Defining the roles of individual residues in the single-stranded DNA binding site of PcrA helicase

Dillingham, Mark S.; Soultanas, Panos; Wiley, Paul F.; Webb, Martin R.; Wigley, Dale B.

doi:10.1073/pnas.131009598

Cited by 97 publications

(91 citation statements)

References 28 publications

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“…4 A and B). Multiple structures of UvrD (36) and PcrA (37)(38)(39), as well as the recent structure of HEL308 (33), suggest a role for this structure in DNA strand separation. In different reaction intermediates, the aromatic residue may stack against the last base pair or with the first unpaired base.…”

Section: Resultsmentioning

confidence: 98%

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: 98%

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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“…The residual activities of the RecB2109CD enzyme likely result from RecD motor activity. recB344 (A68V) contains a mutation in helicase motif 1a of RecB (S. K. Amundsen and G. R. Smith, personal communication), which is known to be involved in the binding and translocation of ssDNA by SF1 helicases (86). This "Tex-class" mutant retains helicase-nuclease activity but is only partially defective for Chi-dependent activities (191), probably because the RecB motor retains partial function.…”

Section: Mutants Of the Recbcd Enzymementioning

confidence: 99%

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

Dillingham

Kowalczykowski

2008

Microbiol Mol Biol Rev

Self Cite

507

599

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SUMMARY The RecBCD enzyme of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. It also degrades linear double-stranded DNA, protecting the bacteria from phages and extraneous chromosomal DNA. The RecBCD enzyme is, however, regulated by a cis-acting DNA sequence known as Chi (crossover hotspot instigator) that activates its recombination-promoting functions. Interaction with Chi causes an attenuation of the RecBCD enzyme's vigorous nuclease activity, switches the polarity of the attenuated nuclease activity to the 5′ strand, changes the operation of its motor subunits, and instructs the enzyme to begin loading the RecA protein onto the resultant Chi-containing single-stranded DNA. This enzyme is a prototypical example of a molecular machine: the protein architecture incorporates several autonomous functional domains that interact with each other to produce a complex, sequence-regulated, DNA-processing machine. In this review, we discuss the biochemical mechanism of the RecBCD enzyme with particular emphasis on new developments relating to the enzyme's structure and DNA translocation mechanism.

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“…In summary, the elucidation of the ATPase cycle has shown a remarkably simple mechanism with a rate-limiting and essentially irreversible cleavage step, greatly accelerated by bound DNA and presumably corresponding to the major transition between structural states 34 . This would fit in with the idea that the translocation is essentially unidirectional.…”

Section: Accepted M Manuscriptmentioning

confidence: 99%