Reconstitution of Human DNA Repair Excision Nuclease in a Highly Defined System

Mu, David; Park, Chi-Hyun; Matsunaga, Tsukasa; Hsu, David S.; Reardon, Joyce T.; Sancar, Aziz

doi:10.1074/jbc.270.6.2415

Cited by 446 publications

(368 citation statements)

References 30 publications

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“…First, rapidly dividing cells are more sensitive to ICL agents than slowly growing cells (Mu et al, 1995;Hartmann et al, 1999). Second, ICL-induced DSB occurs in Saccharomyces cerevisiae only when proliferating .…”

Section: Discussionmentioning

confidence: 99%

Hypersensitivity of Brca1-deficient MEF to the DNA interstrand crosslinking agent mitomycin C is associated with defect in homologous recombination repair and aberrant S-phase arrest

et al. 2005

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Hypersensitivity of Brca1-deficient cells to interstrand crosslinking (ICL) agents such as cisplatin and mitomycin C (MMC) implicates an important role for Brca1 in cellular response to the ICL DNA damage repair. However, the detailed mechanism of how Brca1 is involved in the ICL response remains unclear. In this study, we analysed the cellular response to MMC treatment using isogenic mouse embryonic fibroblasts (MEFs) including wild type, p53 À/À and p53 À/À Brca1 À/À . Marked hypersensitivity of p53 À/À Brca1 À/À MEFs to MMC was found, and the reconstitution of Brca1 expression in these cells restored resistance to MMC. Upon MMC treatment, wild-type MEF was temporarily arrested at G2/M phase but subsequently resumed a normal cell cycle progression. In contrast, Brca1-deficient MEF exhibited a marked time-dependent accumulation of cells arrested at S phase and a prolonged increase in the G2/M population, followed by extensive cell deaths. Importantly, DNA damage-induced Rad51 foci were not formed in these cells, suggesting a defect in homologous recombination. Such defects are fully rescued by reconstitution of Brca1 expression in Brca1-deficient MEF, suggesting that Brca1 directly plays an essential role in ICL repair, which depends on homologous recombination during S phase.

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Section: Discussionmentioning

confidence: 99%

Hypersensitivity of Brca1-deficient MEF to the DNA interstrand crosslinking agent mitomycin C is associated with defect in homologous recombination repair and aberrant S-phase arrest

et al. 2005

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“…The results shown represent one out of a total of six independent experiments that were conducted; in all six experiments, R992A and R992E each exhibited a repair deficiency similar in magnitude to that shown. tion of dual incision (34,35). This indicates that factors in addition to those traditionally considered as constituents of the excision reaction may be needed to effect disassembly of the complex and facilitate reutilization of the constituents at a new damage site.…”

Section: Figmentioning

confidence: 99%

The DNA Repair Endonuclease XPG Binds to Proliferating Cell Nuclear Antigen (PCNA) and Shares Sequence Elements with the PCNA-binding Regions of FEN-1 and Cyclin-dependent Kinase Inhibitor p21

Gary¹,

Ludwig

Cornelius

et al. 1997

Journal of Biological Chemistry

225

165

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Proliferating cell nuclear antigen (PCNA) is a DNA polymerase accessory factor that is required for DNA replication during S phase of the cell cycle and for resynthesis during nucleotide excision repair of damaged DNA. PCNA binds to flap endonuclease 1 (FEN-1), a structure-specific endonuclease involved in DNA replication. Here we report the direct physical interaction of PCNA with xeroderma pigmentosum (XP) G, a structurespecific repair endonuclease that is homologous to FEN-1. We have identified a 28-amino acid region of human FEN-1 (residues 328 -355) and a 29-amino acid region of human XPG (residues 981-1009) that contains the PCNA binding activity. These regions share key hydrophobic residues with the PCNA-binding domain of the cyclin-dependent kinase inhibitor p21 Waf1/Cip1 , and all three competed with one another for binding to PCNA. A conserved arginine in FEN-1 (Arg 339 ) and XPG (Arg 992 ) was found to be crucial for PCNA binding activity. R992A and R992E mutant forms of XPG failed to fully reconstitute nucleotide excision repair in an in vivo complementation assay. These results raise the possibility of a mechanistic linkage between excision and repair synthesis that is mediated by PCNA.Exposure to UV light causes damage to DNA primarily in the form of cyclobutane pyrimidine dimers and (6-4) photoproducts. These types of DNA lesions, as well as bulky adducts produced by some chemical mutagens, are processed by nucleotide excision repair (NER). 1 The human genetic disorder xeroderma pigmentosum (XP) is the result of defects in this DNA damage repair pathway. Symptoms of XP include extreme sensitivity to sunlight exposure and a greatly elevated risk of skin cancer. In the past few years, much progress has been made in understanding the molecular events associated with NER (1). The DNA-binding protein XPA is involved in damage recognition. In concert with replication protein A, which binds singlestranded DNA, and helicases XPB and XPD, a ϳ27-29-base oligonucleotide segment containing the lesion is excised as the result of dual incision by structure-specific endonucleases XPF-ERCC1 and XPG. The XPF-ERCC1 complex cleaves the damaged strand at a 5Ј site about 23 nucleotides from the lesion, whereas XPG cleaves the strand approximately 5 nucleotides to the 3Ј side of the damage. The resultant gap is filled in by the action of DNA polymerase ␦ or ⑀, and then DNA ligase seals the nick to complete repair. The resynthesis step requires proliferating cell nuclear antigen (PCNA; Refs. 2 and 3), a ring-shaped homotrimeric protein that encircles DNA and acts as a "sliding clamp" that links the polymerase to the DNA template (4). PCNA performs the same essential function in replicative DNA synthesis during S phase of the cell cycle. PCNA requires replication factor C, a primer recognition protein that loads the PCNA trimer onto DNA in an ATP-dependent manner (5-7).XPG is homologous to another structure-specific endonuclease, FEN-1. FEN-1 is involved in Okazaki fragment processing during DNA replication (8), and it i...

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“…p127, though not increased after UV-irradiation, is transported from the cytoplasm to the nucleus upon the increase of p48 (Shiyanov et al, 1999;. Since NER is all but completed 48 h after UV-irradiation (Friedberg et al, 1995), and since recent NER reconstitution studies have shown that DDB is not required in vivo (Aboussekhara et al, 1995;Mu et al, 1995;Kazansev et al, 1996), the function, if any, of DDB in NER or DNA repair in general is unclear.…”

Section: Introductionmentioning

confidence: 99%

Abnormal regulation of DDB2 gene expression in xeroderma pigmentosum group E strains

2001

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A damage-speci®c DNA binding protein (DDB) activity is absent from a subset (DDB 7 ) of cells from individuals initially classi®ed as group E of xeroderma pigmentosum (XP), a hereditary, photosensitive disease with a high incidence of skin malignancies. In these cases, mutations have been identi®ed in the DDB2 gene (DDB2 7 ) that codes for the small subunit, p48, of the DDB heterodimer. In four DDB2 7 strains, neither p48 nor DDB activity were observed before or after UVirradiation, despite an unusually strong up-regulation of DDB2 mRNA levels after UV-irradiation. In a ®fth strain, XP82TO, p48 was detectable and both DDB2 mRNA and p48 levels were more up-regulated after UVirradiation than in normal primary cells. Moreover, DDB activity also became apparent after irradiation. XP82TO showed very mild clinical manifestations compared with the other DDB 7 patients. These results, coupled with our ®ndings that most, if not all DDB + cells classi®ed as XP-E were misclassi®ed, suggests a direct correlation between DDB2 levels and the XP-E phenotype. Oncogene (2001) 20, 7041 ± 7050.

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Reconstitution of Human DNA Repair Excision Nuclease in a Highly Defined System

Cited by 446 publications

References 30 publications

Hypersensitivity of Brca1-deficient MEF to the DNA interstrand crosslinking agent mitomycin C is associated with defect in homologous recombination repair and aberrant S-phase arrest

Hypersensitivity of Brca1-deficient MEF to the DNA interstrand crosslinking agent mitomycin C is associated with defect in homologous recombination repair and aberrant S-phase arrest

The DNA Repair Endonuclease XPG Binds to Proliferating Cell Nuclear Antigen (PCNA) and Shares Sequence Elements with the PCNA-binding Regions of FEN-1 and Cyclin-dependent Kinase Inhibitor p21

Abnormal regulation of DDB2 gene expression in xeroderma pigmentosum group E strains

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