Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

MacInnes, Mark A.; Dickson, Juleen; Hernandez, R R; Learmonth, D; Lin, Grace; Mudgett, John S.; Park, M S; Schauer, Susan; Reynolds, Richard J.; Strniste, G.F.

doi:10.1128/mcb.13.10.6393

Cited by 48 publications

(40 citation statements)

References 65 publications

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“…XPG plasmid concentrations were determined by measurement of absorbance at 260 nm and verified by agarose gel electrophoresis and ethidium bromide staining. The XPG-deficient CHO cell line UV135 (16,23) was transfected with a mixture of 150 ng of luciferase plasmid, 150 ng of ␤-galactosidase plasmid (22), and 30 ng of wild-type or mutant XPG plasmid by calcium phosphate precipitation (16). For each XPG transfection set, three to six 60-mm dishes were used.…”

Section: Methodsmentioning

confidence: 99%

“…The amino acid 981-1009 fragment of human XPG was amplified by polymerase chain reaction with forward primer 5Ј-CCAGAATTCTTA-AAGCAACTCGATG-3Ј and reverse primer 5Ј-GTGCTCGAGTTA-ACGTTTAGCATCTTCTTTCTC-3 using plasmid pBSK-XPGA (16,17) as template. The EcoRI/XhoI-digested product was ligated with EcoRI/ XhoI-linearized pGEX-4T-1 to create plasmid GST-XPG that expresses a fusion protein of GST and XPG .…”

Section: Methodsmentioning

confidence: 99%

“…They are hypersensitive to UV-induced cell mortality and are severely defective in the repair of UV-damaged DNA. Both phenotypic defects can be corrected by transfection with an XPG expression plasmid (16,22,26). This system provides a model of NER function in vivo that is dependent upon exogenously supplied XPG.…”

Section: Identification Of a Pcna-binding Domain In Xpg-mentioning

confidence: 99%

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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

224

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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Identification Of a Pcna-binding Domain In Xpg-mentioning

confidence: 99%

See 1 more Smart Citation

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

224

165

View full text Add to dashboard Cite

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“…By introducing human DNA into a Chinese hamster ovary (CHO) mutant (UV135) (14), the human gene (ERCC5) that corrected UV sensitivity of UV135 was cloned (15,16). Recent genetic and biochemical complementation studies have assigned the ERCC5 gene to the human XPG genotype (17)(18)(19).…”

mentioning

confidence: 99%

Characterization of a Putative Helix-Loop-Helix Motif in Nucleotide Excision Repair Endonuclease, XPG

Park

Valdez

Gurley

et al. 1997

Journal of Biological Chemistry

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Complementation group G of xeroderma pigmentosum (XPG) is one of the most rare and pathophysiologically heterogeneous forms of this inherited disease. XPG patients exhibit varying phenotypes, from having a very mild defect in DNA repair to being severely affected, and a few cases are also associated with the neurological degeneracy and growth retardation of Cockayne's syndrome. The XPG gene encodes a 134-kDa nuclear protein that is essential for the incision steps of nucleotide excision repair. XPG protein contains a putative helixloop-helix (HLH) motif in the region that is most conserved among the members of structure-specific endonuclease family. To establish the functional significance of the HLH motif, we used several approaches, including theoretical modeling, functional complementation assay, structure-specific endonuclease assay, and DNA binding assay. A secondary structure of the motif was predicted by energy minimization and the Monte Carlo simulation and empirically proven using the circular dichroism to contain a high content of ␣-helix. When an XPG mutant lacking the HLH was overexpressed in UV135 cells, which have defects in the hamster homolog of XPG, the mutant gene failed to confer to the hamster cells the resistance to UV light. A recombinant XPG protein lacking the HLH motif was purified from insect cells and tested for a structure-specific endonuclease activity. The mutant protein failed to cleave the flap strand. A recombinant peptide containing the HLH (amino acids 758 -871) was expressed in and purified from bacteria, tested for DNA binding activity, and found to bind to a DNA substrate with the flap structure. These results suggest that the HLH motif is required for the catalytic and DNA binding activities of XPG.

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“…While studying tRNA transcription, the laboratory of Stuart Clarkson isolated cDNAs encoding frog and human homologs of the yeast RAD2 gene (the XPG equivalent in Saccharomyces cerevisiae) and demonstrated that the human cDNA was able to complement the UV sensitivity of XP-G cells. 9 XP-G cells were subsequently found to be equivalent to Chinese hamster ovary (CHO) cells from complementation group ERCC5, [10][11][12] and the XPG gene was mapped at chromosome 13q32-33. 13,14 Biochemical properties of the XPG protein Analysis of the primary sequence of XPG quickly revealed that it harbors two nuclease domains placing it in the Fen1 family of structure-specific endonucleases.…”

Section: Discovery and Cloning Of Xpgmentioning

confidence: 99%

XPG: Its Products and Biological Roles

Schärer

Molecular Mechanisms of Xeroderma Pigmentosum

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Xeroderma pigmetosum patients of the complementation group G are rare. One group of XP-G patients displays a rather mild and typical XP phenotype. Mutations in these patients interfere with the function of XPG in the nucleotide excision repair, where it has a structural role in the assembly of the preincision complex and a catalytic role in making the incision 3' to the damaged site in DNA. Another set of XP-G patient is much more severely affected, displaying combined symptoms of xeroderma pigmentosum and Cockayne syndrome, referred to as XP/CS complex. Although the molecular basis leading to the XP/CS complex has not yet been fully established, current evidence suggests that these patients suffer from a mild defect in transcription in addition to a repair defect. Here, the history of how the XPG gene was discovered, the biochemical properties of the XPG protein, and the molecular defects found in XP-G patients and mouse models are reviewed.

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Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Cited by 48 publications

References 65 publications

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

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

Characterization of a Putative Helix-Loop-Helix Motif in Nucleotide Excision Repair Endonuclease, XPG

XPG: Its Products and Biological Roles

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