Accessory proteins assist exonuclease-deficient bacteriophage T4 DNA Polymerase in replicating past an abasic site
Résumé
Replicative DNA polymerases, such as T4 pol, possess both elongation and 3‘-5‘ exonuclease proofreading catalytic activities. They arrest at the base preceding DNA damage on the coding DNA strand and specialized DNA polymerases have evolved to replicate across the lesion by a process known as TransLesion DNA Synthesis (TLS). TLS is considered to take place in two steps that often require different enzymes, insertion of a nucleotide opposite the damaged template base followed by extension from the inserted nucleotide. We and others have observed that inactivation of the 3‘-5‘ exonuclease function of T4 pol enables TLS across a single site-specific abasic (AP) lesion. Here we report a role for auxiliary replicative factors in this reaction. When replication is carried out with a large excess of DNA template over DNA polymerase in absence of auxiliary factors, the exo- polymerase inserts one nucleotide opposite the AP site but does not extend past the lesion. Addition of the clamp processivity factor and the clamp loader complex restores primer extension across an AP lesion on a circular AP-containing DNA substrate by the exo- polymerase, but has no effect on the wt enzyme. Hence T4 DNA pol exhibits a variety of responses to DNA damage. It can behave as a replicative pol or (in the absence of proofreading activity) as a specialized DNA polymerase and carry out TLS. As a specialized polymerase it can function either as an inserter or (with the help of accessory proteins) as an extender. The capacity to separate these distinct functions in a single DNA polymerase provides insight into the biochemical requirements for translesion DNA synthesis.
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