Analysis of the unwinding activity of the dimeric RECQ1 helicase in the presence of human replication protein A

Cui, Sheng; Arosio, Daniele; Doherty, Kevin M.; Brosh, Robert M.; Falaschi, Arturo; Vindigni, Alessandro

doi:10.1093/nar/gkh540

Cited by 103 publications

(146 citation statements)

References 72 publications

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“…Previous biochemical investigations of human RECQ1 used a full-length recombinant protein produced in insect cells. The full-length protein (RECQ1 FL ) appears as a mixture of different oligomeric forms (23,29). In common with other proteins of the RecQ family, RECQ1 FL has DNA-dependent ATPase activity, unwinds forked DNA substrates, resolves HJs, and has a DNA strand-annealing activity.…”

Section: Resultsmentioning

confidence: 99%

“…The proteins were expressed in E. coli and purified by using nickel-affinity purification, followed by preparative gel filtration. Full-length RECQ1 was expressed in insect cells as described previously (29).…”

Section: Methodsmentioning

confidence: 99%

“…The data collection and refinement statistics are summarized in Table S1. DNA helicase, ATPase assays, and analytical size exclusion chromatography were performed as described previously (23,29). The sequences of the oligonucleotides used for the helicase assays are reported in Table S2.…”

Section: Methodsmentioning

confidence: 99%

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Structure of the human RECQ1 helicase reveals a putative strand-separation pin

Pike

Shrestha

Popuri

et al. 2009

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

109

211

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RecQ-like helicases, which include 5 members in the human genome, are important in maintaining genome integrity. We present a crystal structure of a truncated form of the human RECQ1 protein with Mg-ADP. The truncated protein is active in DNA fork unwinding but lacks other activities of the full-length enzyme: disruption of Holliday junctions and DNA strand annealing. The structure of human RECQ1 resembles that of Escherichia coli RecQ, with some important differences. All structural domains are conserved, including the 2 RecA-like domains and the RecQ-specific zinc-binding and winged-helix (WH) domains. However, the WH domain is positioned at a different orientation from that of the E. coli enzyme. We identify a prominent ␤-hairpin of the WH domain as essential for DNA strand separation, which may be analogous to DNA strand-separation features of other DNA helicases. This hairpin is significantly shorter in the E. coli enzyme and is not required for its helicase activity, suggesting that there are significant differences between the modes of action of RecQ family members.DNA helicase ͉ DNA repair ͉ Holliday junction ͉ structural genomics ͉ winged helix T he RecQ helicases are a family of DNA-unwinding enzymes conserved from prokaryotes to mammals that play a key role in the maintenance of genome stability. The RecQ helicase family has 5 representatives in the human genome (1-3): RECQ1 (also known as RECQL or RECQL1), BLM, WRN, RECQ4, and RECQ5. Although these 5 enzymes are similar in their catalytic core, they probably have distinct functions, as indicated by the genetic disorders associated to mutations in the genes of BLM, WRN, and RECQ4. In particular, mutations in the gene encoding for BLM (4) are associated with the Bloom's syndrome (BS), which is manifested as an increased incidence of a wide spectrum of cancers. Werner's syndrome (WS), which is linked to mutations in the WRN (5) gene, involves many signs of premature aging, as well as a predisposition to a more limited spectrum of cancers. Mutations in the gene of RECQ4 are the cause of more varied genetic disease phenotypes, including Rothmund-Thomson (RTS) (6, 7), RAPADILINO (8), and Baller-Gerold (9) syndromes. No disease phenotypes have been associated with mutations in the genes of the other 2 family members, RECQ1 and RECQ5 yet, although they may be responsible for additional cancer predisposition disorders that are distinct from RTS, BS, and WS. In this regard, interesting candidates are patients with a phenotype similar to that of RTS individuals who do not carry any mutations in the RECQ4 gene (7). A possible role of RECQ1 in genome maintenance is suggested by several observations (reviewed in ref 10). Biochemical purification from human embryonic kidney cells recovered RECQ1 as the major Holliday junction (HJ) branch migration activity (11). Knockout of the RECQ1 gene in mice (12) or suppression of its expression in HeLa cells (11) resulted in cellular phenotypes that include chromosomal instability, increased sister chromatid exchange, and hei...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Structure of the human RECQ1 helicase reveals a putative strand-separation pin

Pike

Shrestha

Popuri

et al. 2009

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

109

211

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“…2, C and D); the main products of the Hel112 reaction on the HJ were Y-shaped forks, whereas only a limited amount of ssDNA was occasionally observed. Several mesophilic RecQ family helicases show the ability to unwind synthetic HJs, but in most cases, their reactions proceed up to production of ssDNA and are stimulated by ssDNA-binding proteins (37)(38)(39)(40). We reasoned that this difference might be due to spontaneous reannealing of the ssDNA products at 55°C.…”

Section: Resultsmentioning

confidence: 99%

Synergic and Opposing Activities of Thermophilic RecQ-like Helicase and Topoisomerase 3 Proteins in Holliday Junction Processing and Replication Fork Stabilization

Valenti

Felice

Perugino

et al. 2012

Journal of Biological Chemistry

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Background: RecQ helicases and topoisomerase 3 enzymes play essential functions in all DNA activities, and their malfunctioning is associated with genomic instability. Results: A RecQ-like helicase and topoisomerase 3 from thermophilic archaea interact physically and functionally. Conclusion:The thermophilic enzymes show synergic and antagonistic activities on different DNA substrates. Significance: The results suggest a novel mechanism of modulation of RecQ-like helicases by topoisomerase 3 enzymes.

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“…Yeast Rad54 protein, Srs2 helicase, RECQ1 helicase, human Hop2-Mnd1 complex, and yeast and human RPA were purified to near homogeneity from E. coli cells tailored to express them, as described (Sigurdsson et al 2001;Krejci et al 2003;Cui et al 2004;Van Komen et al 2006;Chi et al 2007). The BLM, RECQ1, and WRN helicase preparations have been used in several of our published studies (e.g., Raynard et al 2006;Hu et al 2007) and they possess a level of DNA-dependent ATPase activity as high as or higher than that reported in the literature (Brosh et al 1999(Brosh et al , 2000Cui et al 2004; data not shown) and, as expected (Brosh et al 1999(Brosh et al , 2000Cui et al 2004), are adept at unwinding a HJ test substrate (Supplemental Fig. 6).…”

Section: Purification Of Other Proteinsmentioning

confidence: 99%

Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination

Prakash¹,

Satory²,

Dray³

et al. 2009

Genes Dev.

232

342

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Eukaryotes possess mechanisms to limit crossing over during mitotic homologous recombination, thus avoiding possible chromosomal rearrangements. We show here that budding yeast Mph1, an ortholog of human FancM helicase, utilizes its helicase activity to suppress spontaneous unequal sister chromatid exchanges and DNA double-strand break-induced chromosome crossovers. Since the efficiency and kinetics of break repair are unaffected, Mph1 appears to channel repair intermediates into a noncrossover pathway. Importantly, Mph1 works independently of two other helicases-Srs2 and Sgs1-that also attenuate crossing over. By chromatin immunoprecipitation, we find targeting of Mph1 to double-strand breaks in cells. Purified Mph1 binds D-loop structures and is particularly adept at unwinding these structures. Importantly, Mph1, but not a helicase-defective variant, dissociates Rad51-made D-loops. Overall, the results from our analyses suggest a new role of Mph1 in promoting the noncrossover repair of DNA double-strand breaks.[Keywords: Genome instability; recombination; DNA helicase; crossing over; Fanconi anemia] Supplemental material is available at http://www.genesdev.org. Received September 8, 2008; revised version accepted November 12, 2008. DNA double-strand breaks (DSBs) that arise during DNA replication or are induced by DNA damaging agents, such as ionizing radiation, are frequently repaired by homologous recombination (HR). In yeast and other eukaryotes, the RAD52 epistasis group of proteins mediate homologous recombination (for reviews, see Paques and Haber 1999;Krogh and Symington 2004). In this process, DSB ends are resected by nucleases to create 39 ssDNA that becomes coated with the recombinase protein Rad51. The Rad51-ssDNA nucleoprotein filament then carries out a search for a homologous donor DNA sequence and promotes strand invasion of the donor molecule to form a D-loop (Sung and Klein 2006). After D-loop formation, there appear to be two alternative pathways that result in DSB repair. One pathway involves the formation of a double Holliday junction (dHJ) that can be resolved by symmetrical strand cleavage into either a crossover or noncrossover gene conversion (Szostak et al. 1983). An alternative mechanism, called synthesis-dependent strand annealing (SDSA), posits formation mostly of noncrossovers, and in most cases does not involve a dHJ intermediate (for review, see Paques and Haber 1999). In support of the SDSA model of gene conversion, we showed recently that both newly synthesized strands are inherited by the broken recipient DNA molecule (Ira et al. 2006). The choice between crossover and noncrossover is tightly regulated in both mitotic and meiotic cells. In meiotic S. cerevisiae cells the correct number of crossovers are required for proper chromosome segregation; the proportion of gene conversion accompanied by crossing over varies between 25% and 50% depending on the locus (Roeder 1995). In mitotic cells, the proportion of crossovers is much lower, ranging between <1% and 7 These authors c...

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Analysis of the unwinding activity of the dimeric RECQ1 helicase in the presence of human replication protein A

Cited by 103 publications

References 72 publications

Structure of the human RECQ1 helicase reveals a putative strand-separation pin

Structure of the human RECQ1 helicase reveals a putative strand-separation pin

Synergic and Opposing Activities of Thermophilic RecQ-like Helicase and Topoisomerase 3 Proteins in Holliday Junction Processing and Replication Fork Stabilization

Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination

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