Functional and physical interaction between WRN helicase and human replication protein A.

The human premature aging disorder Werner syndrome (WS) is associated with a large number of symptoms displayed in normal aging. The WRN gene product, a DNA helicase, has been previously shown to unwind short DNA duplexes (相似文献   

The Werner syndrome protein stimulates DNA polymerase beta strand displacement synthesis via its helicase activity

Werner syndrome is a hereditary premature aging disorder characterized by genomic instability. Genetic analysis and protein interaction studies indicate that the defective gene product (WRN) may play an important role in DNA replication, recombination, and repair. DNA polymerase beta (pol beta) is a central participant in both short and long-patch base excision repair (BER) pathways, which function to process most spontaneous, alkylated, and oxidative DNA damage. We report here a physical interaction between WRN and pol beta, and using purified proteins reconstitute of a portion of the long-patch BER pathway to examine a potential role for WRN in this repair response. We demonstrate that WRN stimulates pol beta strand displacement DNA synthesis and that this stimulation is dependent on the helicase activity of WRN. In addition, a truncated WRN protein, containing primarily the helicase domain, retains helicase activity and is sufficient to mediate the stimulation of pol beta. The WRN helicase also unwinds a BER substrate, providing evidence that WRN plays a role in unwinding DNA repair intermediates. Based on these findings, we propose a novel mechanism by which WRN may mediate pol beta-directed long-patch BER.     

The Werner syndrome gene: the molecular basis of RecQ helicase-deficiency diseases

Werner syndrome (WS) is an autosomal recessive genetic disorder that is manifested by genetic instability and premature onset of age-related diseases, including atherosclerosis and cancer. The gene that is mutated in WS cells (WRN) has been identified recently. Characterizations of the WRN gene product indicate that WRN encodes both a 3'-->5' DNA helicase, belonging to the Escherichiacoli RecQ helicase family, and a 3'-->5' DNA exonuclease. Studies to define the molecular mechanism of WRN-DNA transactions are currently underway in many laboratories. Preliminary results indicate that WRN functions as a key factor in resolving aberrant DNA structures that arise from DNA metabolic processes such as replication, recombination and/or repair, to preserve the genetic integrity in cells.     

Catalytic activities of Werner protein are affected by adduction with 4-hydroxy-2-nonenal

4-Hydroxy-2-nonenal (HNE) is a reactive α,β-unsaturated aldehyde generated during oxidative stress and subsequent peroxidation of polyunsaturated fatty acids. Here, Werner protein (WRN) was identified as a novel target for modification by HNE. Werner syndrome arises through mutations in the WRN gene that encodes the RecQ DNA helicase which is critical for maintaining genomic stability. This hereditary disease is associated with chromosomal instability, premature aging and cancer predisposition. WRN appears to participate in the cellular response to oxidative stress and cells devoid of WRN display elevated levels of oxidative DNA damage. We demonstrated that helicase/ATPase and exonuclease activities of HNE-modified WRN protein were inhibited both in vitro and in immunocomplexes purified from the cell extracts. Sites of HNE adduction in human WRN were identified at Lys577, Cys727, His1290, Cys1367, Lys1371 and Lys1389. We applied in silico modeling of the helicase and RQC domains of WRN protein with HNE adducted to Lys577 and Cys727 and provided a potential mechanism of the observed deregulation of the protein catalytic activities. In light of the obtained results, we postulate that HNE adduction to WRN is a post-translational modification, which may affect WRN conformational stability and function, contributing to features and diseases associated with premature senescence.     

Functional deficit associated with a missense Werner syndrome mutation

Werner syndrome (WS) is a rare autosomal recessive disorder caused by mutations in the WRN gene. WRN helicase, a member of the RecQ helicase family, is involved in various DNA metabolic pathways including DNA replication, recombination, DNA repair and telomere maintenance. In this study, we have characterized the G574R missense mutation, which was recently identified in a WS patient. Our biochemical experiments with purified mutant recombinant WRN protein showed that the G574R mutation inhibits ATP binding, and thereby leads to significant decrease in helicase activity. Exonuclease activity of the mutant protein was not significantly affected, whereas its single strand DNA annealing activity was higher than that of wild type. Deficiency in the helicase activity of the mutant may cause defects in replication and other DNA metabolic processes, which in turn could be responsible for the Werner syndrome phenotype in the patient. In contrast to the usual appearance of WS, the G574R patient has normal stature. Thus the short stature normally associated with WS may not be due to helicase deficiency.     

Enzymatic and DNA binding properties of purified WRN protein: high affinity binding to single-stranded DNA but not to DNA damage induced by 4NQO.

Mutations in the WRN gene result in Werner syndrome, an autosomal recessive disease in which many characteristics of aging are accelerated. A probable role in some aspect of DNA metabolism is suggested by the primary sequence of the WRN gene product. A recombinant His-tagged WRN protein (WRNp) was overproduced in insect cells using the baculovirus system and purified to near homogeneity by several chromatographic steps. This purification scheme removes both nuclease and topoisomerase contaminants that persist following a single Ni(2+)affinity chromatography step and allows for unambiguous interpretation of WRNp enzymatic activities on DNA substrates. Purified WRNp has DNA-dependent ATPase and helicase activities consistent with its homology to the RecQ subfamily of proteins. The protein also binds with higher affinity to single-stranded DNA than to double-stranded DNA. However, WRNp has no higher affinity for various types of DNA damage, including adducts formed during 4NQO treatment, than for undamaged DNA. Our results confirm that WRNp has a role in DNA metabolism, although this role does not appear to be the specific recognition of damage in DNA.     

Identification and biochemical characterization of a Werner's syndrome protein complex with Ku70/80 and poly(ADP-ribose) polymerase-1

Werner's syndrome (WS) is an inherited disease characterized by genomic instability and premature aging. The WS gene encodes a protein (WRN) with helicase and exonuclease activities. We have previously reported that WRN interacts with Ku70/80 and this interaction strongly stimulates WRN exonuclease activity. To gain further insight on the function of WRN and its relationship with the Ku heterodimer, we established a cell line expressing tagged WRN(H), a WRN point mutant lacking helicase activity, and used affinity purification, immunoblot analysis and mass spectroscopy to identify WRN-associated proteins. To this end, we identified three proteins that are stably associated with WRN in nuclear extracts. Two of these proteins, Ku70 and Ku80, were identified by immunoblot analysis. The third polypeptide, which was identified by mass spectrometry analysis, is identical to poly(ADP-ribose) polymerase-1(PARP-1), a 113-kDa enzyme that functions as a sensor of DNA damage. Biochemical fractionation studies and immunoprecipitation assays and studies confirmed that endogenous WRN is associated with subpopulations of PARP-1 and Ku70/80 in the cell. Protein interaction assays with purified proteins further indicated that PARP-1 binds directly to WRN and assembles in a complex with WRN and Ku70/80. In the presence of DNA and NAD(+), PARP-1 poly(ADP-ribosyl)ates itself and Ku70/80 but not WRN, and gel-shift assays showed that poly-(ADP-ribosyl)ation of Ku70/80 decreases the DNA-binding affinity of this factor. Significantly, (ADP-ribosyl)ation of Ku70/80 reduces the ability of this factor to stimulate WRN exonuclease, suggesting that covalent modification of Ku70/80 by PARP-1 may play a role in the regulation of the exonucleolytic activity of WRN.     

The Werner syndrome protein is distinguished from the Bloom syndrome protein by its capacity to tightly bind diverse DNA structures

Loss of Werner syndrome helicase-exonuclease (WRN) or of its homolog Bloom syndrome helicase (BLM) results in different inherited disorders. Whereas Werner syndrome is characterized by premature onset of aging and age-associated diseases, Bloom syndrome involves developmental abnormalities and increased predisposition to diverse malignancies. To identify biochemical differences between WRN and BLM that might contribute to the dissimilar outcomes of their loss, we compared their abilities to unwind and bind in vitro diverse DNA structures. Full-length recombinant WRN and BLM proteins expressed in and purified from Sf9 insect cells unwound to comparable extents and with similar K(m) values partial DNA duplex, splayed arm DNA and G'2 bimolecular quadruplex DNA. However, WRN resolved bubble DNA ～25-fold more efficiently than BLM. The two enzymes were mainly distinguished by their contrasting abilities to bind DNA. WRN bound partial duplexes, bubble and splayed arm DNA and G'2 bimolecular and G4 four-molecular quadruplexes with dissociation constants of 0.25 to 25 nM. By contrast, BLM formed substantial complexes with only G4 quadruplex DNA while binding only marginally other DNA structures. We raise the possibility that in addition to its enzymatic activities WRN may act as a scaffold for the assembly on DNA of additional DNA processing proteins.     

Physical and functional mapping of the replication protein a interaction domain of the werner and bloom syndrome helicases

The single-stranded DNA-binding protein replication protein A (RPA) interacts with several human RecQ DNA helicases that have important roles in maintaining genomic stability; however, the mechanism for RPA stimulation of DNA unwinding is not well understood. To map regions of Werner syndrome helicase (WRN) that interact with RPA, yeast two-hybrid studies, WRN affinity pull-down experiments and enzyme-linked immunosorbent assays with purified recombinant WRN protein fragments were performed. The results indicated that WRN has two RPA binding sites, a high affinity N-terminal site, and a lower affinity C-terminal site. Based on results from mapping studies, we sought to determine if the WRN N-terminal region harboring the high affinity RPA interaction site was important for RPA stimulation of WRN helicase activity. To accomplish this, we tested a catalytically active WRN helicase domain fragment (WRN(H-R)) that lacked the N-terminal RPA interaction site for its ability to unwind long DNA duplex substrates, which the wild-type enzyme can efficiently unwind only in the presence of RPA. WRN(H-R) helicase activity was significantly reduced on RPA-dependent partial duplex substrates compared with full-length WRN despite the presence of RPA. These results clearly demonstrate that, although WRN(H-R) had comparable helicase activity to full-length WRN on short duplex substrates, its ability to unwind RPA-dependent WRN helicase substrates was significantly impaired. Similarly, a Bloom syndrome helicase (BLM) domain fragment, BLM(642-1290), that lacked its N-terminal RPA interaction site also unwound short DNA duplex substrates similar to wild-type BLM, but was severely compromised in its ability to unwind long DNA substrates that full-length BLM helicase could unwind in the presence of RPA. These results suggest that the physical interaction between RPA and WRN or BLM helicases plays an important role in the mechanism for RPA stimulation of helicase-catalyzed DNA unwinding.     

Length-dependent degradation of single-stranded 3' ends by the Werner syndrome protein (WRN): implications for spatial orientation and coordinated 3' to 5' movement of its ATPase/helicase and exonuclease domains

Background  

The cancer-prone and accelerated aging disease Werner syndrome is caused by loss of function of the WRN gene product that possesses ATPase, 3' to 5' helicase and 3' to 5' exonuclease activities. Although WRN has been most prominently suggested to function in telomere maintenance, resolution of replication blockage and/or recombinational repair, its exact role in DNA metabolism remains unclear. WRN is the only human RecQ family member to possess both helicase and exonuclease activity, but the mechanistic relationship between these activities is unknown. In this study, model single-stranded and 3' overhang DNA substrates of varying length and structure were used to examine potential coordination between the ATPase/helicase and exonuclease activities of WRN.     

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.     

Physical and functional interaction of the Werner syndrome protein with poly-ADP ribosyl transferase

Werner's syndrome is a rare disease of premature ageing. The WRN gene product defective in this disorder belongs to the RecQ helicase family and is thought to be involved in DNA metabolism. Another protein, which plays an important role in both DNA replication and repair, is the poly-ADP ribosyl transferase. Here we demonstrate an interaction of these two proteins resulting in ADP-ribosylation of the WRN protein. These results imply that WRN is involved in DNA replication and in DNA repair.     

Modulation of Werner syndrome protein function by a single mutation in the conserved RecQ domain

Naturally occurring mutations in the human RECQ3 gene result in truncated Werner protein (WRN) and manifest as a rare premature aging disorder, Werner syndrome. Cellular and biochemical studies suggest a multifaceted role of WRN in DNA replication, DNA repair, recombination, and telomere maintenance. The RecQ C-terminal (RQC) domain of WRN was determined previously to be the major site of interaction for DNA and proteins. By using site-directed mutagenesis in the WRN RQC domain, we determined which amino acids might be playing a critical role in WRN function. A site-directed mutation at Lys-1016 significantly decreased WRN binding to fork or bubble DNA substrates. Moreover, the Lys-1016 mutation markedly reduced WRN helicase activity on fork, D-loop, and Holliday junction substrates in addition to reducing significantly the ability of WRN to stimulate FEN-1 incision activities. Thus, DNA binding mediated by the RQC domain is crucial for WRN helicase and its coordinated functions. Our nuclear magnetic resonance data on the three-dimensional structure of the wild-type RQC and Lys-1016 mutant proteins display a remarkable similarity in their structures.     

The Werner syndrome helicase/exonuclease (WRN) disrupts and degrades D-loops in vitro

The loss of function of WRN, a DNA helicase and exonuclease, causes the premature aging disease Werner syndrome. A hallmark feature of cells lacking WRN is genomic instability typified by elevated illegitimate recombination events and accelerated loss of telomeric sequences. In this study, the activities of WRN were examined on a displacement loop (D-loop) DNA substrate that mimics an intermediate formed during the strand invasion step of many recombinational processes. Our results indicate that this model substrate is specifically bound by WRN and efficiently disrupted by its helicase activity. In addition, the 3' end of the inserted strand of this D-loop structure is readily attacked by the 3'-->5' exonuclease function of WRN. These results indicate that D-loop structures are favored sites for WRN action. Thus, WRN may participate in DNA metabolic processes that utilize these structures, such as recombination and telomere maintenance pathways.     

Werner syndrome protein is regulated and phosphorylated by DNA-dependent protein kinase

DNA double-strand breaks (DSBs) are a highly mutagenic and potentially lethal damage that occurs in all organisms. Mammalian cells repair DSBs by homologous recombination and non-homologous end joining, the latter requiring DNA-dependent protein kinase (DNA-PK). Werner syndrome is a disorder characterized by genomic instability, aging pathologies and defective WRN, a RecQ-like helicase with exonuclease activity. We show that WRN interacts directly with the catalytic subunit of DNA-PK (DNA-PK(CS)), which inhibits both the helicase and exonuclease activities of WRN. In addition we show that WRN forms a stable complex on DNA with DNA-PK(CS) and the DNA binding subunit Ku. This assembly reverses WRN enzymatic inhibition. Finally, we show that WRN is phosphorylated in vitro by DNA-PK and requires DNA-PK for phosphorylation in vivo, and that cells deficient in WRN are mildly sensitive to ionizing radiation. These data suggest that DNA-PK and WRN may function together in DNA metabolism and implicate WRN function in non-homologous end joining.     

Mutations in the consensus helicase domains of the Werner syndrome gene. Werner's Syndrome Collaborative Group.

Werner syndrome (WS) is an autosomal recessive disease with a complex phenotype that is suggestive of accelerated aging. WS is caused by mutations in a gene, WRN, that encodes a predicted 1,432-amino-acid protein with homology to DNA and RNA helicases. Previous work identified four WS mutations in the 3' end of the gene, which resulted in predicted truncated protein products of 1,060-1,247 amino acids but did not disrupt the helicase domain region (amino acids 569-859). Here, additional WS subjects were screened for mutations, and the intron-exon structure of the gene was determined. A total of 35 exons were defined, with the coding sequences beginning in the second exon. Five new WS mutations were identified: two nonsense mutations at codons 369 and 889; a mutation at a splice-junction site, resulting in a predicted truncated protein of 760 amino acids; a 1-bp deletion causing a frameshift; and a predicted truncated protein of 391 amino acids. Another deletion is >15 kb of genomic DNA, including exons 19-23; the predicted protein is 1,186 amino acids long. Four of these new mutations either partially disrupt the helicase domain region or result in predicted protein products completely missing the helicase region. These results confirm that mutations in the WRN gene are responsible for WS. Also, the location of the mutations indicates that the presence or absence of the helicase domain does not influence the WS phenotype and suggests that WS is the result of complete loss of function of the WRN gene product.     

Interactions between the Werner syndrome helicase and DNA polymerase delta specifically facilitate copying of tetraplex and hairpin structures of the d(CGG)n trinucleotide repeat sequence

Werner syndrome (WS) is an inherited disorder characterized by premature aging and genomic instability. The protein encoded by the WS gene, WRN, possesses intrinsic 3' --> 5' DNA helicase and 3' --> 5' DNA exonuclease activities. WRN helicase resolves alternate DNA structures including tetraplex and triplex DNA, and Holliday junctions. Thus, one function of WRN may be to unwind secondary structures that impede cellular DNA transactions. We report here that hairpin and G'2 bimolecular tetraplex structures of the fragile X expanded sequence, d(CGG)(n), effectively impede synthesis by three eukaryotic replicative DNA polymerases (pol): pol alpha, pol delta, and pol epsilon. The constraints imposed on pol delta-catalyzed synthesis are relieved, however, by WRN; WRN facilitates pol delta to traverse these template secondary structures to synthesize full-length DNA products. The alleviatory effect of WRN is limited to pol delta; neither pol alpha nor pol epsilon can traverse template d(CGG)(n) hairpin and tetraplex structures in the presence of WRN. Alleviation of pausing by pol delta is observed with Escherichia coli RecQ but not with UvrD helicase, suggesting a concerted action of RecQ helicases and pol delta. Our findings suggest a possible role of WRN in rescuing pol delta-mediated replication at forks stalled by unusual DNA secondary structures.     

Molecular cooperation between the Werner syndrome protein and replication protein A in relation to replication fork blockage

The premature aging and cancer-prone disease Werner syndrome is caused by loss of function of the RecQ helicase family member Werner syndrome protein (WRN). At the cellular level, loss of WRN results in replication abnormalities and chromosomal aberrations, indicating that WRN plays a role in maintenance of genome stability. Consistent with this notion, WRN possesses annealing, exonuclease, and ATPase-dependent helicase activity on DNA substrates, with particularly high affinity for and activity on replication and recombination structures. After certain DNA-damaging treatments, WRN is recruited to sites of blocked replication and co-localizes with the human single-stranded DNA-binding protein replication protein A (RPA). In this study we examined the physical and functional interaction between WRN and RPA specifically in relation to replication fork blockage. Co-immunoprecipitation experiments demonstrated that damaging treatments that block DNA replication substantially increased association between WRN and RPA in vivo, and a direct interaction between purified WRN and RPA was confirmed. Furthermore, we examined the combined action of RPA (unmodified and hyperphosphorylation mimetic) and WRN on model replication fork and gapped duplex substrates designed to bind RPA. Even with RPA bound stoichiometrically to this gap, WRN efficiently catalyzed regression of the fork substrate. Further analysis showed that RPA could be displaced from both substrates by WRN. RPA displacement by WRN was independent of its ATPase- and helicase-dependent remodeling of the fork. Taken together, our results suggest that, upon replication blockage, WRN and RPA functionally interact and cooperate to help properly resolve replication forks and maintain genome stability.