A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA

Werner syndrome (WS) is a premature aging disorder where the affected individuals appear much older than their chronological age. The single gene that is defective in WS encodes a protein (WRN) that has ATPase, helicase and 3′→5′ exonuclease activities. Our laboratory has recently uncovered a physical and functional interaction between WRN and the Ku heterodimer complex that functions in double-strand break repair and V(D)J recombination. Importantly, Ku specifically stimulates the exonuclease activity of WRN. We now report that Ku enables the Werner exonuclease to digest through regions of DNA containing 8-oxoadenine and 8-oxoguanine modifications, lesions that have previously been shown to block the exonuclease activity of WRN alone. These results indicate that Ku significantly alters the exonuclease function of WRN and suggest that the two proteins function concomitantly in a DNA damage processing pathway. In support of this notion we also observed co-localization of WRN and Ku, particularly after DNA damaging treatments.     

Characterization of the human and mouse WRN 3'-->5' exonuclease

Werner’s syndrome (WS) is an autosomal recessive disorder in humans characterized by the premature development of a partial array of age-associated pathologies. WRN, the gene defective in WS, encodes a 1432 amino acid protein (hWRN) with intrinsic 3′→5′ DNA helicase activity. We recently showed that hWRN is also a 3′→5′ exonuclease. Here, we further characterize the hWRN exonuclease. hWRN efficiently degraded the 3′ recessed strands of double-stranded DNA or a DNA–RNA heteroduplex. It had little or no activity on blunt-ended DNA, DNA with a 3′ protruding strand, or single-stranded DNA. The hWRN exonuclease efficiently removed a mismatched nucleotide at a 3′ recessed terminus, and was capable of initiating DNA degradation from a 12-nt gap, or a nick. We further show that the mouse WRN (mWRN) is also a 3′→5′ exonuclease, with substrate specificity similar to that of hWRN. Finally, we show that hWRN forms a trimer and interacts with the proliferating cell nuclear antigen in vitro. These findings provide new data on the biochemical activities of WRN that may help elucidate its role(s) in DNA metabolism.     

Ku heterodimer binds to both ends of the Werner protein and functional interaction occurs at the Werner N-terminus

The human Werner syndrome protein, WRN, is a member of the RecQ helicase family and contains 3′→5′ helicase and 3′→5′ exonuclease activities. Recently, we showed that the exonuclease activity of WRN is greatly stimulated by the human Ku heterodimer protein. We have now mapped this interaction physically and functionally. The Ku70 subunit specifically interacts with the N-terminus (amino acids 1–368) of WRN, while the Ku80 subunit interacts with its C-terminus (amino acids 940– 1432). Binding between Ku70 and the N-terminus of WRN (amino acids 1–368) is sufficient for stimulation of WRN exonuclease activity. A mutant Ku heterodimer of full-length Ku80 and truncated Ku70 (amino acids 430–542) interacts with C-WRN but not with N-WRN and cannot stimulate WRN exonuclease activity. This emphasizes the functional significance of the interaction between the N-terminus of WRN and Ku70. The interaction between Ku80 and the C-terminus of WRN may modulate some other, as yet unknown, function. The strong interaction between Ku and WRN suggests that these two proteins function together in one or more pathways of DNA metabolism.     

The Werner syndrome protein operates in base excision repair and cooperates with DNA polymerase beta

Genome instability is a characteristic of cancer and aging, and is a hallmark of the premature aging disorder Werner syndrome (WS). Evidence suggests that the Werner syndrome protein (WRN) contributes to the maintenance of genome integrity through its involvement in DNA repair. In particular, biochemical evidence indicates a role for WRN in base excision repair (BER). We have previously reported that WRN helicase activity stimulates DNA polymerase beta (pol β) strand displacement synthesis in vitro. In this report we demonstrate that WRN exonuclease activity can act cooperatively with pol β, a polymerase lacking 3′–5′ proofreading activity. Furthermore, using small interference RNA technology, we demonstrate that WRN knockdown cells are hypersensitive to the alkylating agent methyl methanesulfonate, which creates DNA damage that is primarily repaired by the BER pathway. In addition, repair assays using whole cell extracts from WRN knockdown cells indicate a defect in long patch (LP) BER. These findings demonstrate that WRN plays a direct role in the repair of methylation-induced DNA damage, and suggest a role for both WRN helicase and exonuclease activities together with pol β during LP BER.     

Physical and functional interactions between Werner syndrome helicase and mismatch-repair initiation factors

Werner syndrome (WS) is a severe recessive disorder characterized by premature aging, cancer predisposition and genomic instability. The gene mutated in WS encodes a bi-functional enzyme called WRN that acts as a RecQ-type DNA helicase and a 3′-5′ exonuclease, but its exact role in DNA metabolism is poorly understood. Here we show that WRN physically interacts with the MSH2/MSH6 (MutSα), MSH2/MSH3 (MutSβ) and MLH1/PMS2 (MutLα) heterodimers that are involved in the initiation of mismatch repair (MMR) and the rejection of homeologous recombination. MutSα and MutSβ can strongly stimulate the helicase activity of WRN specifically on forked DNA structures with a 3′-single-stranded arm. The stimulatory effect of MutSα on WRN-mediated unwinding is enhanced by a G/T mismatch in the DNA duplex ahead of the fork. The MutLα protein known to bind to the MutS α–heteroduplex complexes has no effect on WRN-mediated DNA unwinding stimulated by MutSα, nor does it affect DNA unwinding by WRN alone. Our data are consistent with results of genetic experiments in yeast suggesting that MMR factors act in conjunction with a RecQ-type helicase to reject recombination between divergent sequences.     

Strand exchange of telomeric DNA catalyzed by the Werner syndrome protein (WRN) is specifically stimulated by TRF2

Werner syndrome (WS), caused by loss of function of the RecQ helicase WRN, is a hereditary disease characterized by premature aging and elevated cancer incidence. WRN has DNA binding, exonuclease, ATPase, helicase and strand annealing activities, suggesting possible roles in recombination-related processes. Evidence indicates that WRN deficiency causes telomeric abnormalities that likely underlie early onset of aging phenotypes in WS. Furthermore, TRF2, a protein essential for telomere protection, interacts with WRN and influences its basic helicase and exonuclease activities. However, these studies provided little insight into WRN''s specific function at telomeres. Here, we explored the possibility that WRN and TRF2 cooperate during telomeric recombination processes. Our results indicate that TRF2, through its interactions with both WRN and telomeric DNA, stimulates WRN-mediated strand exchange specifically between telomeric substrates; TRF2''s basic domain is particularly important for this stimulation. Although TRF1 binds telomeric DNA with similar affinity, it has minimal effects on WRN-mediated strand exchange of telomeric DNA. Moreover, TRF2 is displaced from telomeric DNA by WRN, independent of its ATPase and helicase activities. Together, these results suggest that TRF2 and WRN act coordinately during telomeric recombination processes, consistent with certain telomeric abnormalities associated with alteration of WRN function.     

Selective blockage of the 3'-->5' exonuclease activity of WRN protein by certain oxidative modifications and bulky lesions in DNA

Individuals with mutations in the WRN gene suffer from Werner syndrome, a disease with early onset of many characteristics of normal aging. The WRN protein (WRNp) functions in DNA metabolism, as the purified polypeptide has both 3′→5′ helicase and 3′→5′ exonuclease activities. In this study, we have further characterized WRNp exonuclease activity by examining its ability to degrade double-stranded DNA substrates containing abnormal and damaged nucleo­tides. In addition, we directly compared the 3′→5′ WRNp exonuclease activity with that of exo­nuclease III and the Klenow fragment of DNA polymerase I. Our results indicate that the presence of certain abnormal bases (such as uracil and hypoxanthine) does not inhibit the exonuclease activity of WRNp, exo­nuclease III or Klenow, whereas other DNA modifications, including apurinic sites, 8-oxoguanine, 8-oxoadenine and cholesterol adducts, inhibit or block WRNp. The ability of damaged nucleo­tides to inhibit exonucleolytic digestion differs significantly between WRNp, exonuclease III and Klenow, indicating that each exonuclease has a distinct mechanism of action. In addition, normal and modified DNA substrates are degraded similarly by full-length WRNp and an N-terminal fragment of WRNp, indicating that the specificity for this activity lies mostly within this region. The biochemical and physiological significance of these results is discussed.     

An essential DnaB helicase of Bacillus anthracis: identification, characterization, and mechanism of action

We have described a novel essential replicative DNA helicase from Bacillus anthracis, the identification of its gene, and the elucidation of its enzymatic characteristics. Anthrax DnaB helicase (DnaB_BA) is a 453-amino-acid, 50-kDa polypeptide with ATPase and DNA helicase activities. DnaB_BA displayed distinct enzymatic and kinetic properties. DnaB_BA has low single-stranded DNA (ssDNA)-dependent ATPase activity but possesses a strong 5′→3′ DNA helicase activity. The stimulation of ATPase activity appeared to be a function of the length of the ssDNA template rather than of ssDNA binding alone. The highest specific activity was observed with M13mp19 ssDNA. The results presented here indicated that the ATPase activity of DnaB_BA was coupled to its migration on an ssDNA template rather than to DNA binding alone. It did not require nucleotide to bind ssDNA. DnaB_BA demonstrated a strong DNA helicase activity that required ATP or dATP. Therefore, DnaB_BA has an attenuated ATPase activity and a highly active DNA helicase activity. Based on the ratio of DNA helicase and ATPase activities, DnaB_BA is highly efficient in DNA unwinding and its coupling to ATP consumption.     

DNA secondary structure of the released strand stimulates WRN helicase action on forked duplexes without coordinate action of WRN exonuclease

Werner syndrome (WS) is an autosomal recessive premature aging disorder characterized by aging-related phenotypes and genomic instability. WS is caused by mutations in a gene encoding a nuclear protein, Werner syndrome protein (WRN), a member of the RecQ helicase family, that interestingly possesses both helicase and exonuclease activities. Previous studies have shown that the two activities act in concert on a single substrate. We investigated the effect of a DNA secondary structure on the two WRN activities and found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. These results imply that WRN helicase and exonuclease activities can act independently, and we propose that the uncoordinated action may be relevant to the in vivo activity of WRN.     

WRN Exonuclease activity is blocked by specific oxidatively induced base lesions positioned in either DNA strand

Werner syndrome (WS) is a premature aging disorder caused by mutations in the WS gene (WRN). Although WRN has been suggested to play an important role in DNA metabolic pathways, such as recombination, replication and repair, its precise role still remains to be determined. WRN possesses ATPase, helicase and exonuclease activities. Previous studies have shown that the WRN exonuclease is inhibited in vitro by certain lesions induced by oxidative stress and positioned in the digested strand of the substrate. The presence of the 70/86 Ku heterodimer (Ku), participating in the repair of double-strand breaks (DSBs), alleviates WRN exonuclease blockage imposed by the oxidatively induced DNA lesions. The current study demonstrates that WRN exonuclease is inhibited by several additional oxidized bases, and that Ku stimulates the WRN exonuclease to bypass these lesions. Specific lesions present in the non-digested strand were shown also to inhibit the progression of the WRN exonuclease; however, Ku was not able to stimulate WRN exonuclease to bypass these lesions. Thus, this study considerably broadens the spectrum of lesions which block WRN exonuclease progression, shows a blocking effect of lesions in the non-digested strand, and supports a function for WRN and Ku in a DNA damage processing pathway.     

Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks

Pif-1 proteins are 5′→3′ superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3′ ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity.     

DNA2 Cooperates with the WRN and BLM RecQ Helicases to Mediate Long-range DNA End Resection in Human Cells

The 5′-3′ resection of DNA ends is a prerequisite for the repair of DNA double strand breaks by homologous recombination, microhomology-mediated end joining, and single strand annealing. Recent studies in yeast have shown that, following initial DNA end processing by the Mre11-Rad50-Xrs2 complex and Sae2, the extension of resection tracts is mediated either by exonuclease 1 or by combined activities of the RecQ family DNA helicase Sgs1 and the helicase/endonuclease Dna2. Although human DNA2 has been shown to cooperate with the BLM helicase to catalyze the resection of DNA ends, it remains a matter of debate whether another human RecQ helicase, WRN, can substitute for BLM in DNA2-catalyzed resection. Here we present evidence that WRN and BLM act epistatically with DNA2 to promote the long-range resection of double strand break ends in human cells. Our biochemical experiments show that WRN and DNA2 interact physically and coordinate their enzymatic activities to mediate 5′-3′ DNA end resection in a reaction dependent on RPA. In addition, we present in vitro and in vivo data suggesting that BLM promotes DNA end resection as part of the BLM-TOPOIIIα-RMI1-RMI2 complex. Our study provides new mechanistic insights into the process of DNA end resection in mammalian cells.     

Requirements for the nucleolytic processing of DNA ends by the Werner syndrome protein-Ku70/80 complex

Werner syndrome (WS) is an inherited disease characterized by premature onset of aging, increased cancer incidence, and genomic instability. The WS gene encodes a protein with helicase and exonuclease activities. Our previous studies indicated that the Werner syndrome protein (WRN) interacts with Ku, a heterodimeric factor of 70- and 80-kDa subunits implicated in the repair of double strand DNA breaks. Moreover, we demonstrated that Ku70/80 strongly stimulates and alters WRN exonuclease activity. In this report, we investigate further the association between WRN and Ku70/80. First, using various WRN deletion mutants we show that 50 amino acids at the amino terminus are required and sufficient to interact with Ku70/80. In addition, our data indicate that the region of Ku80 between amino acids 215 and 276 is necessary for binding to WRN. Then, we show that the amino-terminal region of WRN from amino acid 1 to 388, which comprise the exonuclease domain, can be efficiently stimulated by Ku to degrade DNA substrates, indicating that the helicase domain and the carboxyl-terminal tail are not required for the stimulatory process. Finally, using gel shift assays, we demonstrate that Ku recruits WRN to DNA. Taken together, these results suggest that Ku-mediated activation of WRN exonuclease activity may play an important role in a cellular pathway that requires processing of DNA ends.     

Collaboration of Werner syndrome protein and BRCA1 in cellular responses to DNA interstrand cross-links

Cells deficient in the Werner syndrome protein (WRN) or BRCA1 are hypersensitive to DNA interstrand cross-links (ICLs), whose repair requires nucleotide excision repair (NER) and homologous recombination (HR). However, the roles of WRN and BRCA1 in the repair of DNA ICLs are not understood and the molecular mechanisms of ICL repair at the processing stage have not yet been established. This study demonstrates that WRN helicase activity, but not exonuclease activity, is required to process DNA ICLs in cells and that WRN cooperates with BRCA1 in the cellular response to DNA ICLs. BRCA1 interacts directly with WRN and stimulates WRN helicase and exonuclease activities in vitro. The interaction between WRN and BRCA1 increases in cells treated with DNA cross-linking agents. WRN binding to BRCA1 was mapped to BRCA1 452–1079 amino acids. The BRCA1/BARD1 complex also associates with WRN in vivo and stimulates WRN helicase activity on forked and Holliday junction substrates. These findings suggest that WRN and BRCA1 act in a coordinated manner to facilitate repair of DNA ICLs.     

Biochemical Characterization of Adeno-Associated Virus Rep68 DNA Helicase and ATPase Activities

The adeno-associated virus (AAV) nonstructural proteins Rep68 and Rep78 are site-specific DNA binding proteins, ATP-dependent site-specific endonucleases, helicases, and ATPases. These biochemical activities are required for viral DNA replication and control of viral gene expression. In this study, we characterized the biochemical properties of the helicase and ATPase activities of homogeneously pure Rep68. The enzyme exists as a monomer in solution at the concentrations used in this study (<380 nM), as judged by its mobility in sucrose density gradients. Using a primed single-stranded (ss) circular M13 substrate, the helicase activity had an optimum pH of 7 to 7.5, an optimum temperature of 45°C, and an optimal divalent-cation concentration of 5 mM MgCl₂. Several nucleoside triphosphates could serve as cofactors for Rep68 helicase activity, and the order of preference was ATP = GTP > CTP = dATP > UTP > dGTP. The K_m values for ATP in both the DNA helicase reaction and the site-specific trs endonuclease reaction were essentially the same, approximately 180 μM. Both reactions were sigmoidal with respect to ATP concentration, suggesting that a dimer or higher-order multimer of Rep68 is necessary for both DNA helicase activity and terminal resolution site (trs) nicking activity. Furthermore, when the enzyme itself was titrated in the trs endonuclease and ATPase reactions, both activities were second order with respect to enzyme concentration. This suggests that a dimer of Rep68 is the active form for both the ATPase and nicking activities. In contrast, DNA helicase activity was linear with respect to enzyme concentration. When bound to ssDNA, the enzyme unwound the DNA in the 3′-to-5′ direction. DNA unwinding occurred at a rate of approximately 345 bp per min per monomeric enzyme molecule. The ATP turnover rate was approximately 30 to 50 ATP molecules per min per enzyme molecule. Surprisingly, the presence of DNA was not required for ATPase activity. We estimated that Rep translocates processively for more than 1,300 bases before dissociating from its substrate in the absence of any accessory proteins. DNA helicase activity was not significantly stimulated by substrates that have the structure of a replication fork and contain either a 5′ or 3′ tail. Rep68 binds only to ssDNA, as judged by inhibition of the DNA helicase reaction with ss or double-stranded (ds) DNA. Consistent with this observation, no helicase activity was detected on blunt-ended ds oligonucleotide substrates unless they also contained an ss 3′ tail. However, if a blunt-ended ds oligonucleotide contained the 22-bp Rep binding element sequence, Rep68 was capable of unwinding the substrate. This means that Rep68 can function both as a conventional helicase for strand displacement synthesis and as a terminal-repeat-unwinding protein which catalyzes the conversion of a duplex end to a hairpin primer. Thus, the properties of the Rep DNA helicase activity suggest that Rep is involved in all three of the key steps in AAV DNA replication: terminal resolution, reinitiation, and strand displacement.     

The enzymatic activities of the Werner syndrome protein are disabled by the amino acid polymorphism R834C

The Werner syndrome protein, WRN, is a member of the RecQ family of DNA helicases. It possesses both 3'-->5' DNA helicase and 3'-->5' DNA exonuclease activities. Mutations in WRN are causally associated with a rare, recessive disorder, Werner syndrome (WS), distinguished by premature aging and genomic instability; all are reported to result in loss of protein expression. In addition to WS-linked mutations, single nucleotide polymorphisms, with frequencies that exceed those of WS-associated mutations, are also present in WRN. We have initiated studies to determine if six of these polymorphisms affect the enzymatic activities of WRN. We show that two common polymorphisms, F1074L and C1367R, and two infrequent polymorphisms, Q724L and S1079L, exhibit little change in activity relative to wild-type WRN; the polymorphism, T172P, shows a small but consistent reduction of activity. However, an infrequent polymorphism, R834C, located in the helicase domain dramatically reduces WRN helicase and helicase-coupled exonuclease activity. The structure of the E. coli helicase core suggests that R834 may be involved in interactions with ATP. As predicted, substitution of Arg with Cys interferes with ATP hydrolysis that is absolutely required for unwinding DNA. R834C thus represents the first missense amino acid polymorphism in WRN that nearly abolishes enzymatic activity while leaving expression largely unaffected.     

Biochemical and kinetic characterization of the DNA helicase and exonuclease activities of werner syndrome protein

The WRN gene, defective in the premature aging and genome instability disorder Werner syndrome, encodes a protein with DNA helicase and exonuclease activities. In this report, cofactor requirements for WRN catalytic activities were examined. WRN helicase performed optimally at an equimolar concentration (1 mm) of Mg(2+) and ATP with a K(m) of 140 microm for the ATP-Mg(2+) complex. The initial rate of WRN helicase activity displayed a hyperbolic dependence on ATP-Mg(2+) concentration. Mn(2+) and Ni(2+) substituted for Mg(2+) as a cofactor for WRN helicase, whereas Fe(2+) or Cu(2+) (10 microm) profoundly inhibited WRN unwinding in the presence of Mg(2+).Zn(2+) (100 microm) was preferred over Mg(2+) as a metal cofactor for WRN exonuclease activity and acts as a molecular switch, converting WRN from a helicase to an exonuclease. Zn(2+) strongly stimulated the exonuclease activity of a WRN exonuclease domain fragment, suggesting a Zn(2+) binding site in the WRN exonuclease domain. A fluorometric assay was used to study WRN helicase kinetics. The initial rate of unwinding increased with WRN concentration, indicating that excess enzyme over DNA substrate improved the ability of WRN to unwind the DNA substrate. Under presteady state conditions, the burst amplitude revealed a 1:1 ratio between WRN and DNA substrate, suggesting an active monomeric form of the helicase. These are the first reported kinetic parameters of a human RecQ unwinding reaction based on real time measurements, and they provide mechanistic insights into WRN-catalyzed DNA unwinding.