis (Vertical line = Average)
They scored 52% on Tinability, higher than 82% of your peers.
All possible test results
Deffinately not!
Please leave. You scare me. Read more
Maybe Friends
You seem okay but I don't think we're meant to be. Here take this dead fish as a token of our new friendship. Wait. Where are you going? Read more
How you doin?
I think there is some deffinate potential here. E-mail me or something and I might let you take me out. You have to pay though. Just kidding. Or am I? No, no. I am. Well no I'm not actually. O... Read more
Marry me?
I'll take your silence as a yes. I'll start making arrangements. But no. Seriously. E-mail me or something. You might be the woman of my dreams. Read more
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St. Thomas, VI (Sports Network) - Ryan Boatright had 16 points, seven rebounds and five assists as No. 23 Connecticut took a 77-71 win over Wake Forest in the first round of the Paradise Jam.
Shabazz Napier also had 16 points for the Huskies (3-0), who will take on Quinnipiac in the second round on Saturday. Enosch Wolf posted 12 points and seven rebounds in the win.
Codi Miller-McIntyre had 21 points, C.J. Harris added 17 points and Travis McKie gave 16 points and eight rebounds for the Demon Deacons (1-1), who had opened their season with a win against Radford last Friday.
"Shabazz finally showed up for us those last (10) minutes," UConn head coach Kevin Ollie said.
Wake Forest had trailed by six about five minutes into the second half, but a 10-0 run capped on 3-pointers from McKie and Arnaud William Adala Moto gave the Deacons a 50-46 lead with 12 minutes to play.
However, the Huskies answered with a 9-0 run a few minutes later to take a lead they never relinquished. Napier capped the burst with a 3-point play and a 58-52 lead with under eight minutes to play.
A few minutes later, McKie hit one of two from the line and later buried a 3- pointer to cut the Deacons' deficit to 73-69 with under a minute to play.
The Huskies ran the clock down to 28 seconds before Napier was fouled and he hit both of his attempts for a 75-69 lead. McKie followed with a layup, but Napier hit two from the line with six seconds left to put the game away.
It took a little bit for the offense to get started in the game, but an 8-0 run by the Huskies saw the team take a 13-6 lead off a Niels Giffey tip-in with 11 1/2 minutes left.
An 8-2 run later in the half by the Deacons saw the team take a 25-22 lead when Miller-McIntyre hit a jumper with 5 1/2 minutes to play.
DeAndre Daniels, though, hit a 3-pointer a few minutes later to give Connecticut the lead back and the team took a 36-32 advantage into the halftime break.
This was the third time these two schools met and the Huskies have won two ...
Connecticut shot 47.2 percent from the floor and 24-of-30 from the free throw
line ... Wake Forest made 49.1 percent of its floor shots, but just 12-of-22
from the stripe.
The overall growth of super conferences across the FCS has gone largely unnoticed in the last decade.
NASCAR placed Sprint Cup Series crew chief Slugger Labbe on suspension for three races and fined him $50,000 for a rules infraction discovered during last Friday's opening-day inspection at Auto Club Speedway in Fontana, California.
Sometimes we all have to jump through a few hoops.
The Cincinnati Reds acquired infielder Dan Johnson from the Houston Astros for a player to be named later or cash considerations on Thursday.
On the day the Houston Rockets are getting Dwight Howard back in the lineup, the team received some concerning news about another of its starting players.
The Buffalo Sabres activated forward Patrick Kaleta from injured reserve on Thursday.


text:
Henrico County news
Business in brief
Citizen Staff Reports 03/26/2015 BusinessBusiness in Brief
Shine Blowdry Bar, Richmond's first salon devoted exclusively to hairstyling and makeup services, opens at 5227 W. Broad St. near Willow Lawn this April. With 10 styling stations, the 1,440 square foot, modern salon is designed to be walk-in friendly. There is no need to make an appointment, with the exception of booking large/private parties or visits outside of regular business hours. At Shine, the experience typically starts with a complimentary cup of coffee or glass of wine (for patrons over 21) and a peek at a "Look Book" for inspiration on various styles, ranging from the bouncy "Starlet" to the sleek "Executive Sweet." Basic blowouts include a shampoo, blowdry and styling at a flat fee $40, no matter the hair length or level of difficulty. Kids under 10 are eligible for "The Twinkle" styling at $25. Updos, braids and on-location styling can be scheduled at additional cost. Hair repair and massage treatments and a variety of makeup services are also available. > Read more.
Governor creates center for mental health services
By Margo Maier, Capital News Service 03/26/2015 General AssemblyGeneral Assembly 2015
Gov. Terry McAuliffe has signed an executive directive to create a center that will foster collaboration among government agencies in providing behavioral health and justice services for people with mental illnesses or other problems.
McAuliffe announced the establishment of the Center for Behavioral Health and Justice during the final meeting of the Governor's Task Force for Improving Mental Health Services and Crisis Response.
"Nearly a year ago, I asked this task force to continue its work and develop bold ideas to help the commonwealth address the gaps in our behavioral health system," McAuliffe said Monday. > Read more.
Logistical excellence
Photos by Roger Walk for the Henrico Citizen 02/05/2015 Business
A Henrico County venue hosted the first RVA LogistXGames Jan. 29, pitting supply chain and logistics industry employees together in a friendly competition designed to judge the teamwork, talent, efficiency and speed of participating teams as they assembled and stacked boxes, moved them through relay courses on pallets and performed other warehouse-type tasks.
The Fort Lee – Joint Strike Group won the event and claimed the "Golden Pallet." The event was sponsored by CBRE Richmond, Liberty Property Trust, the Community College Workforce Alliance and the Virginia Port Authority. The event was held at a Liberty Property Trust facility on Eastport Boulevard in Sandston. > Read more.
Scott earns national Head Start award
Citizen Staff Reports 02/05/2015 EducationGovernment
Congressman Bobby Scott, whose Third District includes a portion of Henrico County, recently received the National Head Start Association's PROMISE (PRotecting Opportunities for our Most Important Students Early) Award for his commitment to advance the needs of Head Start children and families. Scott is the ranking member of the U.S. House Committee on Education and the Workforce.
More than 300 Head Start teachers gathered in Washington, D.C. recently to celebrate the 50th Anniversary of the Head Start program, an early childhood education program. > Read more.
HCPS counselor honored at White House ceremony
Citizen Staff Reports 02/05/2015 Education
Dumbarton Elementary School counselor Liz Parker was honored at a White House ceremony Jan. 30 as a national finalist for 2015 School Counselor of the Year. The American School Counselor Association selects an annual winner for the award.
First Lady Michelle Obama presided over the ceremony honoring the five finalists for the award, as well as semifinalists. Also present was actress Connie Britton, who portrayed a school counselor on the TV show "Friday Night Lights."
The judges considered such criteria as counseling innovations, effective programs, leadership skills and contributions to student advancement. > Read more.
Uber, Lyft soon will be legal in Virginia
By Kevin Lata, Capital News Service 02/05/2015 General AssemblyGeneral Assembly 2015
After a yearlong ban, Uber, Lyft and other app-based ride-sharing services soon will have a law allowing them to operate legally in Virginia.
The House of Delegates on Friday joined the Senate in passing a bill that would set licensing requirements for such services' drivers. The legislation is similar to an interim agreement approved by state officials last August after the Virginia Department of Motor Vehicles sent cease-and-desist letters banning the taxicab-like service from the state.
The House voted 67-28 in favor of HB 1662, sponsored by Del. Tom Rust, R-Herndon. > Read more.
Henrico County property transactions, Mar. 2-8
'All Henrico Reads' author Tayari Jones to speak at Glen Allen High School
Freeman students 'Read Aloud'
Legislative session a mixed bag for immigrants
Vegetable gardening workshop planned
- More News
Print Edition
Mar. 19, 2015Click here to read the print edition or see the archive.
One beauty of a charmer
'An Evening of Country' scheduled April 9-10
Restaurant watch
Weekend Top 10
Weekend Top 10
- More Entertainment
Obituary – Roxana Woodman Leiby
Obituary – William E. Durham
Obituary – Thomas G. Roseme
- More Obituaries
Local couple wins wedding at Lewis Ginter
Fourth-annual Healy Gala planned
Ruritan Club holding Brunswick stew sale
- More Community
Cabin fever cures - and blessings
Getting schooled
Time to give spanking the boot
- More Opinions
Local bowlers win awards at tourney
Athlete of the Week – March 20: Annemarie Beran
Athlete of the Week - March 13: Justice Williams
- More Sports
Weekend Top 10
By Sarah Story, Citizen Events Editor 01/25/2013
Henrico has several family-friendly events to offer this weekend: a mini career day at Virginia Center Commons, a pajama party to benefit Commonwealth Parenting and a family game night at Dorey Recreation Center. Whether you have children or not, saving money these days is vital – a Couponing 101 workshop on Saturday will help you get started! Click here for more! > Read more.
Rec and Parks registration planned
Citizen Staff Reports 01/24/2013
Henrico County Division of Recreation & Parks will host its Winter/Spring 2013 Program Registration on Saturday, Jan. 26 from 8 a.m. to 11 a.m. at the Henrico Training Center, 7701 East Parham Rd., and the Eastern Henrico Recreation Center, 1440 N. Laburnum Ave.
Look for your "At Your Leisure" program guide in the mail or at any Henrico County Public Library. > Read more.
Look for your "At Your Leisure" program guide in the mail or at any Henrico County Public Library. > Read more.
Weekend Top 10
By Sarah Story, Citizen Events Editor 01/18/2013
The Modlin Center for the Arts at the University of Richmond has three events this weekend: the Mark Morris Dance Group, NT Live and "The Ugly Duckling & The Tortoise and the Hare." It's also opening weekend for CAT Theatre's "The 13th of Paris." For all our top picks this weekend, click here! > Read more.
Page 37 of 124 pages ‹ First < 35 36 37 38 39 > Last ›
Restaurant watch
Find out how your favorite dining establishments fared during their most recent inspections by the Virginia Department of Health. > Read more.
From many, one voice
Members of the Shenandoah Valley Children Choir with conductor Jo-Anne Van Der Vat-Chromy perform at the St. Michael Catholic Church in Glen Allen during a jazz concert of the One Voice Chorus.
The concert, entitled "A Little Jazz'll Do Ya !" entertained the audience with "cool contemporary and classic jazz favorites (including J. Hendricks, G. Gershwin, A. Jobim and R. Rogers). It was a joint performance of the One Voice Chorus, the Russell Wilson Jazz Combo and the Shenandoah Valley Children Choir. > Read more.
Weekend Top 10
Don't give Dad another tie. Give him an experience to remember! Dads who like beer and meat will love the inaugural Moo and Brew taking place tomorrow. Dads who like cars will appreciate the 45th annual Richmond AACA Car Show and Swap Meet at RIR. Dads who love ice cream are sure to enjoy Walkerton Tavern's ice cream social on Sunday. And Dads that just need a little down time can relax with music, food and family activities at Lewis Ginter Botanical Garden. For all our top picks this weekend, click here! > Read more.
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ClassifiedsPROFLOWERS. Send Flowers for Every Occasion! Anniversary, Birthday, Just Because. Starting at just $19.99. Go to http://www.proflowers.com/Celebrate to receive an extra 20 percent off any order over $29.99 or… Full text
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CalendarThe Latin Ballet will present "Macondo," a performing arts interpretation based on the novel "Cien Anos de Soledad" (One Hundred Years of Solitude) written by Nobel prize-winning author Gabriel Garcia… Full text


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Books Worth Reading
Books Worth Reading
- 8 Pins
Related Boards
Interesting and informative breakdown
Book Lovers, Reading Infographic, Infographic Book, Flow Charts, Reading Books, Random House, Book Reading, Books To Read, Reading Lists
A few more to add to my stupidly long reading list.
What Do You Feel Like Reading? #books #reading
find a book to read
what do you feel like reading flow chart
What Do You Feel Like Reading? #Infographic #books #reading Random House!
This infographic can help you to buy for your book lovers this holiday! #infographic #giftguide #booklover #christmas #holiday #gifts
What Do I Feel Like Reading [Infographic]
A literary thumbprint: the books that have defined your life fill out your thumbprint.
Book Lovers, Literary Thumbprint, Book Title, Thumb Prints, Cherylsorg, Life Filling, Thumbprint Portraits, Fingerprints Art, Cheryl Sorg
Custom Literary Thumbprint by cherylsorg via bookpatrol: The books that have defined your life fill out your thumbprint. You send your thumbprint and list of up to 50 book titles and the artist creates your 3ft-tall print: 400.00
Could be neat to have students make an identity thumb print! or Character traits thumbprint! Idea inspired by this: Custom Literary Thumbprint by cherylsorg via bookpatrol: The books that have defined your life fill out your thumbprint. #Portrait #Thumbprint #Books
Personalized Literary Thumbprint: Cheryl Sorg creates a meter-high portrait from book titles that have shaped your worldview.
Custom Book Lover Thumbprint Portrait by cherylsorg on Etsy,
Fingerprint Art
Get Your Literary Thumbprint - Book Patrol
If you're a YA reader looking into adult books or an adult looking to try YA, check out this "crossover" guide for a recommendation on where to start!
Website, Young Adult, Addict Guide, Adult Book, Adult Fiction, Book Infographic, Crossover Book, Infographic Reading, Book Addict
"Crossover" Books: Young Adult and Adult books that can appeal to many ages! [An Infographic] #infographic #youngadult #ya #books (from The Book Addict's Guide)
Looking to cross over from YA to contemporary adult fiction? This is a great infographic from The Book Addict's Guide!
Crossover Books Infographic Reading Guide
The Book Addict's GUIDE… to "Crossover" Books!
11 books to read after college
Book Lists, College Graduation, Gotta Reading, Future Reference, Books For College Graduates, Book To Reading, Books To Read In Your 20S, 11 Book, Career Books
#Books You Need to Read in Your 20s | | career books, careeradvice, books
Gotta read these!!
For future reference
Some awesome picks. 11 books to read after college graduation.
THIS ONE ACTUALLY HAS THE BOOK LIST 11 books to read after college
Hot Teas, Life, Quotes, Cups Of Coffe, Sweets Teas, True, Hot Chocolates, Cup Of Coffee, Good Books
Or tea. . . or hot chocolate . . . or sweet tea . . . or beer . . . .or a glass of wine . . . .just so long as it is with a book
Alas, my good book will be replaced by a text book and a cup of coffee will turn into 10. Got to love the life of a uni student!!
Or a cup of hot tea. Or hot chocolate. With Marshmallows. Ohhhh yeah!
I am perfectly content with a good book and a hot cup of coffe.
my life in pictures quotes - Google Search
In short supply
Inspiration, Life, Quotes, Limited Editing, Funny, True, Things, I M Limited, Weird
It's true. #weird #limited #edition #typography
Inspiring image quotes, live, Best, weird, like, life #1595694 by lovely_jessy - Resolution 386x377px - Find the image to your taste
When I was in grade school some of my classmates used to tell me I was weird. When I was in high school the same girl and her new friends would say the same thing. Far from it, I am not weird, I'm unique, I'm special, because I am nothing like you! And I'm nothing like those you hang out with..totally different and still loving it!
Im not weird. Im limited edition. #funny #quote #thisissome
i'm not weird. i'm limited edition. #quote #inspiration #positive #quote #strength #unique
I m limited edition
Herds of Nerds, and I among them.
Quentin Blake, Book Lovers, Book Worms, Funny Pictures, New Book, Roald Dahl, Funny Quotes, Bookworm, True Stories
I had some great new #books for Christmas pressies - can't wait to morph into bookworm mode!
Bookworms will rule the world! ...As soon as we finish one more chapter... If you're a book worm, you'll notice that that is Matilda!!!
Bookworms! (And Roald Dahl!)
Quentin Blake - This is from Matilda by Roald Dahl.
Matilda - Roald Dahl. On books and book lovers
Bookworms. True story.
Dump A Day dumpaday, funny pictures, funny quotes (1) - Dump A Day
A great way to teach points of view in writing...and not to mention, it's funny!


text:
David Shrum, the president of the board of directors; and Toni Elliot, the executive director, both of the Haven Men's Shelter in Rock Hill, join Manning Kimmel on Straight Talk, along with Calvin, one of the Haven's success stories.
Podcast: Play in new window | Download | Embed
Download our new mobile apps for iOS and Android Devices. Find out more here.
Click here to sign up for eAlerts from WRHI.


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Reward Points
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Log in to see how many reward points you have already earned
I must say, I was quite impressed with MJM Magic my first time ordering through them! A friend of mine referred me to the site, and at first I was...
Great Service
I always kick myself for wasting time shopping around. I KNOW MJM always has what I need and for a better price. I can't say enough about how much I...
MJM rocks
Everything about MJM is awesome. From the free shippping over $20, to the 3% off just about everything in the store and the 10% lifetime discount....
Highly Recommended Highly Recommended
Item received great service and fast shipping, communication with this store is really great! Thanks from 'Italy
Best Magic Site!
I have placed various orders with MJM Magic and everytime, my order got here really fast, the items were packaged well, everything just as...


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(904): View more from Florida
I keep pulling short curlies out of my mouth. Not cool
Tap Here to view the Mobile Optimized TFLN
I keep pulling short curlies out of my mouth. Not cool
Anddd after the worst sex of my life, he said.."do you mind taking off the condom, tying it up, and throwing it at the door?" Weird.
His second form of ID was an emergency room wristband from an hour ago. What the fuck is going on right now
Haha yeah he had an allergic reaction to the alcohol earlier. He thinks that if he only drinks vodka he will be ok...
i make up for being a shitty girlfriend by being amazing in bed.
he bit the head off a dead goose for 5 beers. this is my future boyfriend.
Also, new rule: You are no longer allowed to send me a text with the word "dildo" in it before 10am.
I don't remember anything but yelling at the ref in Spanish.
I feel like I have streams of color and coldness wrapping around my body.
FYI I'm about to upload a vid of you to facebook of you screaming "SNACK ATTACK" and throwing cheetos at everyone playing pong...
Wish i knew who the f is sending me pics of asian newborns.
I wanna die of smoke inhalation. In a huge teepee. Or one of those big things kids in kindergarten have that you throw up in the air then sit inside of.
She is the epitome of a puke & rally. She picked a random hott guy at the bar & made him pinky promise not to leave while she took a power nap. She went & passed out in her friends car & apparently puked just outside the bar. She stumbled in & found the randome guy again & claimed she was golden. Made it to the after party & stayed up til 6 doing body shots off every girl she saw & hooked up with the random from the bar. I love her life
I gave him a bunch of ideas to use to spice up their sex life. Say what you will, I am the best 'other' woman ever!
Would I be a horrible mom if I got a babysitter at 6am so I could go get laid.


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Page 1
Conformations of p53 response elements in solution
deduced using site-directed spin labeling and
Monte Carlo sampling
Xiaojun Zhang1, Ana Carolina Dantas Machado2, Yuan Ding1, Yongheng Chen2,
Yan Lu2, Yankun Duan2, Kenneth W. Tham1, Lin Chen1,2, Remo Rohs1,2,* and
Peter Z. Qin1,2,*
1Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA and2Department of
Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA
Received June 8, 2013; Accepted November 4, 2013
recognizing diverse p53 response elements (REs).
Understanding the mechanisms of p53-DNA inter-
action requires structural information on p53 REs.
However, such information is limited as a 3D
structure of any RE in the unbound form is not avail-
able yet. Here, site-directed spin labeling was used
to probe the solution structures of REs involved in
p53 regulation of the p21 and Bax genes. Multiple
nanometer distances in the p21-RE and BAX-
nitroxideprobe and
resonance spectroscopy, were used to derive
molecular models of unbound REs from pools of
all-atom structures generated by Monte-Carlo simu-
lations, thus enabling analyses to reveal sequence-
dependent DNA shape features of unbound REs
in solution. The data revealed distinct RE con-
core domain, and support the hypothesis that
sequence-dependent properties encoded in REs
are exploited by p53 to achieve the energetically
most favorablemode
quently enhancing binding specificity. This work
reveals mechanisms of p53-DNA recognition, and
approach for studying DNA shape in solution that
has far-reaching implications for studying protein–
DNA interactions.
tumor suppressor
The tumor suppressor protein p53 plays various essential
roles in maintaining the integrity of the human genome.
Sequence-specific binding of the p53 core DNA-binding
domain (DBD) to its response elements (REs) is a key
component of the regulation of a large number of signal-
ing pathways (1). The importance of DNA recognition by
p53 is highlighted by the fact that >80% of missense
mutations of p53found
located within the DBD (2), and many cancer hot-spot
mutants have been shown to impair recognition of target
DNAs (3).
The p53 REs are defined by two closely spaced
A,T; Y=C,T; n=spacer of length 0–20 base pairs
(bp)], and hundreds of them have been validated in
human and mouse (1). The mechanisms by which p53 spe-
cifically recognizes its REs have been a long-standing
question (1,3). p53 is known to recognize REs using
base readout in the major groove, as exemplified by the
bidentate hydrogen bonds between Arg280 and the
conserved guanines in the CWWG core (3). The import-
ance of DNA shape readout has also been noted: Arg248,
the most frequently mutated residue in human cancers,
recognizes its DNA target through readout of minor
groove geometry and electrostatic potential (4); similarly,
another cancer hot-spot residue, Arg273, plays a role in
maintaining DNA shape (5).
Intriguingly, several recent crystal structures of tetra-
meric p53DBDs bound to full REs have revealed
various deformations of the bound DNA (4,6–9), suggest-
ing a propensity of DNA conformational change upon
formation of the p53/RE complex. However, it remains
inhumancancers are
*To whom correspondence should be addressed. Tel: +1 213 821 2461; Fax: +1 213 740 0930; Email: firstname.lastname@example.com
Correspondence may also be addressed to Remo Rohs. Tel: +1 213 740 0552; Fax: +1 213 821 4257; Email: firstname.lastname@example.com
Published online 30 November 2013 Nucleic Acids Research, 2014, Vol. 42, No. 42789–2797
? The Author(s) 2013. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial
re-use, please contact email@example.com
at University of Southern California on March 18, 2014
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Page 2
unclear to what degree the observed DNA deformation
may be biased by crystal packing, and more importantly,
how inherent variations of the shape of REs impact the
mode of conformational change upon p53 binding.
Answering these important questions requires a compari-
son of the bound and unbound RE conformations in
solution. The latter is severely lacking—no atomic reso-
lution structure of any unbound p53 RE has yet been
The challenge of deducing DNA conformation in
solution is not limited to p53 REs. Current knowledge
on sequence-dependent DNA shape, particularly for free
DNA, is rather inadequate despite its important role in
protein–DNA recognition (10). For instance, sampling of
sequence versatility in structure databases is insufficient,
with the Dickerson dodecamer accounting for ?10% of all
DNA entries in the Nucleic Acid Database (11), and
analyses of short non-coding DNA sequences in several
eukaryotic genomes conclude that none of the abundant
sequences have had their structures determined (12). These
limited experimental data on intrinsic DNA shape is due,
in large part, to the difficulty of obtaining unbiased struc-
tural information of 'naked' DNA. Whereas high-reso-
lution structures of free DNA have been obtained by
X-ray crystallography and NMR spectroscopy, their
number is small compared with available data for
protein–DNA complexes (13). In addition, X-ray crystal-
lography studies are hindered by crystal-packing biases,
and NMR studies are constrained by the size of the DNA.
Here, we introduce a new experimental/computational
pipeline, in which the method of site-directed spin labeling
(SDSL) is combined with all-atom Monte Carlo (MC)
simulations to derive atomic resolution data representing
the sequence-dependent conformation of DNA duplexes
in solution. SDSL uses electron paramagnetic resonance
(EPR) spectroscopy to monitor nitroxide radicals (i.e. the
spin labels) attached at specific sites of biomolecules, and
has matured as a tool for studying the structure and
dynamics of proteins and nucleic acids (14,15). The MC
simulation technique was shown to enable efficient con-
formational sampling and was extensively validated using
massive experimental data from X-ray crystallography,
NMR spectroscopy and hydroxyl radical cleavage experi-
ments (13,16). In our new SDSL-MC scheme, a pulsed
EPR technique,double-electron-electron-resonance
(DEER) (17), is applied to measure distances between
nitroxide pairs attached to the target DNA duplex.
These distances are then used as constraints to query a
large pool of all-atom models generated by MC simula-
tions (18,19), thereby identifying those that conform to the
experimental measurements. The SDSL-MC approach is
not limited by the size of the DNA or the requirement of
crystalline samples, and provides a new method for
examining the sequence-dependent shape of DNA in
This work presents studies on two prototypic naturally
occurring p53 REs (1): the p21-RE with no spacer (0-bp)
between the two half-sites; and the BAX-RE with a 1-bp
insertion between the two half-sites (Figure 1A). Using the
SDSL-MC approach, conformations of the unbound p21-
REandBAX-REwere determined insolution.
Comparing the unbound and bound DNA revealed con-
formational changes in the central region between the two
half-sites on protein binding, which allow formation of
key protein–DNA and protein–protein contacts. The
modes of conformational
between the two REs, could be linked to properties that
are encoded in the individual nucleotide sequences, sug-
gesting a possible means to achieve binding specificity
through sequence-dependent conformational changes.
DNA spin labeling
DNA oligonucleotides were synthesized by solid-phase
chemicalsynthesis (Integrated
Coralville, IA, USA). Following previously reported
protocols, the R5 spin labels (1-oxyl-2,2,5,5-tetramethyl-
pyrroline, Figure 1B) were attached to specific DNA sites
using the phosphorothioate scheme, and the labeled DNA
was purified by HPLC (20). Concentrations of labeled
DNA were determined by absorbance at 260nm. In this
work, the Rp and Sp phosphorothioate diastereomers
present at each attachment site were not separated.
Previous studies using model systems have validated the
use of Rp/Spmixtures in DEER measurement and estab-
lished an appropriatemethod
measured inter-nitroxide distances (21–23).
forinterpreting the
EPR sample preparation and DEER spectroscopy
measurements of inter-spin distances
described in (9). Each DEER sample contains 50–
100mM of labeled DNA or p53DBD-DNA complex,
50mM HEPES (pH 7.5), 100mM NaCl, 5mM MgCl2
and 40% (v/v) glycerol. DEER measurements were
carried out at 80K on a Bruker ELEXSYS E580
X-band spectrometer equipped with a MD4 resonator.
Previously reported acquisition parameters and proced-
ures (24) were used. Inter-spin distance distributions
were computed from the resulting dipolar evolution data
using DEERanalysis2011 (25). From these distance distri-
bution profiles, the average distance (r0) and the width of
distance distribution (?) were calculated as reported pre-
viously (21). Repeated measurements indicated that errors
in the measured r0are less than 1A˚.
sampleswerepreparedfollowing procedures
Generation of DNA models using Monte Carlo
A previously published protocol was used to generate an
MC ensemble of all-atom DNA models with sequence-de-
pendent shape (18,26). Simulations started from idealized
B-DNA structures generated without any sequence-de-
pendent structural features. The MC sampling was based
on collective and internal degrees of freedom using the
AMBER force field implemented as previously described
Jacobians(19) andexplicit
combined with an implicit solvent description (27). MC
sampling was performed >1 million cycles with random
2790 Nucleic Acids Research, 2014,Vol.42, No. 4
at University of Southern California on March 18, 2014
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Page 3
conformational changes of all degrees of freedom in each
cycle. All-atom structures were recorded every 100 MC
cycles, forming an MC ensemble of 10000 structures for
each simulation.
Computation of expected inter-R5 distances
Our previously validated NASNOX program (20,22) was
used to calculate expected inter-R5 distances (rmodel).
Briefly, with each DNA model, the program modeled R5
at the target site, then identified sterically allowed R5 con-
formers using the following search parameters [see (20) for
details on these parameters]: t1 steps: 3; t2 steps: 6; t3
steps: 6; fine search: on; t1 starting values: 180?; t2
starting values: 180?; t3 starting values: 180?; and no add-
itional conformer search criterion. Searches were carried
out separately for the Rpand Spdiastereomers (i.e. R5
attached to the O1P or O2P atom), and the results were
combined to yield the ensemble of allowable R5 confor-
mers at the given labeling site. The rmodelbetween two-
specific labeling sites was then calculated by averaging
all inter-R5 distances between the two corresponding R5
ensembles. Controls showed that varying the search par-
ameters resulted in <1A˚difference in rmodel.
For the bound REs, expected inter-R5 distances (rcrystal)
were computed based on the reported crystal structures:
PDB ID 3TS8 for the p21-RE (8); and 4HJE for the
BAX-RE (9). A modified version of the NASNOX
program was used to account for the presence of protein
Characterization of DNA duplex models
For a given model j of a DNA duplex, we defined a
scoring function Ptas:
where i designates a particular distance in the DNA
duplex, rmodel-j is the NASNOX computed expected
distance for the model j, r0 is the DEER measured
average distance and ? is the measured distance distribu-
tion width. Heavy atom root-mean-square-deviations
between DNA models (RMSDstruct) were calculated
using the program VMD (28). Calculations included,
unless otherwise stated, only the interior of the duplex
andexcluded twobase-pairs
DNA structuralparameters
? ðri
model?j? ri
DEER-measured distances reveal that p53DBD binding
induces RE conformational changes
To examine the conformation of REs, a pair of R5 probes
(Figure 1B) were attached at specific DNA sites using a
Figure 1. Experimental design and example DEER results. (A) Nucleotide sequences of the REs. The p21-RE has no spacer between the two half-
sites and is located 50upstream of the promoter of the CDKN1A gene involved in controlling cell cycle arrest. The BAX-RE has a 1-bp spacer
(indicated by the lower-case letter) and is present at the promoter of the Bax gene. For each RE, the numbering scheme of the phosphates is shown
next to each strand. The two half-sites are noted, with the CWWG core of each half-site shown in bold and the dotted box marking the central
region between the two half-sites. Colored dotted lines and corresponding phosphates designate, as examples, the measured distance sets shown in
(C). (B) Chemical configuration of the R5 nitroxide probe. (C) Examples of DEER data. Each data set is designated by the RE and the corres-
ponding labeling sites. DEER spectra measured in the absence (straight line) and presence (dashed line) of p53DBD are shown, with black dotted
lines included to aid comparison. See Supplementary Figures S3 and S5 for additional DEER data and analyses.
Nucleic Acids Research,2014, Vol.42, No. 4 2791
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previously established phosphorothioate scheme (20,21),
and inter-R5 distances were measured by DEER. Each
measured distance was designated by the corresponding
labeling site numbers, for example, data sets [14; 34] in
p21-RE represents the distance measured with a pair of
R5 attached to the phosphorothioates of nucleotides C14
and C34(Figure 1A). For all double labeled REs, the
measured dipolar evolution traces showed an oscillating
decay pattern (see Figure 1C for examples), from which
the inter-R5 distances were determined. Control meas-
urements on single-labeled
(Supplementary Figure S1), thus ensuring that the dis-
tances measured in the double-labeled DNA were not
biased by spin interactions due to undesired sample aggre-
gation. In addition, previous studies have demonstrated
that R5 did not significantly distort the DNA duplex,
and the measured distances accurately reported on the
native structure (21,22,30).
We measured multiple distances in REs in the absence
and presence of p53DBD to examine protein-induced
DNA conformational changes. In these measurements,
we chose labeling sites with minimal perturbation to p53
binding and we confirmed p53DBD-DNA complex for-
mation by gel shift assays (Supplementary Figure S2).
For the p21-RE, 2 of the 6 data sets, [14; 34]
(Figure 1C) and [13; 34] (Supplementary Figure S3D),
showed clear differences in the measured echo evolution
traces on p53DBD binding. The differences in r0between
the unbound and bound DNA were 5A˚and 3A˚, respect-
ively (Supplementary Table S1), which are well beyond the
error of r0 measurements (±1A˚). In addition, control
studies indicated that changes in R5 conformers on
p53DBD binding were minimal and were unlikely to
account for the observed r0 changes (Supplementary
Figure S4). As such, distance changes observed in data
sets [14; 34] and [13; 34] clearly revealed p21-RE conform-
ational changes on p53DBD binding. On the other hand,
the remaining four datasets, including [9; 24] shown in
Figure 1C, gave superimposable dipolar evolution traces
in the absence and presence of p53DBD, resulting in little
orno changesinr0
Supplementary Figure S3D).
For the BAX-RE, 10 sets of distances were measured in
unbound and bound DNA (Supplementary Table S1,
Supplementary Figure S5). Interestingly, the observed
distance changes were much smaller than those observed
for the p21-RE. The only noticeable distance changes were
from data sets [15; 36] (Figure 1C) and [14; 36]
(Supplementary Figure S5D), both at 2A˚, whereas the
remaining 8 data sets showed little or no changes in r0
(Supplementary Table S1, Supplementary Figure S5D).
Overall, in both the p21- and BAX-RE, p53 induced
distance changes were detected by DEER, thus unambigu-
ously revealing RE conformational changes on interacting
with p53. However, with the presence of both variable and
invariable distances, it was difficult to intuitively deduce
the mode of RE conformational changes, even though
crystal structures of both bound DNAs were available
(8,9). This motivated us to 'solve' the conformation of
the unbound REs in solution.
REsgave flattraces
Conformations of unbound REs in solution
To obtain all-atom models of unbound REs, we imple-
mented a strategy that has been successfully used in
SDSL mapping of an RNA junction (24), namely, to use
the DEER measured distances to select sterically accept-
able models from a large pool of 3D structures. In our
approach, these structures were generated by unrestrained
MC simulations (18). For each structural model, the
NASNOX program (20) was used to select sterically
allowed R5 conformers at the respective labeling sites,
from which the relevant average inter-R5 distances
(rmodel) were obtained. We then computed a scoring
function Pt for each model (Equation 1, 'Materials
measured and predicted average distances (r0
rmodel) and the width of the measured distance distribu-
tion (?). Pt represents, under the assumption of an
idealized normal distribution, the effective probability
of a given set of rmodel values that match the corres-
ponding r0values, with a perfect match resulting in a
maximum Ptscore of 1.
For the p21-RE, we used 16 sets of DEER-measured
distance (Supplementary Figure S3, Supplementary Table
S2) to compute Ptfor a pool of 10000 models obtained
from 1 million MC cycles. The top-ranked model had a Pt
score of 0.64 (Figure 2A, Supplementary Table S2), cor-
responding to on average a 97% probability of matching
each rmodelto the corresponding r0(0.9716&0.64). For this
between rmodel and r0 (RMSDdeer) was 0.81A˚, and the
largest difference between a corresponding set of r0and
rmodelwas 1.8A˚(Supplementary Table S2). Given that r0
and rmodeleach might incur errors of ±1A˚, differences
below 2A˚were deemed insignificant. The results indicated
that the top-ranked MC model satisfies all the measured
To characterize variations in EPR-derived p21-RE
models, we adopted a commonly used approach in
NMR studies and further analyzed the 20 MC models
with the highest Pt scores. The pairwise RMSDstruct
among this top-20 ensemble was (1.0±0.3) A˚
age±standard deviation, same below). This suggested
that models that conformed to the measured distances
were structurally similar (Supplementary Figure S6). In
addition, we carried out a search using only 14 of the 16
measured distances, which yielded the same top-ranked
model and a similar top-20 ensemble (Supplementary
Table S3). Overall, the data indicated that within the reso-
lution of the method, the 16 measured distances were suf-
ficient to identify a set of all-atom MC models with
convergent conformations.
We also examined models of generic B- and A-form
(Figure 2B and Supplementary Table S2), which therefore
exhibited uniform shapes without sequence-dependent
characteristics. As expected, a uniform 20-bp A-form
duplex, which drastically differed from the top-ranked
p21-RE model with an RMSDstruct of 5.0A˚, fitted
poorly to the measured distances (Pt=1.0?10?17;
RMSDdeer=8.5A˚) (Supplementary Table S2). More
takingintoaccount the
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importantly, a uniform 20-bp B-form duplex (Figure 2B)
also yielded a low Pt score (3.3?10?6) and a high
RMSDdeer value of 3.9A˚
indicating that this generic B-DNA did not conform to
distances measured for the p21-RE. The generic B-DNA
had an RMSDstructof 2.9A˚when compared with the top-
ranked p21-RE model, which was larger than the variation
among the top-20 EPR-derived models (Figure 2C).
Therefore, the 16 sets of distances yielded a converged con-
formation of the unbound p21-RE with a sequence-de-
pendent shape.
Using the SDSL-MC approach, we also obtained
all-atom models for the unbound BAX-RE with 18
sets of measured distances (Supplementary Figure S5,
Supplementary Table S4). The top-ranked BAX-RE
model(Figure 2D)had
(Supplementary Table S4), i.e. an average 98% probabil-
ity of matching each rmodelto the corresponding r0. This
top-ranked MC model again differed from a 21-bp generic
B-form duplex (Figure 2E) and satisfied all the DEER
measureddistances (Supplementary
addition, the pair-wise RMSDstruct among the top-20
BAX-RE models was (1.0±0.3) A˚, indicating a high
degree of structural similarity (Supplementary Figure
S6). Furthermore, control studies showed that the use of
18 sets of distances was sufficient to identify a set of
(Supplementary Table S2),
score of0.67
TableS4). In
all-atom models with convergent conformations for the
unbound BAX-RE (Supplementary Table S5).
Assessing p53DBD bound RE conformations in solution
To evaluate the bound RE conformation in solution, we
compared the expected average inter-R5 distances based
on the crystal structures (rcrystal) with the corresponding
DEER measured r0values (Supplemental Table S6). For
the p21-RE, the four distances spanning the central region
between the two CATG cores showed differences ranging
between ?1.4 and 1.0A˚, which were within the variability
range of 2A˚in our measurements. This indicated that
within the resolution accessible to our method, the
crystal structure of the bound p21-RE central region ac-
curately reflected its conformation in solution. The same
conclusion has also been previously drawn regarding the
central region of the bound BAX-RE (9) (see also
Supplementary Table S6).
Despite the general agreement observed at the central
region, four equivalent distances, each one measured
across one of the four CWWG cores presented in the
p21- and BAX-RE, showed that the measured r0values
exceeded the corresponding
(SupplementaryTable S6).
(Supplementary Figure S2), although r0did not change
Figure 2. Characterization of the unbound p21-RE (A–C) and BAX-RE (D-F). (A) Top-ranked MC model of the unbound p21-RE. (B) Uniform
20-bp B-DNA model constructed with a standard set of base-pair parameters (Helix twist: 35.9?; X-displacement; ?0.66A˚; and C2'-endo sugar
pucker). (C) Data mining of MC-generated unbound p21-RE models using EPR-derived distances. X-axis: Ptcomputed based on 16 measured
distances in the unbound p21-RE duplex. Y-axis: RMSDstructcomputed against the top-ranked MC model. Data points corresponding to uniform
B-DNA (cyan) and bound DNA from PDB ID 3TS8 (red) are also included. (D) Top-ranked MC model of the unbound BAX-RE. (E) Uniform
21-bp B-DNA model. (F) Data mining of MC-generated unbound BAX-RE models using 18 EPR-measured distances. All color codes are the same
as those in (C), except that the bound DNA data point (red) was obtained using PDB ID 4HJE.
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upon p53DBD binding (Supplementary Table S1). In
addition, these r0values were substantially larger than
the corresponding rcrystaldistances obtained from other
crystal structures (4–6). Taken together, the data indicated
that in solution the CWWG core conformation in the
bound REs likely differed from conformations captured
in the crystal (see 'Discussion'). At this point, however,
extensive protein–DNA contacts involved in the CWWG
regions severely limited our ability to measure informative
distances to derive an EPR-based model, and bound con-
formations of the CWWG core regions of the REs could
not be deduced with certainty.
Distinct conformational changes at the central region of
the REs on p53DBD binding
The unbound p21-RE conformation derived from the
reported in the crystal structure with an RMSDstructof
2.6A˚, which was beyond variations among the top-20
models (Figure 2C, Supplementary Figure S7A). We
modeledthe unboundDNAs
complex by aligning half-site 1 (i.e. nucleotides A3-C10/
G31-T38; Figure 1A). This half-site could be aligned rea-
sonably well between the bound and unbound DNAs,
with an RMSD of 1.4A˚between heavy atoms of the
aligned nucleotides (Figure 3A). However, with half-site
1 aligned, the bound and unbound DNAs deviated at half-
site 2 (i.e. C11–T18/A23–G30) with an RMSD of 8.4A˚
(Figure 3A). Apparently, if the protein tetramer were
maintained, the unbound DNA conformation would not
allow proper protein–DNA contacts (e.g. R280 to G7,
G17, G27and G37, Figure 3A) to form simultaneously at
both half-sites. This likely caused a deformation at the
central region of the bound p21-RE, resulting in a previ-
ously noted displacement between the helix axes of the two
half-sites (Supplementary Figure S8) (8).
For the BAX-RE, the RMSDstructbetween the unbound
and bound DNA was 2.1A˚ (Figure 2F). This is smaller
than that of the p21-RE, and indicated that the BAX-RE
underwent a more subtle p53-induced conformational
change (Figure 3). When we aligned the unbound and
bound BAX-RE based on half-site 1 (i.e. nucleotides
A3-A10/T33-T40), the bound DNA was superimposed
bytheensemble oftop-20
(Supplementary Figure S7B), and half-site 2 (i.e. nucleo-
tides A12-C19/G24-T31) of the top-ranked unbound BAX-
RE deviated from the corresponding segment of the
bound DNA with an RMSD of 3.9A˚(Figure 3B). These
observations were consistent with the small distance
changes observed at the central region of BAX-RE on
p53 binding (Supplementary Table S1), and might
suggest that the unbound BAX-RE was poised to
interact with the p53 tetramer due to its sequence-depend-
ent shape (Figure 3B). Most noticeably, for the 9-bp
centralregion spanning
(Figure 1A), the unbound BAX-RE was under-wound
by ?15?compared with a generic B-DNA. This facilitated
the transition into the p53 bound form, in which further
unwinding at the central region has been observed (9).
from the bound DNA
intothe tetrameric
unboundRE models
We established a new SDSL-MC approach to study con-
formations of two prototypic p53 REs in solution. The
unbound RE conformations, obtained using multiple
measured nanometer distances as constraints, were within
the B-DNA family while exhibiting sequence-dependent
structural properties distinct from a uniform B-DNA. In
both REs, p53-induced DNA deformations were detected
at the central region between the two half-sites. The results
indicate that sequence-dependent shapes of the unbound
RE influence the mode of DNA conformational changes
upon interacting with p53, which thereby may serve as a
mechanism to achieve p53-RE binding specificity.
Sequence-dependent conformational changes of REs on
p53DBD binding
Previous biochemical and computational studies have sug-
gested changes of RE conformations upon p53 binding
(31–33). However, the molecular details of the DNA con-
formational changes and their relationship to individual
RE sequences remained rather unclear. Earlier work sug-
gested that the bound REs undergo bending at the
CWWG region (31). However, recent structural (4–9)
and biochemical (33) studies of p53DBD bound REs
Figure 3. Conformational changes in REs upon p53 binding. In each
panel, shown on the left is the superimposition of the SDSL-MC-
derived unbound RE onto the corresponding co-crystal structure of
the complex, with the blue sticks representing the Arg280 residues;
and the CPK representation denoting the G7, G17, G27and G37nu-
cleotides in the RE. Shown on the right are schematic representations
of the bound and unbound REs. (A) p21-RE, with DNAs aligned at
nucleotides A3-C10/G31-T38(dashed box). In this work, the p53 con-
struct included only the wild-type DBD, whereas the co-crystal struc-
ture of the complex (PDB ID 3TS8) included the DBD covalently
linked to the oligomerization domain without the wild-type linker
present, but with mutations in both domains (7,8). (B) BAX-RE,
with the DNAs aligned at nucleotides A3-A10/T33-T40(dashed box).
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showed generally rather small bending. Instead, in a
number of crystal structures, deviations from canonical
B-DNA characteristics were noted at a confined location
between the two half-sites (i.e. the central region) (4,6–9).
In this work, SDSL measured distances unambiguously
demonstrated that in solution p53DBD binding induces
conformational changes at the central region of the p21-
and BAX-RE (Figure 1C, Supplementary Table S1).
Perhaps unexpectedly, the degree of p53 induced DNA
alteration was more subtle in the 1-bp-spacer BAX-RE as
compared with that in the 0-bp-spacer p21-RE (Figure 3),
whereas the p53DBD bound complexes exhibited a similar
tetrameric scaffold for both REs (9). This provides a hint
that sequence-dependent structural properties encoded in a
particular DNA target are exploited by p53 to achieve the
energetically most favorable mode of deformation. This hy-
pothesis is further supported by structural analyses of
unbound REs, which were enabled by the all-atom
models provided by the new SDSL-MC method. The
analyses revealed RE shape variations and suggested
tangible connections between structural features in the
unbound and bound DNA (Figure 4). For the BAX-RE,
larger positive Roll of the T9pA10 base pair step was
already apparent in the unbound DNA (Figure 4C). Such
intrinsic property of the TpA step facilitated widening of
the minor groove (10), which was observed in the bound
form (Figure 4A). In addition, the unbound BAX-RE was
under-wound at the central region (Figures 3B and 4), thus
facilitating further unwinding to accommodate the 9-bp
central region into the same volume occupied by 8 bp in
other REs with 0-bp spacers (9). On the other hand, in the
unbound p21-RE the relative positioning of the two
CWWG cores deviated significantly from the bound
form, necessitating a shift of the helix axis at a 'hinge'
located at the interface between two half-sites (Figure 3).
Further analyses indicated that conformational changes at
the central region of the REs occurred to facilitate proper
protein–DNA interactions, while at the same time main-
taining the intra- and inter-dimer protein contacts (Figure
3). As the collective protein–DNA and protein–protein
contacts give rise to cooperative binding, sequence-depend-
ent conformational changes at the central region of REs
thus may modulate cooperativity in p53-RE interactions,
thereby contributing to specific RE recognition.
For both bound p21- and BAX-RE, the SDSL data
indicated thatsolution-state
CWWG cores deviated to a certain degree compared
with the corresponding crystal structures despite the
good agreement in the central regions (Supplementary
Table S6). Although we cannot rule out the possibility
that this discrepancy might be due to the fact that the
CWWG cores and the central region were differentially
impacted by differences in experimental conditions (e.g.
frozen solution versus crystal; difference in constructs,
see Figure 3 caption), the SDSL data may also reflect an
intrinsic variability of the CWWG core as suggested by
previous studies (4–6,9,31,33). In particularly, the ApT
steps within the CWWG cores have been reported in
conformations ofthe
Figure 4. Analyses of p53 RE structures. The DNA shape parameters (A) minor groove width, (B) helix twist and (C) roll are shown for the p21-RE
(left panel) and the BAX-RE (right panel). Structural features were derived from the crystal structures of the complexes (red), the top-ranked MC
models (green) and the averages of the top-20 MC models (blue). The error bars indicate the standard deviations of structural parameters among the
top-20 models, demonstrating an efficient conformational sampling. The structural parameters indicate that the conformations observed in the crystal
structures of the bound forms were partially apparent in the intrinsic DNA shape of the unbound forms. Examples for this observation are the low
helix twist values at the C10pC11step of the p21-RE and the A10pG11step of the BAX-RE, as well as the negative Roll at the A12pA13step of the
p21-RE and the positive Roll at the T9pA10step of the BAX-RE.
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either Watson–Crick (6) or Hoogsteen configuration (4).
geometry, which are associated with base flipping (34),
may account for the longer distances measured in
solutionas reportedhere.
p53DBD binding induced a larger degree of deformation
at the central region of p21-RE as compared with that of
the BAX-RE, the p21-RE is known to bind tighter to p53
(35). One of the possible explanations for this apparently
puzzling observation is that the respective CWWG cores
responded differently to p53 binding. Further investiga-
tion of the CWWG core, particularly in the bound state,
is therefore required.
Finally, p53/DNA interactions can be impacted by
regions beyond the DBD and RE (3). Whereas biophysical
studies focusing on folded p53 fragments have provided a
wealth of information regarding p53 structure and
function, expanding beyond these 'truncated' systems is
highly desirable. The SDSL-MC approach, which is
capable ofproviding molecular
non-crystalline complexes, is particularly suited for these
detailsin large
Mapping sequence-dependent DNA shape using the
SDSL-MC approach
Whereas early SDSL studies used DNA duplexes as model
systems (21,36,37), recent reports have emerged in which
SDSL measured distances were used to study DNA duplex
conformation in response to base lesion (38), mismatches
(39) and protein binding (40). In addition, SDSL has also
been used to study higher order DNA structures such as
quadruplexes (41) and four-way junctions (42). In this
work, using the R5 probe that can be attached to any nu-
cleotide within a target sequence, multiple distances were
readily measured, and they directly revealed conform-
ational changes between the bound and unbound DNA.
In addition, synergistic integration with MC sampling
allowed us to derive atomic models of the target DNA
with sequence-dependent shape. In each top-20 ensemble
of unbound REs, the models are: (i) structurally highly
similar; and (ii) clearly different from a uniform B-DNA
(Figures 2 and 4). This demonstrates that the SDSL-MC
pipeline has the ability to provide detailed structural infor-
mation of DNA duplexes. The bound p21-RE structure
differed from the top-ranked model of the unbound
DNA by an RMSDstructof 2.6A˚
DNA by only 1.8A˚. As such, obtaining the sequence-de-
pendent shape of the unbound DNA has a profound
impact on properly assessing protein induced deformations
of DNA targets. Furthermore, intrinsic DNA shape
features revealed by the SDSL-MC approach will benefit
a broad range of efforts, such as prediction of transcription
factor binding specificities based on regression models that
combine DNA sequence and shape (43–45).
Nevertheless, further studies are needed to explore the
utility and limitation of the SDSL-MC method. For
example, the 'resolution' that can be achieved by this
approach remains to be investigated. In addition, DEER
measures distances in a frozen solution state, whereas MC
simulations target solution-state equilibrium at ambient
and from uniform B-
temperature. It is not clear how unique aspects of each
methodology impact the interpretation of the resulting
DNA shape.
In summary, results reported here clearly demonstrate
that the SDSL-MC approach reveals sequence-dependent
shapes of p53 REs that advance our understanding of p53/
DNA recognition. The method is not limited by the size of
the system and allows parallel examination of DNA shape
in both the unbound and protein-bound states. This is a
step forward toward uncovering the role of intrinsic
DNA shape on protein–DNA recognition on a general
Supplementary Data are available at NAR Online.
The authors thank I. Haworth for the continued develop-
ment of the NASNOX program. R.R. is an Alfred
P. Sloan Research Fellow. See Supplementary Data for
detailed author contributions.
U01GM103804 and R01HG003008 (in part to R.R.);
and R01GM064642 and 2U54RR022220 (to L.C.)]; and
NSF [MCB-0546529 and CHE-1213673 (to P.Z.Q.)].
Funding for open access charge: NSF, NIH.
[GM069557 andRR028992 (to P.Z.Q.);
Conflict of interest statement. None declared.
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