Abstract
DNA Polymerase δ (Pol δ) and the Werner syndrome protein, WRN, are involved in maintaining cellular genomic stability. Pol δ synthesizes the lagging strand during replication of genomic DNA and also functions in the synthesis steps of DNA repair and recombination. WRN is a member of the RecQ helicase family, loss of which results in the premature aging and cancer-prone disorder, Werner syndrome. Both Pol δ and WRN encode 3' → 5' DNA exonuclease activities. Pol δ exonuclease removes 3'-terminal mismatched nucleotides incorporated during replication to ensure high fidelity DNA synthesis. WRN exonuclease degrades DNA containing alternate secondary structures to prevent formation and enable resolution of stalled replication forks. We now observe that similarly to WRN, Pol δ degrades alternate DNA structures including bubbles, four-way junctions, and D-loops. Moreover, WRN and Pol δ form a complex with enhanced ability to hydrolyze these structures. We also present evidence that WRN can proofread for Pol δ; WRN excises 3'-terminal mismatches to enable primer extension by Pol δ. Consistent with our in vitro observations, we show that WRN contributes to the maintenance of DNA synthesis fidelity in vivo. Cells expressing limiting amounts (∼10% of normal) of WRN have elevated mutation frequencies compared with wild-type cells. Together, our data highlight the importance of WRN exonuclease activity and its cooperativity with Pol δ in preserving genome stability, which is compromised by the loss of WRN in Werner syndrome.
Highlights
WRN and DNA polymerase ␦ are involved in DNA replication and repair
We report the following novel observations. (i) Pol ␦ can exonucleolytically digest blunt duplexes containing non-canonical DNA structures, and that WRN co-operates with Pol ␦ to degrade these structures. (ii) WRN exonuclease can substitute for Pol ␦ exonuclease to remove single 3Ј-terminal mismatched nucleotides. (iii) The spontaneous random point mutation frequency is higher in human cells depleted of WRN and, deficient in WRN helicase and exonuclease
DNA Pol ␦ encodes 3Ј 3 5Ј proofreading exonuclease activity that ensures high fidelity DNA synthesis during cellular DNA replication; it does so by removing 3Ј-terminal misincorporated nucleotides that impair the addition of subsequent nucleotides
Summary
WRN and DNA polymerase ␦ are involved in DNA replication and repair. Results: WRN synergizes with Pol ␦ to degrade alternate DNA structures. High fidelity DNA synthesis by Pol ␦ is ensured through selectivity at the level of nucleotide incorporation and by hydrolysis of mismatched nucleotides partitioned from the polymerase active site to the proofreading exonu-. By virtue of its helicase activity, we showed that WRN resolves DNA structural roadblocks such as hairpins and quadruplexes to alleviate stalling and enable synthesis by Pol ␦ [19]. In previous studies on the functional interaction of WRN with Pol ␦, we utilized polymerase holoenzyme from Saccharomyces cerevisiae, a heterotrimeric complex comprising of Pol 3p, Pol 31p, and Pol32p subunits [21] It lacks the fourth smallest subunit, homologous to the p12 subunit that is present in human Pol ␦, and that modulates both the rate and fidelity of DNA synthesis by human Pol ␦ holoenzyme [22]. We hypothesize that WRN and Pol ␦ co-operate to maintain the fidelity of DNA transactions and, genomic stability
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