Analysis of helicase activity and substrate specificity of Drosophila RECQ5

Özsoy, A. Zeynep; Ragonese, Heather M.; Matson, Steven W.

doi:10.1093/nar/gkg243

Cited by 49 publications

(44 citation statements)

References 35 publications

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“…The implication that Recql5 can prevent impeded replication forks from collapsing after CPT treatment is in agreement with this recent finding and suggests that a similar scenario also exist in mammalian cells. A role of Recql5 in maintaining the integrity of stalled replication forks to prevent fork collapse is in agreement with previous reports that link this helicase (Garcia et al, 2004;Kanagaraj et al, 2006) and its homologues (Ozsoy et al, 2003) to DNA replication. Inter- First, a pair of arrows in opposite orientations is used to indicate the highly reversible nature of a Topo I-DNA complex associated with an on-going DNA replication fork (1F and 1R).…”

Section: Discussionsupporting

confidence: 92%

Recql5 Plays an Important Role in DNA Replication and Cell Survival after Camptothecin Treatment

Zhou

et al. 2009

MBoC

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Disruption of replication can lead to loss of genome integrity and increase of cancer susceptibility in mammals. Thus, a replication impediment constitutes a formidable challenge to these organisms. Recent studies indicate that homologous recombination (HR) plays an important role in suppressing genome instability and promoting cell survival after exposure to various replication inhibitors, including a topoisomerase I inhibitor, camptothecin (CPT). Here, we report that the deletion of RecQ helicase Recql5 in mouse ES cells and embryonic fibroblast (MEF) cells resulted in a significant increase in CPT sensitivity and a profound reduction in DNA replication after the treatment with CPT, but not other DNAdamaging agents. This CPT-induced cell death is replication dependent and occurs primarily after the cells had exited the first cell cycle after CPT treatment. Furthermore, we show that Recql5 functions nonredundantly with Rad51, a key factor for HR to protect mouse ES cells from CPT-induced cytotoxicity. These new findings strongly suggest that Recql5 plays an important role in maintaining active DNA replication to prevent the collapse of replication forks and the accumulation of DSBs in order to preserve genome integrity and to prevent cell death after replication stress as a result of topoisomerase I poisoning. INTRODUCTIONMammalian RecQ helicases share a high degree of homology with the RecQ helicase of Escherichia coli (Nakayama, 2002). These helicases have important roles in maintaining the integrity of the genomes and promoting cell survival in response to various types of genotoxic stress (Hickson, 2003). In addition, some of them have also been implicated in DNA replication (Oakley et al., 2002;Cheok et al., 2005). The only RecQ helicase in E. coli, RecQ, plays an important role in the recovery of genomic replication after DNA damage Hanawalt, 1999, 2001). In both budding yeast and fission yeast, there exist also just a single RecQ helicase. In both species, the RecQ helicase is involved in a number of DNA transaction processes, including repair, recombination and chromosome segregation (Hickson, 2003). Interestingly, these enzymes do not appear to be essential for processive DNA synthesis. Rather, they are required to prevent genomic instability by coordinating replication and recombination events at stalled replication forks (Lambert et al., 2007). Unlike unicellular organisms which have a single RecQ helicase, mammals possess a family of helicases encoded by five different genes, i.e., human RECQL, BLM, WRN, RECQL4, and RECQL5 (Hickson, 2003). Each of these five genes encodes one or multiple polypeptides with distinctive C-and/or N-terminal domains, suggesting that they have related but nonoverlapping functions.In particular, mutations in three of the five RecQ homologues, RECQL4, WRN, and BLM give rise to three distinct genomic instability and cancer-prone genetic disorders: Rothmund-Thomson, Werner, and Bloom syndromes, respectively (Ellis et al., 1995;Yu et al., 1996;Kitao et al., 1999). However, th...

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Section: Discussionsupporting

confidence: 92%

Recql5 Plays an Important Role in DNA Replication and Cell Survival after Camptothecin Treatment

Zhou

et al. 2009

MBoC

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“…2). In contrast, DmRecQ5 preferentially unwinds forked duplexes and is less active on HJ and bubble substrates (14). RecQ helicases likely have some complementary and distinct roles in vivo.…”

Section: Biochemical Characteristics Of Recq Helicasesmentioning

confidence: 97%

“…Similar to WRN, BLM, and Sgs1, the small isoform of DmRecQ5 helicase from Drosophila melanogaster (14) and human RecQ1 (RecQL) (15) are inactive on 5Ј ssDNA tailed or blunt-ended duplexes but do unwind some junction-containing substrates (Fig. 2).…”

Section: Biochemical Characteristics Of Recq Helicasesmentioning

confidence: 99%

Junction of RecQ Helicase Biochemistry and Human Disease

Opresko

Cheng

Bohr

2004

Journal of Biological Chemistry

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RecQ helicases are a family of conserved enzymes required for maintaining the genomic integrity, that function as suppressors of inappropriate recombination. Mutations in Escherichia coli RecQ and in the Saccharomyces cerevisiae RecQ homolog, Sgs1, result in an increased frequency of illegitimate recombination (1). In humans, defects in three RecQ family proteins are associated with rare autosomal-recessive disorders characterized by genomic instability and increased cancer susceptibility. Mutations in WRN, BLM, and RECQ4 give rise to the disorders Werner syndrome (WS), 1 Bloom syndrome (BS), and Rothmund-Thomson syndrome (RTS), respectively, the clinical features of which have been reviewed elsewhere (2). Briefly, BS patients are predisposed to many types of cancer with the mean age of onset of 24. WS patients are especially predisposed to sarcomas, premature aging, and ageassociated diseases. RTS patients have a characteristic rash, poikiloderma, and are predisposed to osteosarcomas and some features of premature aging. The molecular basis of genomic instability and premature aging is not well understood.The RecQ family is named after E. coli RecQ helicase, a well characterized prototypical member (Fig. 1). Helicases separate complementary strands of nucleic acids in a reaction coupled to NTP hydrolysis. RecQ helicases have a common helicase domain, which binds and hydrolyzes ATP. Most RecQ helicases have a highly conserved multifunctional RecQ C-terminal region (RQC) and a helicase RNase D C-terminal (HRDC) domain (Fig. 1). A recent report of the x-ray crystal structure for the E. coli RecQ catalytic core indicates that the RQC domain contains DNA and protein binding motifs (3). Consistent with this, the RQC domain of WRN binds to various DNA substrates and mediates interactions with other proteins involved in DNA metabolism (4). The E. coli RecQ HRDC domain is required for stable DNA binding but not for catalytic activity (5). Similarly, the HRDC domain of human WRN also binds DNA substrates but is not required for catalytic activity (4). Two RecQ family proteins WRN and Xenopus laevis FFA-1 also have an exonuclease domain. Bacteria and yeast have a single RecQ family member, and up to five RecQ members have been found in mammals.To better define the precise roles of RecQ helicases in vivo, significant research effort has been devoted to characterizing the biochemical properties of RecQ helicases and to identifying important protein interactions between RecQ helicases and other well characterized proteins. This review will focus primarily on these aspects and on the most well characterized RecQ helicases, due to space limitations. RecQ helicase genetic studies in yeast have been recently reviewed elsewhere (6). Biochemical Characteristics of RecQ HelicasesAll RecQ helicases purified and characterized to date unwind duplex DNA in a 3Ј to 5Ј direction with respect to the DNA strand bound by the helicase. Substrate preferences are determined by comparing product amounts, reaction kinetics, and/or protein affini...

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“…DNA Substrates-The sequences of oligonucleotides used in preparation of various substrates for gel mobility shift assays have been described elsewhere (14). These oligomers were annealed in appropriate combinations to obtain the DNA forms shown in Figs.…”

Section: Cloning Of Claspin Into Expression Vectors-we Isolated Humanmentioning

confidence: 99%

Human Claspin Is a Ring-shaped DNA-binding Protein with High Affinity to Branched DNA Structures

Sar

Lindsey-Boltz

Subramanian

et al. 2004

Journal of Biological Chemistry

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Claspin is an essential protein for the ATR-dependent activation of the DNA replication checkpoint response in Xenopus and human cells. Here we describe the purification and characterization of human Claspin. The protein has a ring-like structure and binds with high affinity to branched DNA molecules. These findings suggest that Claspin may be a component of the replication ensemble and plays a role in the replication checkpoint by directly associating with replication forks and with the various branched DNA structures likely to form at stalled replication forks because of DNA damage.

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Analysis of helicase activity and substrate specificity of Drosophila RECQ5

Cited by 49 publications

References 35 publications

Recql5 Plays an Important Role in DNA Replication and Cell Survival after Camptothecin Treatment

Recql5 Plays an Important Role in DNA Replication and Cell Survival after Camptothecin Treatment

Junction of RecQ Helicase Biochemistry and Human Disease

Human Claspin Is a Ring-shaped DNA-binding Protein with High Affinity to Branched DNA Structures

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