Unwinding of a DNA Triple Helix by the Werner and Bloom Syndrome Helicases

Brosh, Robert M.; Majumdar, Angshuman; Desai, Shital; Hickson, Ian D.; Bohr, Vilhelm A.; Seidman, Michael M.

doi:10.1074/jbc.m006784200

Cited by 119 publications

(103 citation statements)

References 95 publications

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“…This regulation may depend upon the specific phosphorylation sites that are involved locally in different protein interactions, and there are three phosphorylation sites in WRN. We found the greatest increase in WRN helicase activity by dephosphorylation on the forked duplex substrate, a preferred substrate for WRN (52). Our finding that the phosphorylation state of WRN protein affects its helicase activity on that substrate suggests that WRN engages this structure during specific events such as replication fork block.…”

Section: Wrn Is Phosphorylated In Vivo After Dna Damagementioning

confidence: 69%

Werner Protein Is a Target of DNA-dependent Protein Kinase in Vivo and in Vitro, and Its Catalytic Activities Are Regulated by Phosphorylation

Karmakar¹,

Piotrowski²,

Brosh³

et al. 2002

Journal of Biological Chemistry

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150

133

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Human Werner Syndrome is characterized by early onset of aging, elevated chromosomal instability, and a high incidence of cancer. Werner protein (WRN) is a member of the recQ gene family, but unlike other members of the recQ family, it contains a unique 335 exonuclease activity. We have reported previously that human Ku heterodimer interacts physically with WRN and functionally stimulates WRN exonuclease activity. Because Ku and DNA-PKcs, the catalytic subunit of DNAdependent protein kinase (DNA-PK), form a complex at DNA ends, we have now explored the possibility of functional modulation of WRN exonuclease activity by DNA-PK. We find that although DNA-PKcs alone does not affect the WRN exonuclease activity, the additional presence of Ku mediates a marked inhibition of it. The inhibition of WRN exonuclease by DNA-PKcs requires the kinase activity of DNA-PKcs. WRN is a target for DNA-PKcs phosphorylation, and this phosphorylation requires the presence of Ku. We also find that treatment of recombinant WRN with a Ser/Thr phosphatase enhances WRN exonuclease and helicase activities and that WRN catalytic activity can be inhibited by rephosphorylation of WRN with DNA-PK. Thus, the level of phosphorylation of WRN appears to regulate its catalytic activities. WRN forms a complex, both in vitro and in vivo, with DNA-PKC. WRN is phosphorylated in vivo after treatment of cells with DNA-damaging agents in a pathway that requires DNA-PKcs. Thus, WRN protein is a target for DNA-PK phosphorylation in vitro and in vivo, and this phosphorylation may be a way of regulating its different catalytic activities, possibly in the repair of DNA dsb. Werner syndrome (WS)1 is a human autosomal recessive disorder characterized by early onset of premature aging characteristics including graying and loss of hair, wrinkling and ulceration of skin, atherosclerosis, osteoporosis, and cataracts. In addition, WS patients exhibit an increased incidence of diabetes mellitus type 2, hypertension, and malignancies (1). The gene (WRN), defects in which are responsible for WS, encodes a 1,432-amino acid protein (WRN) (2) that has both 3Ј35Ј helicase and 3Ј35Ј exonuclease activities (3-7). Although WRN appears to play an important role in DNA metabolism, the precise cellular roles of both the helicase and exonuclease activities of WRN remain to be determined.Cells from patients with WS show premature replicative senesence compared with cells derived from normal individuals (8). The WS cellular phenotype suggests correlations among faulty DNA metabolism, genomic instability, and senescence. WS cells show hypersensitivity to selected DNA-damaging agents including 4-nitroquinoline-1-oxide (4NQO) (9), topoisomerase inhibitors (10), and certain DNA cross-linking agents (11). Compared with normal cells, WS cells also exhibit increased genomic instability including higher levels of DNA deletions, translocations, and chromosomal breaks (12, 13). These studies suggest that WRN plays an important role in DNA metabolism possibly by participating in DNA repair, re...

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Section: Wrn Is Phosphorylated In Vivo After Dna Damagementioning

confidence: 69%

Werner Protein Is a Target of DNA-dependent Protein Kinase in Vivo and in Vitro, and Its Catalytic Activities Are Regulated by Phosphorylation

Karmakar¹,

Piotrowski²,

Brosh³

et al. 2002

Journal of Biological Chemistry

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150

133

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“…This parallel suggests a model in which WH domain dynamics in BLM and bacterial RecQs could allow the flexibility needed to accommodate diverse DNA substrates, whereas the fixed position of the WH domain in RecQ1 limits its activity to a narrower range of substrates. This model further suggests that additional RecQ family members, such as WRN, that also can unwind diverse DNA structures (31,32) would have dynamic WH domains as well.…”

Section: Discussionmentioning

confidence: 94%

“…S1) (10)(11)(12). The structure-specific DNA-unwinding properties of these enzymes also vary: BLM and EcRecQ process diverse DNA structures, including duplex, triplex, and quadruplex DNA, but RecQ1 is far more restricted, unwinding only duplex and Holliday junction DNA (13,(28)(29)(30)(31). This parallel suggests a model in which WH domain dynamics in BLM and bacterial RecQs could allow the flexibility needed to accommodate diverse DNA substrates, whereas the fixed position of the WH domain in RecQ1 limits its activity to a narrower range of substrates.…”

Section: Discussionmentioning

confidence: 99%

Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

Manthei

Hill

Burke

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. Here, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3′ single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ∼90°to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. This bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.helicase | RecQ | mechanism | aromatic-rich loop | DNA bending H elicases are motor proteins that convert the chemical energy of nucleoside triphosphate (NTP) hydrolysis into the mechanical energy needed to separate nucleic acid strands (1). The largest and most diverse helicase superfamilies, SF1 and SF2, use conserved sequence motifs (I, Ia, II-VI) within their helicase domains to couple NTP hydrolysis to conformational changes that mediate DNA translocation and unwinding (1, 2). Although the DNA-unwinding mechanisms of SF1 helicases have been examined extensively, far less is known about SF2 enzymes, in part because of the smaller number of available helicase/substrate complex structures.RecQ DNA helicases are SF2 enzymes with broad roles in promoting genomic stability in eubacterial and eukaryotic species (3). Their importance is underscored by the multiple genomic instability diseases caused by mutations in human recQ genes (4-8). At a structural level, most RecQ helicases share a similar domain architecture that includes a helicase domain, a RecQ C-terminal (RQC) element comprised of Zn 2+ -binding and winged-helix (WH) domains, and a helicase and RNaseD C-terminal (HDRC) domain (Fig. 1A) (9, 10). The helicase and RQC domains combine to form a catalytic core that is sufficient for DNA-unwinding activity in many RecQ proteins. Crystal structures of several RecQ catalytic cores have been determined, including Escherichia coli RecQ (EcRecQ), human RecQ1, and human Bloom syndrome protein (BLM) [refs. 10-12 and unpublished structures (4CDG, 2WWY, and 4CGZ) available through the Protein Data Bank (PDB)]. A comparison of these structures reveals strong similarities among domains within the catalytic core but also differences in the relative positioning of these domains among RecQ proteins. The EcRecQ and antibody-bound BLM structures form an open arrangement in which the WH domain is centered relative to the helicase and Zn 2+ -binding domains, but in RecQ1 and DNA-bound BLM a closed arrangement is observed with the WH domain positioned laterally to the helicase domain (Fig. 1B and Fig. S1) (10-12). The functional relevance of...

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“…Helicase reactions with the forked duplex substrates were terminated with the same buffer and 3.3 nM unlabeled oligonucleotide. Reactions were electrophoresed on 12% polyacrylamide 1ϫ Tris borate/ EDTA gels and analyzed as described (23).…”

Section: Fig 1-continuedmentioning

confidence: 99%

Colocalization, Physical, and Functional Interaction between Werner and Bloom Syndrome Proteins

Kobbe¹,

Karmakar²,

Dawut³

et al. 2002

Journal of Biological Chemistry

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104

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The RecQ helicase family comprises a conserved group of proteins implicated in several aspects of DNA metabolism. Three of the family members are defective in heritable diseases characterized by abnormal growth, premature aging, and predisposition to malignancies. These include the WRN and BLM gene products that are defective in Werner and Bloom syndromes, disorders which share many phenotypic and cellular characteristics including spontaneous genomic instability. Here, we report a physical and functional interaction between BLM and WRN. These proteins were coimmunoprecipitated from a nuclear matrix-solubilized fraction, and the purified recombinant proteins were shown to interact directly. Moreover, BLM and WRN colocalized to nuclear foci in three human cell lines. Two regions of WRN that mediate interaction with BLM were identified, and one of these was localized to the exonuclease domain of WRN. Functionally, BLM inhibited the exonuclease activity of WRN. This is the first demonstration of a physical and functional interaction between RecQ helicases. Our observation that RecQ family members interact provides new insights into the complex phenotypic manifestations resulting from the loss of these proteins. Werner syndrome (WS)1 is a hallmark premature aging syndrome associated with increased malignancies and genomic instability (1). The protein defective in WS, Werner syndrome protein (WRN), is an ATP-dependent 3Ј-5Ј-helicase and also has a 3Ј-5Ј-exonuclease activity (2). Recently, a number of protein partners for WRN have been identified. These include proliferating cell nuclear antigen (3), replication protein A (RPA) (4), DNA topoisomerase I (3), the Ku heterodimer (5, 6), DNA polymerase ␦ (7), and p53 (8). Some of these interactions are not only physical but are also functional. Each of these binding proteins is involved in some form of DNA metabolism, such as DNA recombination, replication, and repair, and in the resolution of alternative DNA structures (1, 2). This suggests that WRN plays a role in a number of key DNA metabolic pathways. Bloom syndrome (BS) is a highly cancer-prone disease associated with increased genomic instability and elevated sister chromatid exchanges. The protein defective in this disorder, Bloom syndrome protein (BLM), is a 3Ј-5Ј-helicase (9). BLM binds to RPA, p53, topoisomerase III␣, and RAD51 and is a component of the BRCA1-associated genome surveillance complex (10). This links BLM with proteins implicated in several aspects of DNA metabolism (DNA recombination, replication, and repair).BLM and WRN both belong to the RecQ family of helicases, which are conserved from Escherichia coli to human (11). WRN is unique among the human RecQ helicases in having an exonuclease domain in the N-terminal region of the protein. A number of the RecQ helicases have been purified, and their biochemical properties have been characterized (11). There is considerable interest in the substrate specificities of each of these enzymes and in possible differences between them. However, so far there h...

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Unwinding of a DNA Triple Helix by the Werner and Bloom Syndrome Helicases

Cited by 119 publications

References 95 publications

Werner Protein Is a Target of DNA-dependent Protein Kinase in Vivo and in Vitro, and Its Catalytic Activities Are Regulated by Phosphorylation

Werner Protein Is a Target of DNA-dependent Protein Kinase in Vivo and in Vitro, and Its Catalytic Activities Are Regulated by Phosphorylation

Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

Colocalization, Physical, and Functional Interaction between Werner and Bloom Syndrome Proteins

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