The Block of DNA Polymerase δ Strand Displacement Activity by an Abasic Site Can Be Rescued by the Concerted Action of DNA Polymerase β and Flap Endonuclease 1

Abstract

Abasic (AP) sites are very frequent and dangerous DNA lesions. Their ability to block the advancement of a replication fork has been always viewed as a consequence of their inhibitory effect on the DNA synthetic activity of replicative DNA polymerases (DNA pols). Here we show that AP sites can also affect the strand displacement activity of the lagging strand DNA pol ␦, thus preventing proper Okazaki fragment maturation. This block can be overcome through a polymerase switch, involving the combined physical and functional interaction of DNA pol ␤ and Flap endonuclease 1. Our data identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism and suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling.Loss of purine and pyrimidine bases is a significant source of DNA damage in prokaryotic and eukaryotic organisms. Abasic (apurinic and apyrimidinic) lesions occur spontaneously in DNA; in eukaryotes it has been estimated that about 10 4 depurination and 10 2 depyrimidation events occur per genome per day. An equally important source of abasic DNA lesions results from the action of DNA glycosylases, such as uracil glycosylase, which excises uracil arising primarily from spontaneous deamination of cytosines (1). Although most AP sites are removed by the base excision repair (BER) 5 pathway, a small fraction of lesions persists, and DNA with AP lesions presents a strong block to DNA synthesis by replicative DNA polymerases (DNA pols) (2, 3). Several studies have been performed to address the effects of AP sites on the template DNA strand on the synthetic activity of a variety of DNA pols. The major replicative enzyme of eukaryotic cells, DNA pol ␦, was shown to be able to bypass an AP lesion, but only in the presence of the auxiliary factor proliferating cell nuclear antigen (PCNA) and at a very reduced catalytic efficiency if compared with an undamaged DNA template (4). On the other hand, the family X DNA pols ␤ and were shown to bypass an AP site but in a very mutagenic way (5). Recent genetic evidence in Saccharomyces cerevisiae cells showed that DNA pol ␦ is the enzyme replicating the lagging strand (6). According to the current model for Okazaki fragment synthesis (7-9), the action of DNA pol ␦ is not only critical for the extension of the newly synthesized Okazaki fragment but also for the displacement of an RNA/DNA segment of about 30 nucleotides on the pre-existing downstream Okazaki fragment to create an intermediate Flap structure that is the target for the subsequent action of the Dna2 endonuclease and the Flap endonuclease 1 (Fen-1). This process has the advantage of removing the entire RNA/DNA hybrid fragment synthesized by the DNA pol ␣/primase, potentially containing nucleotide misincorporations caused by the lack of a proofreading exonuclease activity of DNA pol ␣/primase. This results in a more accurate copy synthesized by DNA pol ␦. The intrinsic strand displacement activity of DNA pol ␦, in conjunction with F...

Oxidative DNA Damage Modulates Trinucleotide Repeat Instability Via DNA Base Excision Repair

“…It was reported that proliferating cell nuclear antigen (PCNA) and replication protein A (RPA) can stimulate pol β bypass of an AP site during DNA replication that occurs in an open template as well as Okazaki fragment maturation (267). In addition, pol β bypass of an AP site opposite to a 1-nt gap is facilitated by FEN1 and RPA (267).…”

“…Similar to the Y family translesion synthesis polymerases, pol β can bypass an apurinic/apyrimidinic site (AP site) by inserting an adenosine opposite to the damage (266). In addition, pol β can switch with pol  and pol ε to bypass an AP site (267,268). During Okazaki fragment maturation, pol β can bypass an AP site that was annealed with a downstream primer by switching with pol  in a stranddisplacement synthesis dependent manner (267).…”

“…In addition, pol β can switch with pol  and pol ε to bypass an AP site (267,268). During Okazaki fragment maturation, pol β can bypass an AP site that was annealed with a downstream primer by switching with pol  in a stranddisplacement synthesis dependent manner (267). Moreover, because pol β tends to skip over a TNR hairpin structure on the template (256,257), we further hypothesize that pol β may also skip over a cdPu lesion located at a TNR tract to bypass the lesion during DNA replication and BER, and this may subsequently cause TNR instability.…”

“…Moreover, pol β can switch with replication polymerases to bypass a template AP site during DNA leading and lagging strand synthesis (267,268). It has been shown that pol β coordinates with FEN1 and performs an efficient AP site bypass synthesis to facilitate Okazaki fragment maturation (267). Because previous studies showed that pol β DNA synthesis efficiency varied during BER of an oxidized base lesion that was located at different locations in a TNR tract (197,200).…”

“…Previous studies also showed that pol β can efficiently bypass a template AP site by following the "A rule" (266). Moreover, pol β can switch with replication polymerases to bypass a template AP site during DNA leading and lagging strand synthesis (267,268). It has been shown that pol β coordinates with FEN1 and performs an efficient AP site bypass synthesis to facilitate Okazaki fragment maturation (267).…”