Analysis of the <i>Xenopus</i> Werner syndrome protein in DNA double-strand break repair

Yan, Hong; McCane, Jill E.; Toczylowski, Thomas; Chen, Chin-Yi

doi:10.1083/jcb.200502077

Cited by 30 publications

(41 citation statements)

References 65 publications

(80 reference statements)

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“…The nonhomologous tails are then trimmed off, and the two ends are ligated. Repair by SSA does not require Rad51, which mediates the strand invasion step of classical HR, but does involve the MRN complex (17,66). This is consistent with the requirements for Mre11 and NBS1, but not for Rad51c, XRCC2, and XRCC3, which are all specific for classical HR, in our circularization assays in deficient cell lines.…”

Section: Discussionsupporting

confidence: 74%

“…WRN colocalizes with both DSB repair and DNA replication sites and may function in restarting stalled or collapsed replication forks. A recent report suggests that WRN does not participate in NHEJ but has an essential role in single-strand annealing (SSA), an alternate form of HR (28,66). In SSA, the 5Ј DNA ends are unwound or degraded to expose 3Ј single-strand tails, as in classical HR, but instead of forming a synapse with a sister chromatid, the two exposed ends are base paired with one another via small regions of chance homology.…”

Section: Discussionmentioning

confidence: 99%

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Host Cell DNA Repair Pathways in Adeno-Associated Viral Genome Processing

Choi

McCarty

Samulski

2006

J Virol

119

133

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Recent studies have shown that wild-type and recombinant adeno-associated virus (AAV and rAAV) genomes persist in human tissue predominantly as double-stranded (ds) circular episomes derived from input linear single-stranded virion DNA. Using self-complementary recombinant AAV (scAAV) vectors, we generated intermediates that directly transition to ds circular episomes. The scAAV genome ends are palindromic hairpin-structured terminal repeats, resembling a double-stranded break repair intermediate. Utilizing this substrate, we found cellular DNA recombination and repair factors to be essential for generating circular episomal products. To identify the specific cellular proteins involved, the scAAV circularization-dependent vector was used as a reporter in 19 mammalian DNA repair-deficient cell lines. The results show that RecQ helicase family members (BLM and WRN), Mre11 and NBS1 of the Mre11-Rad50-Nbs1 (MRN) complex, and ATM are required for efficient scAAV genome circularization. We further demonstrated that the scAAV genome requires ATM and DNA-PK CS , but not NBS1, to efficiently convert to a circular form in nondividing cells in vivo using transgenic mice. These studies identify specific pathways involved for further elucidating viral and cellular mechanisms of DNA maintenance important to the viral life cycle and vector utilizations. Adeno-associated virus (AAV) and AAV-derived vectors (recombinant AAV [rAAV]) deliver a single-stranded DNA genome, which must be converted into double-stranded DNA (dsDNA) by the host cell. This process is normally carried out by utilizing the hairpinned inverted terminal repeats (TRs) in a self-priming replication mechanism (3). After this conversion, the vector genome undergoes additional changes mediated by host cell recombination factors acting on the AAV TR ends (30). Most of the vector DNA circularizes, but when the dose is high enough to deliver multiple genomes to the nucleus, they can join end to end to form concatemers, which also can circularize (11-13). A small number of vector genomes are integrated into the host chromosome, putatively through similar recombination reactions between the TRs and chromosomal double-strand breaks (DSBs) (30,32,34,45). Though relatively little is known about the cellular factors that participate in these reactions, these recombinations ultimately govern the stability of rAAV DNA and the potential for adverse interactions with genomic DNA sequences. Persistence of the vector genome in the host cell is a critical parameter for successful use of rAAV for gene delivery. In preclinical and clinical trials, rAAV genomes have been documented to persist in monkeys and humans for over 6 and 3.7 years, respectively (41). More recently, the molecular fate of both wild-type (wt) AAV in humans and rAAV in animals has been demonstrated to be persistence as episomal circles (5,47,48). This observation strongly suggests that wt and vector AAV genomes are utilizing similar host recombination pathways. While the correlation between long-term persistenc...

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Section: Discussionsupporting

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Host Cell DNA Repair Pathways in Adeno-Associated Viral Genome Processing

Choi

McCarty

Samulski

2006

J Virol

119

133

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“…In agreement with these findings, it has been observed that cells depleted of both BLM and EXO1 show a reduction in the formation of RPA foci in response to DSBs and are defective in DSB repair by HR (16,19). However, studies using Xenopus egg extracts and purified proteins have shown that Dna2 mediates DNA end resection together with WRN rather than BLM (21)(22)(23). This discrepancy prompted us to investigate the role of WRN in DNA end resection in human cells.…”

mentioning

confidence: 66%

DNA2 Cooperates with the WRN and BLM RecQ Helicases to Mediate Long-range DNA End Resection in Human Cells

Sturzenegger

Burdová

Kanagaraj

et al. 2014

Journal of Biological Chemistry

171

164

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Background: DNA end resection is a critical step in the homology-directed repair of DNA double strand breaks (DSBs). Results: Human WRN helicase stimulates the DNA2-catalyzed resection of DNA ends and acts in concert with DNA2 to promote DSB repair by single strand annealing. Conclusion: DNA2 cooperates with WRN or BLM to mediate the resection of DSBs in mammalian cells. Significance: Defects in DNA end resection might, in part, account for the genomic instability phenotype of Werner syndrome.

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“…As a hallmark, mutants of eukaryotic RecQ helicases, such as WRN, BLM, or SGS1, show enhanced sister chromatid exchange (SCE) frequencies (6,(29)(30)(31)(32)(33). SCEs are a specific type of HR events based on crossovers between sister chromatids.…”

Section: Resultsmentioning

confidence: 99%

Two closely related RecQ helicases have antagonistic roles in homologous recombination and DNA repair in Arabidopsis thaliana

Hartung

Suer

Puchta

2007

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

133

261

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RecQ helicases are involved in the processing of DNA structures arising during replication, recombination, and repair throughout all kingdoms of life. Mutations of different RecQ homologues are responsible for severe human diseases, such as Blooms (BLM) or Werner (WRN) syndrome. The loss of RecQ function is often accompanied by hyperrecombination caused by a lack of crossover suppression. In the Arabidopsis genome seven different RecQ genes are present. Two of them (AtRECQ4A and 4B) arose because of a recent duplication and are still nearly 70% identical on a protein level. Knockout of these genes leads to antagonistic phenotypes: the RECQ4A mutant shows sensitivity to DNA-damaging agents, enhanced homologous recombination (HR) and lethality in a mus81 background. Moreover, mutation of RECQ4A partially suppresses the lethal phenotype of an AtTOP3␣ mutant, a phenomenon that had previously been demonstrated for RecQ homologues of unicellular eukaryotes only. Together, these facts strongly suggest that in plants RECQ4A is functionally equivalent to SGS1 of Saccharomyces cerevisiae and the mammalian BLM protein. In stark contrast, mutants of the closely related RECQ4B are not mutagen-sensitive, not viable in a mus81 background, and unable to suppress the induced lethality caused by loss of TOP3␣. Moreover, they are strongly impaired in HR. Thus, AtRECQ4B is specifically required to promote but not to suppress crossovers, a role in which it differs from all eukaryotic RecQ homologues known.Blooms syndrome ͉ crossover suppression ͉ Holliday Junction ͉ Mus81 ͉ topoisomerase G enes coding for RecQ helicases are present in all pro-and eukaryotes investigated. The data available so far from completed genome sequences indicate that the number of RecQ genes rises from organisms with low complexity to those of higher complexity. Single RecQ genes are present in Escherichia coli and Saccharomyces cerevisiae, and five to eight RecQ genes are found in mammals and plants, respectively (1, 2). Therefore, the function of RecQ helicases seems to have adapted to the complexity of genomes present in higher eukaryotes by increasing their number. Sequence duplications were followed by subsequent differentiation of known functions or possible acquisition of new ones. In most cases knockout of RecQ genes results in a hyperrecombination phenotype in various organisms, such as bacteria, yeasts, mammals, or plants (3-6). Mutations in the BLM, WRN, and RECQ4 genes are the cause of severe diseases, such as Blooms, Werner, and Rothmund-Thomson syndromes, respectively. Furthermore, a mutant of the mammalian RECQ5 gene shows a synergistic increase of sister chromatid exchange in a blm background (7,8).A prominent function of several RecQ helicases is the processing of double-holliday junctions (dHJs) that occur as intermediates during replication, DNA repair, or recombination and dissolve them in a manner which prevents deleterious crossover recombination (9-11). The respective RecQ homologue (e.g., BLM, SGS1, or RQH1) acts in concert with topoi...

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Analysis of the Xenopus Werner syndrome protein in DNA double-strand break repair

Cited by 30 publications

References 65 publications

Host Cell DNA Repair Pathways in Adeno-Associated Viral Genome Processing

Host Cell DNA Repair Pathways in Adeno-Associated Viral Genome Processing

DNA2 Cooperates with the WRN and BLM RecQ Helicases to Mediate Long-range DNA End Resection in Human Cells

Two closely related RecQ helicases have antagonistic roles in homologous recombination and DNA repair in Arabidopsis thaliana

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