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A novel function for the Mre11-Rad50-Xrs2 complex in base excision repair. Nucleic Acids Res
, 2010
"... The Mre11/Rad50/Xrs2 (MRX) complex in Saccharomyces cerevisiae has well-characterized functions in DNA double-strand break processing, checkpoint activation, telomere length maintenance and meiosis. In this study, we demonstrate an involvement of the complex in the base excision repair (BER) pathway ..."
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The Mre11/Rad50/Xrs2 (MRX) complex in Saccharomyces cerevisiae has well-characterized functions in DNA double-strand break processing, checkpoint activation, telomere length maintenance and meiosis. In this study, we demonstrate an involvement of the complex in the base excision repair (BER) pathway. We studied the repair of methyl-methanesulfonate-induced heat-labile sites in chromosomal DNA in vivo and the in vitro BER capacity for the repair of uracil- and 8-oxoG-containing oligonucleotides in MRX-deficient cells. Both approaches show a clear BER deficiency for the xrs2 mutant as compared to wildtype cells. The in vitro analyses revealed that both subpathways, long-patch and short-patch BER, are affected and that all components of the MRX complex are simi-larly important for the new function in BER. The investigation of the epistatic relationship of XRS2 to other BER genes suggests a role of the MRX complex downstream of the AP-lyases Ntg1 and Ntg2. Analysis of individual steps in BER showed that base recognition and strand incision are not affected by the MRX complex. Reduced gap-filling activity and the missing effect of aphidicoline treat-ment, an inhibitor for polymerases, on the BER effi-ciency indicate an involvement of the MRX complex in providing efficient polymerase activity.
Involvement of Werner syndrome protein in MUTYH-mediated repair of oxidative DNA damage
, 2011
"... Reactive oxygen species constantly generated as by-products of cellular metabolism readily attack genomic DNA creating mutagenic lesions such as 7,8-dihydro-8-oxo-guanine (8-oxo-G) that promote aging. 8-oxo-G:A mispairs arising during DNA replication are eliminated by base excision repair initiated ..."
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Reactive oxygen species constantly generated as by-products of cellular metabolism readily attack genomic DNA creating mutagenic lesions such as 7,8-dihydro-8-oxo-guanine (8-oxo-G) that promote aging. 8-oxo-G:A mispairs arising during DNA replication are eliminated by base excision repair initiated by the MutY DNA glycosylase homologue (MUTYH). Here, by using formaldehyde crosslinking in mammalian cell extracts, we demonstrate that the WRN helicase/exonuclease defective in the premature aging disorder Werner syndrome (WS) is recruited to DNA duplex containing an 8-oxo-G:A mispair in a manner dependent on DNA polymerase j (Polj) that catalyzes accurate DNA synthesis over 8-oxo-G. Similarly, by immunofluorescence, we show that Polj is required for accumulation of WRN at sites of 8-oxo-G lesions in human cells. Moreover, we show that nuclear focus formation of WRN and Polj induced by oxidative stress is de-pendent on ongoing DNA replication and on the presence of MUTYH. Cell viability assays reveal that depletion of MUTYH suppresses the hypersen-sitivity of cells lacking WRN and/or Polj to oxidative stress. Biochemical studies demonstrate that WRN binds to the catalytic domain of Polj and specifically stimulates DNA gap filling by Polj over 8-oxo-G followed by strand displacement synthesis. Our results suggest that WRN promotes long-patch DNA repair synthesis by Polj during MUTYH-initiated repair of 8-oxo-G:A mispairs.
DNA Interstrand Crosslinks Repair in Mammalian Cells
, 2008
"... We studied the formation of double strand breaks (DSBs) as intermediates in the repair of DNA interstrand crosslinks (ICLs) by homologous recombination (HR). The plasmid EGFP-N1 was crosslinked with trioxsalen to give one ICL per plasmid on average. HeLa cells were transfected with the crosslinked ..."
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We studied the formation of double strand breaks (DSBs) as intermediates in the repair of DNA interstrand crosslinks (ICLs) by homologous recombination (HR). The plasmid EGFP-N1 was crosslinked with trioxsalen to give one ICL per plasmid on average. HeLa cells were transfected with the crosslinked plasmids and the ICL repair was monitored by following the restoration of the GFP expression. It was accompanied by γ-H2AX foci formation suggesting that DSBs were formed during the process. However, the same amount of γ-H2AX foci was observed when cells were transfected with native plasmid, which indicated that γ-H2AX foci appearance could not be used to determine the amount of DSBs connected with the ICL repair in this system. For this reason we further monitored the DSB formation by determining the amount of linearized plasmids, since having one crosslink per plasmid on average, any ICL-driven DSB formation would lead to plasmid linearization. Native and crosslinked plasmids were incubated in repair-competent cell-free extracts from G1 and S phase HeLa cells. Although a considerable part of the ICLs was repaired, no linearization of the plasmids was observed in the extracts, which was interpreted that DSBs were not formed as intermediates in the process of ICL repair. In another set of experiments HRproficient HeLa and HR-deficient irs3 cells were transfected with native and crosslinked plasmids, and 6 h and 12 h later the plasmid DNA was isolated and analyzed by electrophoresis. The same amount of linear plasmid molecules was observed in both cell lines, regardless of whether they were transfected with native or crosslinked pEGFP-N1, which further confirmed that DSB formation was not an obligatory step in the process of ICL repair by HR.
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"... This work deals with the problem of estimating the intrinsic dimension of noisy, highdimensional point clouds. A general class of sets which are locally well-approximated by k dimensional planes but which are embedded in a D ≫ k dimensional Euclidean space are considered. Assuming one has samples fr ..."
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This work deals with the problem of estimating the intrinsic dimension of noisy, highdimensional point clouds. A general class of sets which are locally well-approximated by k dimensional planes but which are embedded in a D ≫ k dimensional Euclidean space are considered. Assuming one has samples from such a set, possibly corrupted by high-dimensional noise, if the data is linear the dimension can be recovered using PCA. However, when the data is non-linear, PCA fails, overestimating the intrinsic dimension. A multiscale version of PCA is thus introduced which is robust to small sample size, noise, and non-linearities in the data. iv For Trevor, whose constant friendship and support made this work possible. v Contents
doi:10.1093/nar/gkp1217 Human RECQL5b stimulates flap endonuclease 1
, 2009
"... Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated with elevated cancer risk. RECQL1 and RECQL5 have not been linked to any human disorder yet; cells ..."
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Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated with elevated cancer risk. RECQL1 and RECQL5 have not been linked to any human disorder yet; cells devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5b, with the human flap endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5b dramatically stimulates the rate of FEN1 cleavage of flap DNA substrates. Moreover, we show that RECQL5b and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4’s stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes. This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.
doi:10.1093/nar/gkm1008 SURVEY AND SUMMARY Human premature aging, DNA repair and
, 2007
"... Genomic instability leads to mutations, cellular dysfunction and aberrant phenotypes at the tissue and organism levels. A number of mechanisms have evolved to cope with endogenous or exogenous stress to prevent chromosomal instability and maintain cellular homeostasis. DNA helicases play important r ..."
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Genomic instability leads to mutations, cellular dysfunction and aberrant phenotypes at the tissue and organism levels. A number of mechanisms have evolved to cope with endogenous or exogenous stress to prevent chromosomal instability and maintain cellular homeostasis. DNA helicases play important roles in the DNA damage response. The RecQ family of DNA helicases is of particular interest since several human RecQ helicases are defective in diseases associated with premature aging and cancer. In this review, we will provide an update on our understanding of the specific roles of human RecQ helicases in the maintenance of genomic stability through their catalytic activities and protein interactions in various pathways of cellular nucleic acid metabolism with an emphasis on DNA replication and repair. We will also discuss the clinical features of the premature aging disorders associated with RecQ helicase deficiencies and how they relate to the molecular defects.
Comparative assessment of plasmid and
, 2007
"... oligonucleotide DNA substrates in measurement of in vitro base excision repair activity ..."
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oligonucleotide DNA substrates in measurement of in vitro base excision repair activity
Nucleic Acids Research, 2007, 1–18 doi:10.1093/nar/gkm1008 Human premature aging, DNA repair and
, 2007
"... Genomic instability leads to mutations, cellular dysfunction and aberrant phenotypes at the tissue and organism levels. A number of mechanisms have evolved to cope with endogenous or exogenous stress to prevent chromosomal instability and maintain cellular homeostasis. DNA helicases play important r ..."
Abstract
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Genomic instability leads to mutations, cellular dysfunction and aberrant phenotypes at the tissue and organism levels. A number of mechanisms have evolved to cope with endogenous or exogenous stress to prevent chromosomal instability and maintain cellular homeostasis. DNA helicases play important roles in the DNA damage response. The RecQ family of DNA helicases is of particular interest since several human RecQ helicases are defective in diseases associated with premature aging and cancer. In this review, we will provide an update on our understanding of the specific roles of human RecQ helicases in the maintenance of genomic stability through their catalytic activities and protein interactions in various pathways of cellular nucleic acid metabolism with an emphasis on DNA replication and repair. We will also discuss the clinical features of the premature aging disorders associated with RecQ helicase deficiencies and how they relate to the molecular defects.
