Abstract
Mutagenesis is one of major consequences of irradiation of ionizing radiation. Oxidative DNA damage produced by ionizing radiation inhibits replicative DNA polymerases. Cells have a function to restore DNA replication called transleion DNA synthesis, in which specialized DNA polymerases could extend the primer termini by bypass synthesis of damage bases. It has been considered that the bypass synthesis is an error-prone process inducing mutations. To elucidate molecular mechanisms of polymerase switches, we reconstituted DNA replication in vitro using purified proteins including DNA polymerase δ (pol δ), replication factor C (RFC), proliferating cell nuclear antigen (PCNA) and replication protein A (RPA). Here we describe dynamic properties of these proteins in the elongation step on single-stranded M13 template, providing evidence that pol δ has a distributive nature over the 7 kb of the template, repeating a frequent dissociation-association cycle at growing 3'-hydroxyl ends. Some PCNA could remain at the primer terminus during this cycle, while the remainder could slide out of the primer terminus or be unloaded once pol δ has dissociated. RFC remains around the primer terminus through the elongation phase, and could probably hold PCNA from which pol δ has detached, or reload PCNA from solution to restart DNA synthesis. Based on these observations, we discuss the molecular mechanism of polymerase switches.
