Werner syndrome cells are sensitive to DNA cross‐linking drugs

Poot, Martin; Yom, Jin Song; Whang, Susan H.; Kato, Jackson T.; Gollahon, Katherine A.; Rabinovitch, Peter S.

doi:10.1096/fj.00-0611fje

Cited by 149 publications

(139 citation statements)

References 38 publications

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“…The DNA damaging agent chosen for our analyses was cis-Pt, one of several DNA cross-linking agents to which WS patient cells and cell lines are selectively hypersensitive [21]. In contrast to several other cross-linking agents, cis-Pt does not require metabolic activation, and has a well-defined DNA damage and mutational spectrum profile (see, for example, [22]).…”

Section: Resultsmentioning

confidence: 99%

“…Transient transfections were normalized for the percent transfected cells as revealed by use of a co-transfected bacterial ␤-galactosidase reporter plasmid. Recombination frequencies were corrected for the intrinsic difference in CFE between WRN-deficient and control cells, and for the selective sensitivity of WRN cells to cis-Pt cytotoxicity ( [13,21] and additional unpublished results). The statistical significance of differences in survival or recombinant colony generation after DNA damage as a function of genotype and WRN protein expression was determined as previously described [13].…”

Section: Cell Survival and Recombination Assaysmentioning

confidence: 99%

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The Werner syndrome protein has separable recombination and survival functions☆

et al. 2004

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The Werner syndrome (WS) protein WRN is unique in possessing a 3 to 5 exonuclease activity in addition to the 3 to 5 helicase activity characteristic of other RecQ proteins. In order to determine in vivo functions of the WRN catalytic activities and their roles in Werner syndrome pathogenesis, we quantified cell survival and homologous recombination after DNA damage in cells expressing WRN missense-mutant proteins that lacked exonuclease and/or helicase activity. Both WRN biochemical activities were required to generate viable recombinant daughter cells. In contrast, either activity was sufficient to promote cell survival after DNA damage in the absence of recombination. These results indicate that WRN has recombination and survival functions that can be separated by missense mutations. Two implications are that Werner syndrome most likely results from the loss of both activities and their associated functions from patient cells, and that WRN missense mutations or polymorphisms could promote genetic instability and cancer in the general population by selectively interfering with recombination in somatic cells.

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Section: Resultsmentioning

confidence: 99%

Section: Cell Survival and Recombination Assaysmentioning

confidence: 99%

The Werner syndrome protein has separable recombination and survival functions☆

et al. 2004

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“…WS cells display elevated chromosomal aberrations (2-4), replication defects (3,(5)(6)(7)(8), abnormal recombination (9,10), altered telomere dynamics (11), and hypersensitivity to DNA-damaging agents (12)(13)(14)(15)(16). The gene defective in WS, designated WRN (17), encodes a protein with DNA helicase (18,19) and exonuclease (20 -22) activities which presumably functions in DNA metabolism to preserve genome integrity.…”

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confidence: 99%

The Exonucleolytic and Endonucleolytic Cleavage Activities of Human Exonuclease 1 Are Stimulated by an Interaction with the Carboxyl-terminal Region of the Werner Syndrome Protein

Sharma

Sommers

Driscoll

et al. 2003

Journal of Biological Chemistry

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Exonuclease 1 (EXO-1), a member of the RAD2 family of nucleases, has recently been proposed to function in the genetic pathways of DNA recombination, repair, and replication which are important for genome integrity. Although the role of EXO-1 is not well understood, its 5 to 3 -exonuclease and flap endonuclease activities may cleave intermediates that arise during DNA metabolism. In this study, we provide evidence that the Werner syndrome protein (WRN) physically interacts with human EXO-1 and dramatically stimulates both the exonucleolytic and endonucleolytic incision functions of EXO-1. The functional interaction between WRN and EXO-1 is mediated by a protein domain of WRN which interacts with flap endonuclease 1 (FEN-1). Thus, the genomic instability observed in WRN؊/؊ cells may be at least partially attributed to the lack of interactions between the WRN protein and human nucleases including EXO-1.Werner syndrome (WS) 1 is a hereditary premature aging disorder characterized by genome instability (1). WS cells display elevated chromosomal aberrations (2-4), replication defects (3, 5-8), abnormal recombination (9, 10), altered telomere dynamics (11), and hypersensitivity to DNA-damaging agents (12-16). The gene defective in WS, designated WRN (17), encodes a protein with DNA helicase (18,19) and exonuclease (20 -22) activities which presumably functions in DNA metabolism to preserve genome integrity. To understand the basis of WS, a number of groups have investigated WRN protein interactions (for review, see Ref. 23). The collective work indicates that WRN interacts functionally with proteins implicated in replication and DNA repair including replication protein A (RPA), p53, Ku, and polymerase ␦. These studies have enabled researchers to speculate about pathways of DNA metabolism in which WRN might participate; however, the precise functions of the WRN gene product in vivo are not well understood.Recently we reported a novel interaction of WRN protein with the human 5Ј-flap endonuclease/5Ј to 3Ј-exonuclease (FEN-1) (24), a DNA structure-specific nuclease implicated in DNA replication, recombination, and repair (25). WRN protein stimulates FEN-1 cleavage activity by a physical interaction with a COOH-terminal domain of the WRN protein (24). Another member of the RAD2 family to which FEN-1 belongs is human exonuclease 1 (EXO-1) (26). Like FEN-1, EXO-1 is a structure-specific endonuclease as well as an exonuclease (27, 28). EXO-1 has been implicated in a number of DNA metabolic pathways including mismatch correction, mitotic and meiotic recombination, and double strand break repair (29 -34). A role for EXO-1 in Okazaki fragment processing during DNA replication has been suggested based on its structure-specific endonuclease and RNase H activities that are similar to those of FEN-1 (30). Functional overlap between EXO-1 and FEN-1 (Rad27 in Saccharomyces cerevisiae) has been proposed from observations in yeast that exoI: rad27 double mutants are inviable (35) and that overexpression of yeast EXO-1 or human EXO-1 co...

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“…WRN may also play a role in base excision repair as WRN stimulates DNA polymerase b-strand displacement synthesis via its helicase activity (Harrigan et al, , 2006. The sensitivity of WS cells to cross-linking drugs (Poot et al, 2001) and a very recent report on the WRN response to UV treatment (Guay et al, 2006) suggest that WRN might also be involved in the repair process that removes cross-linking induced lesions. Increasing evidence also supports the hypothesis that WRN plays a direct role in telomere maintenance (Opresko et al, 2004a;Laud et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

The Werner syndrome protein is required for recruitment of chromatin assembly factor 1 following DNA damage

et al. 2006

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The Werner syndrome protein (WRN) and chromatin assembly factor 1 (CAF-1) are both involved in the maintenance of genome stability. In response to DNAdamaging signals, both of these proteins relocate to sites where DNA synthesis occurs. However, the interaction between WRN and CAF-1 has not yet been investigated. In this report, we show that WRN interacts physically with the largest subunit of CAF-1, hp150, in vitro and in vivo. Although hp150 does not alter WRN catalytic activities in vitro, and the chromatin assembly activity of CAF-1 is not affected in the absence of WRN in vivo, this interaction may have an important role during the cellular response to DNA replication fork blockage and/or DNA damage signals. In hp150 RNA-mediated interference (RNAi) knockdown cells, WRN partially formed foci following hydroxyurea (HU) treatment. However, in the absence of WRN, hp150 did not relocate to form foci following exposure to HU and ultraviolet light. Thus, our results demonstrate that WRN responds to DNA damage before CAF-1 and suggest that WRN may recruit CAF-1, via interaction with hp150, to DNA damage sites during DNA synthesis.

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Werner syndrome cells are sensitive to DNA cross‐linking drugs

Cited by 149 publications

References 38 publications

The Werner syndrome protein has separable recombination and survival functions☆

The Werner syndrome protein has separable recombination and survival functions☆

The Exonucleolytic and Endonucleolytic Cleavage Activities of Human Exonuclease 1 Are Stimulated by an Interaction with the Carboxyl-terminal Region of the Werner Syndrome Protein

The Werner syndrome protein is required for recruitment of chromatin assembly factor 1 following DNA damage

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