 The investigators will transfect U2OS cells with plasmids expressing relevant proteins then determine their occupancy in origin sequences using ChIP assays. Plasmids expressing epitope tagged WT HBO1 or mutant derivatives along with plasmids expressing WT or mutant E1A proteins will be expressed in U2OS cells, then occupancy of these proteins on origin regions will be quantified using antibodies as appropriate. Typically in these experiments, using relevant antibodies, ChIP assays are performed with primer pairs encompassing origin regions and also regions that are far from origin. Occupancy of initiation factors are increased several fold in the origin region as compared to that of 2KB upstream or downstream regions. Loading of MCM complex along with Cdt1 onto the origins is an indication that initiation of replication occurs in that origin and usually assayed in ChIP-re-ChIP assays as follows: First, loading of HBO1 to the origins will be confirmed using epitope specific antibodies in the first ChIP. The anti-HBO1 precipitates will be re-ChIPed with anti-MCM3 antibodies. Loading of MCM3 helicase to origins occurs after Cdt1 binding and depends on HBO1 HAT activity. Normal amounts of MCM3 will be detected after re-ChIP-ing in E1A control samples. If reduced amount of MCM3 is recovered in reChIP assays when mutant E1A or HBO1 mutant e.g. HBO1 G435A is used, it would indicate that stimulation of HAT activity by E1A is critical for maximal origin activity in E1A cells. This type of assay has considerable flexibility in that mutant proteins can be rapidly assayed. This ChIP-reChiP assays will repeated in different combinations to determine the E1A loading. 