16. Summary

13.16 Summary

DNA synthesis occurs by semidiscontinuous replication, in which the leading strand of DNA growing 5′ V3′ is extended continuously, but the lagging strand that grows overall in the opposite 3′ V5′ direction is made as short Okazaki fragments, each synthesized 5′ V3′. The leading strand and each Okazaki fragment of the lagging strand initiate with an RNA primer that is extended by DNA polymerase. Bacteria and eukaryotes each possess more than one DNA polymerase activity. DNA polymerase III synthesizes both lagging and leading strands in E. coli. Many proteins are required for DNA polymerase III action and several constitute part of the replisome within which it functions.

The replisome contains an asymmetric dimer of DNA polymerase III; each new DNA strand is synthesized by a different core complex containing a catalytic (α) subunit. Processivity of the core complex is maintained by the β clamp, which forms a ring round DNA. The looping model for the replication fork proposes that, as one half of the dimer advances to synthesize the leading strand, the other half of the dimer pulls DNA through as a single loop that provides the template for the lagging strand. The transition from completion of one Okazaki fragment to the start of the next requires the lagging strand catalytic subunit to dissociate from DNA and then to reattach to a β clamp at the priming site for the next Okazaki fragment.

DnaB provides the helicase activity at a replication fork; this depends on ATP cleavage. DnaB may function by itself in oriC replicons to provide primosome activity by interacting periodically with DnaG, which provides the primase that synthesizes RNA.

Phage T4 codes for a sizeable replication apparatus, consisting of 7 proteins: DNA polymerase, helicase, single-strand binding protein, priming activities, and accessory proteins. Similar functions are required in other replication systems, including a HeLa cell system that replicates SV40 DNA. Different enzymes, DNA polymerase α and DNA polymerase δ, initiate and elongate the new strands of DNA.

The common mode of origin activation involves an initial limited melting of the double helix, followed by more general unwinding to create single strands. Several proteins act sequentially at the E. coli origin. DnaA binds to a series of 9 bp repeats and 13 bp repeats, forming an aggregate of 20 V40 monomers with DNA in which the 13 bp repeats are melted. The helicase activity of DnaB, together with DnaC, unwinds DNA further. Similar events occur at the lambda origin, where phage proteins O and P are the counterparts of bacterial proteins DnaA and DnaC, respectively. In SV40 replication, several of these activities are combined in the functions of T antigen.

The φX priming event also requires DnaB, DnaC, and DnaT. PriA is the component that defines the primosome assembly site (pas) for φX replicons; it displaces SSB from DNA in an action that involves cleavage of ATP. PriB and PriC are additional components of the primosome.

Several sites that are methylated by the Dam methylase are present in the E. coli origin, including those of the 13-mer binding sites for DnaA. The origin remains hemimethylated and is in a sequestered state for ~10 minutes following initiation of a replication cycle. During this period it is associated with the membrane, and reinitiation of replication is repressed.

After cell division, nuclei of eukaryotic cells have a licensing factor that is needed to initiate replication. Its destruction after initiation of replication prevents further replication cycles from occurring in yeast. Licensing factor cannot be imported into the nucleus from the cytoplasm, and can be replaced only when the nuclear membrane breaks down during mitosis.