The Werner Syndrome Exonuclease Facilitates DNA Degradation and High Fidelity DNA Polymerization by Human DNA Polymerase δ

Abstract

<jats:p>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<jats:italic>in vitro</jats:italic>observations, we show that WRN contributes to the maintenance of DNA synthesis fidelity<jats:italic>in vivo</jats:italic>. 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.</jats:p>

The Werner Syndrome Helicase Coordinates Sequential Strand Displacement and FEN1-Mediated Flap Cleavage during Polymerase δ Elongation

“…Unlike the helicase activity, the exonuclease activity of WRN does not seem to play a prominent role in any of the processes examined in this study. We cannot rule out the possibility that this enzymatic activity contributes to telomere replication in certain situations, such as the removal of mismatched or damaged nucleotides incorporated during processive DNA synthesis, as others have suggested (61). Our data show that WRN is not unique in promoting Pol␦ extension and strand displacement synthesis through the telomeric G-rich repeats, since BLM performs as well as WRN in these reactions.…”

Definitive diagnosis of mandibular hypoplasia, deafness, progeroid features and lipodystrophy (MDPL) syndrome caused by a recurrent <i>de novo</i> mutation in the <i>POLD1</i> gene

“…It plays a critical role in genome maintenance through its involvement in replicative DNA synthesis and multiple synthetic repair processes [27,28]. POLD1 cooperates with WRN, which a gene that is typically associated with Werner syndrome [25,29]. Moreover, the age-related decrease in POLD1 expression has been shown to be involved in the DNA repair pathway in vitro [29][30][31].…”

“…POLD1 cooperates with WRN, which a gene that is typically associated with Werner syndrome [25,29]. Moreover, the age-related decrease in POLD1 expression has been shown to be involved in the DNA repair pathway in vitro [29][30][31]. The mutation encoding p.Ser605del exists in a highly conserved region of the polymerase δ catalytic subunit, and leads to a failure to catalyze DNA polymerization [11,27].…”

Sphingosine, a Modulator of Human Translesion DNA Polymerase Activity

“…CPD Bypass Activity in Cells-CPD bypass activity in cells was measured using a modification of our recently established oligonucleotide retrieval assay (27,28). A synthetic oligonucleotide containing a site-specific CPD lesion was extended by ligating oligonucleotides, 5Ј-AmMC6ACGGAGGGAATC-GGAGGTCGC-3Ј and 5Ј-CCTTCCACCTCCCATTCCTGA-TTCAGTCACTG-ddC-3Ј, at the 5Ј and 3Ј termini, respectively.…”

“…We transfected this substrate into SV40-immortalized fibroblasts (27,28) and monitored bypass activity in the absence or presence of SO treatment by qPCR, as described under "Experimental Procedures." The apoptotic response triggered by SO precluded the use of high concentrations.…”