separation (SSB and RPA factors) Priming of the leading and lagging template strands (primase or DNA polymerase alpha) Ensuring processivity (clamp loading factors, ring-shaped clamp proteins, strand binding proteins) High-fidelity DNA replication (DNA polymerase III, DNA polymerase delta, DNA polymerase epsilon. All have intrinsically low error rates because of their structure and chemistry.) Error correction (replicative polymerase active sites sense errors; 3' to 5' exonuclease domains of replicative polymerases fix errors) Synchronised polymerisation of leading and lagging strands despite anti-parallel structure (replication fork structure, dimerisation of replicative polymerases) Primer removal (DNA polymerase I, RNAse H, flap endonucleases such as FEN1, or other DNA repair factors) Formation of phosphodiester bonds at gaps between Okazaki fragments (ligase) In general, the challenges of DNA replication involve the structure of the molecules, the chemistry of the molecules, and, from a systems perspective, the underlying relationships between the structure and the chemistry. Solving the challenges of DNA replication Many of the structural and chemical problems associated with DNA replication are managed by molecular machinery that is highly conserved across organisms. This section discusses how replisome factors solve the structural and chemical challenges of DNA replication. Replisome assembly DNA replication begins at sites called origins of replication. In organisms with small genomes and simple chromosome structure, such as bacteria, there may be only a few origins of replication on each chromosome. Organisms with large genomes and complex chromosome structure, such as humans, may have hundreds, or even thousands, of origins of replication spread across multiple chromosomes. DNA structure varies with time, space, and sequence, and it is thought that these variations, in addition to their role in gene expression, also play active roles in replisome assembly during DNA synthesis. Replisome assembly at an origin of replication is roughly divided into three phases. For bacteria: Formation of pre-replication complex. DnaA binds to the origin recognition complex and separates the duplex. This attracts DnaB helicase and DnaC, which maintain the replication bubble. Formation of pre-initiation complex. SSB binds to the single strand and then gamma (clamp loading factor) binds to SSB. Formation of initiation complex. Gamma deposits the sliding clamp (beta) and attracts DNA polymerase III. For eukaryotes: Formation of pre-replication complex. MCM factors bind to the origin recognition complex and separate the duplex, forming a replication bubble. Formation of pre-initiation complex. Replication protein A (RPA) binds to the single stranded DNA and then RFC (clamp loading factor)