Molecular cloning and biological characterization of a human gene, ERCC2, that corrects the nucleotide excision repair defect in CHO UV5 cells

1988 ◽  
Vol 8 (3) ◽  
pp. 1137-1146
Author(s):  
C A Weber ◽  
E P Salazar ◽  
S A Stewart ◽  
L H Thompson
Keyword(s):  
Cell Line ◽  
Nucleotide Excision Repair ◽  
Human Gene ◽  
Excision Repair ◽  
Hybrid Cell ◽  
Repetitive Sequences ◽  
Chinese Hamster ◽  
Wild Type ◽  
Wild Type Level ◽  
Nucleotide Excision

The UV-sensitive Chinese hamster ovary (CHO) cell line UV5, which is defective in the incision step of nucleotide excision repair, was used to identify and clone a complementing human gene, ERCC2, and to study the repair process. Genomic DNA from a human-hamster hybrid cell line was sheared and cotransferred with pSV2gpt plasmid DNA into UV5 cells to obtain five primary transformants. Transfer of sheared DNA from one primary transformant resulted in a secondary transformant expressing both gpt and ERCC2. The human repair gene was identified with a probe for Alu-family repetitive sequences. For most primary, secondary, and cosmid transformants, survival after UV exposure showed a return to wild-type levels of resistance. The levels of UV-induced mutation at the aprt locus for secondary and cosmid transformants varied from 50 to 130% of the wild-type level. Measurements of the initial rate of UV-induced strand incision by alkaline elution indicated that, whereas the UV5 rate was 3% of the wild-type level, rates of cosmid-transformed lines were similar to that of the wild type, and the secondary transformant rate was about 165% of the wild-type rate. Analysis of overlapping cosmids determined that ERCC2 is between 15.5 and 20 kilobases and identified a closely linked gpt gene. Cosmids were obtained with functional copies of both ERCC2 and gpt. ERCC2 corrects only the first of the five CHO complementation groups of incision-defective mutants.

scholarly journals Molecular cloning and biological characterization of a human gene, ERCC2, that corrects the nucleotide excision repair defect in CHO UV5 cells.

1988 ◽  
Vol 8 (3) ◽  
pp. 1137-1146 ◽  
Author(s):  
C A Weber ◽  
E P Salazar ◽  
S A Stewart ◽  
L H Thompson
Keyword(s):  
Cell Line ◽  
Nucleotide Excision Repair ◽  
Human Gene ◽  
Excision Repair ◽  
Hybrid Cell ◽  
Repetitive Sequences ◽  
Chinese Hamster ◽  
Wild Type ◽  
Wild Type Level ◽  
Nucleotide Excision

The UV-sensitive Chinese hamster ovary (CHO) cell line UV5, which is defective in the incision step of nucleotide excision repair, was used to identify and clone a complementing human gene, ERCC2, and to study the repair process. Genomic DNA from a human-hamster hybrid cell line was sheared and cotransferred with pSV2gpt plasmid DNA into UV5 cells to obtain five primary transformants. Transfer of sheared DNA from one primary transformant resulted in a secondary transformant expressing both gpt and ERCC2. The human repair gene was identified with a probe for Alu-family repetitive sequences. For most primary, secondary, and cosmid transformants, survival after UV exposure showed a return to wild-type levels of resistance. The levels of UV-induced mutation at the aprt locus for secondary and cosmid transformants varied from 50 to 130% of the wild-type level. Measurements of the initial rate of UV-induced strand incision by alkaline elution indicated that, whereas the UV5 rate was 3% of the wild-type level, rates of cosmid-transformed lines were similar to that of the wild type, and the secondary transformant rate was about 165% of the wild-type rate. Analysis of overlapping cosmids determined that ERCC2 is between 15.5 and 20 kilobases and identified a closely linked gpt gene. Cosmids were obtained with functional copies of both ERCC2 and gpt. ERCC2 corrects only the first of the five CHO complementation groups of incision-defective mutants.

scholarly journals ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs.

1996 ◽  
Vol 16 (11) ◽  
pp. 6553-6562 ◽  
Author(s):  
K W Brookman ◽  
J E Lamerdin ◽  
M P Thelen ◽  
M Hwang ◽  
J T Reardon ◽  
...  
Keyword(s):  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Cosmid Library ◽  
Protein Levels ◽  
Recombination Proteins ◽  
Acid Protein ◽  
Nucleotide Excision ◽  
Eukaryotic Dna

ERCC4 is an essential human gene in the nucleotide excision repair (NER) pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER genes, ERCC4 and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC1-ERCC4 heterodimer, like the homologous Rad10-Rad1 complex, was recently found to possess an endonucleolytic activity that incises on the 5' side of damage. The ERCC4 gene, assigned to chromosome 16p13.1-p13.2, was previously isolated by using a chromosome 16 cosmid library. It corrects the defect in Chinese hamster ovary (CHO) mutants of NER complementation group 4 and is implicated in complementation group F of the human disorder xeroderma pigmentosum. We describe the ERCC4 gene structure and functional cDNA sequence encoding a 916-amino-acid protein (104 kDa), which has substantial homology with the eukaryotic DNA repair and recombination proteins MEI-9 (Drosophila melanogaster), Rad16 (Schizosaccharomyces pombe), and Rad1 (Saccharomyces cerevisiae). ERCC4 cDNA efficiently corrected mutants in rodent NER complementation groups 4 and 11, showing the equivalence of these groups, and ERCC4 protein levels were reduced in mutants of both groups. In cells of an XP-F patient, the ERCC4 protein level was reduced to less than 5%, consistent with XPF being the ERCC4 gene. The considerable identity (40%) between ERCC4 and MEI-9 suggests a possible involvement of ERCC4 in meiosis. In baboon tissues, ERCC4 was expressed weakly and was not significantly higher in testis than in nonmeiotic tissues.

Cloning and Molecular Characterization of the Chinese Hamster ERCC2 Nucleotide Excision Repair Gene

Genomics ◽  
1994 ◽  
Vol 23 (3) ◽  
pp. 592-599 ◽  
Author(s):  
J.M. Kirchner ◽  
E.P. Salazar ◽  
J.E. Lamerdin ◽  
M.A. Montgomery ◽  
A.V. Carrano ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Chinese Hamster ◽  
Repair Gene ◽  
Nucleotide Excision Repair Gene ◽  
Nucleotide Excision

scholarly journals Single Nucleotide Polymorphisms in MiRNA Binding Sites of Nucleotide Excision Repair-Related Genes Predict Clinical Benefit of Oxaliplatin in FOLFOXIRI Plus Bevacizumab: Analysis of the TRIBE Trial

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1742
Author(s):  
Mitsukuni Suenaga ◽  
Marta Schirripa ◽  
Shu Cao ◽  
Wu Zhang ◽  
Dongyun Yang ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Nucleotide Excision Repair ◽  
Binding Sites ◽  
Excision Repair ◽  
Dependent Manner ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
First Line ◽  
Single Nucleotide ◽  
Nucleotide Excision

Background: The nucleotide excision repair (NER) pathway participates in platinum-induced DNA damage repair. Single nucleotide polymorphisms (SNPs) in miRNA-binding sites in the NER genes RPA2 and GTF2H1 are associated with the risk of colorectal cancer (CRC). Here, we analyzed whether RPA2 and GTF2H1 SNPs predict the efficacy of oxaliplatin in metastatic CRC (mCRC) patients. Patients and methods: Genomic DNA was extracted from blood samples from 457 patients with mCRC enrolled in the TRIBE trial, which compared first-line FOLFOXIRI plus bevacizumab (BEV) (n = 230, discovery cohort) and first-line FOLFIRI plus BEV (n = 227, control cohort). SNPs were analyzed by PCR-based direct sequencing. Results: In the FOLFOXIRI + BEV-treated cohort expressing wild-type KRAS, progression-free survival (PFS) was shorter for the RPA2 rs7356 C/C variant subgroup than the any T allele subgroup in univariate analysis (9.1 versus 13.3 months respectively, hazard ratio (HR) 2.32, 95% confidence interval (CI): 1.07–5.03, p = 0.020) and this remained significant in multivariable analysis (HR 2.97, 95%CI: 1.27–6.94, p = 0.012). A similar trend was observed for overall survival. In contrast, patients expressing mutant RAS and RPA2 rs7356 C/C variant had longer PFS with FOLFOXIRI + BEV than with FOLFIRI + BEV (12.1 versus 7.6 months, HR 0.23, 95%CI: 0.09–0.62, p = 0.002) but no superiority of FOLFOXIRI + BEV was observed for the RAS mutant, RPA2 rs7356 any T variant subgroup (11.7 versus 9.6 months, HR 0.77, 95%CI: 0.56–1.07, p = 0.12) or the RAS wild-type, RPA2 rs7356 C/C variant subgroup. Conclusion: RPA2 SNPs may serve as predictive and prognostic markers of oxaliplatin responsiveness in a RAS status-dependent manner in mCRC patients receiving FOLFOXIRI + BEV.

Nucleotide excision repair and photolyase repair of UV photoproducts in nucleosomes: assessing the existence of nucleosome and non-nucleosome rDNA chromatin in vivoThis paper is one of a selection of papers published in this Special Issue, entitled 29th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.

2009 ◽  
Vol 87 (1) ◽  
pp. 337-346 ◽  
Author(s):  
Maxime Tremblay ◽  
Martin Toussaint ◽  
Annie D’Amours ◽  
Antonio Conconi
Keyword(s):  
Dna Repair ◽  
Rna Polymerase ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Repetitive Sequences ◽  
High Rate ◽  
Rrna Genes ◽  
Nucleotide Excision ◽  
Nuclear Domains ◽  
Uv Photoproducts

The genome is organized into nuclear domains, which create microenvironments that favor distinct chromatin structures and functions (e.g., highly repetitive sequences, centromeres, telomeres, noncoding sequences, inactive genes, RNA polymerase II and III transcribed genes, and the nucleolus). Correlations have been drawn between gene silencing and proximity to a heterochromatic compartment. At the other end of the scale are ribosomal genes, which are transcribed at a very high rate by RNA polymerase I (~60% of total transcription), have a loose chromatin structure, and are clustered in the nucleolus. The rDNA sequences have 2 distinct structures: active rRNA genes, which have no nucleosomes; and inactive rRNA genes, which have nucleosomes. Like DNA transcription and replication, DNA repair is modulated by the structure of chromatin, and the kinetics of DNA repair vary among the nuclear domains. Although research on DNA repair in all chromosomal contexts is important to understand the mechanisms of genome maintenance, this review focuses on nucleotide excision repair and photolyase repair of UV photoproducts in the first-order packing of DNA in chromatin: the nucleosome. In addition, it summarizes the studies that have demonstrated the existence of the 2 rDNA chromatins, and the way this feature of the rDNA locus allows for direct comparison of DNA repair in 2 very different structures: nucleosome and non-nucleosome DNA.

Chromosomal model for analysis of a long CTG/CAG tract stability in wild-type Escherichia coli and its nucleotide excision repair mutants

2007 ◽  
Vol 53 (7) ◽  
pp. 860-868 ◽  
Author(s):  
Sylwia T. Szwarocka ◽  
Paweł Stączek ◽  
Paweł Parniewski
Keyword(s):  
Escherichia Coli ◽  
Nucleotide Excision Repair ◽  
Trinucleotide Repeat ◽  
Excision Repair ◽  
Repetitive Sequences ◽  
Lambda Phage ◽  
E Coli ◽  
Repeat Sequences ◽  
Nucleotide Excision ◽  
The Stability

Many human hereditary neurological diseases, including fragile X syndrome, myotonic dystrophy, and Friedreich’s ataxia, are associated with expansions of the triplet repeat sequences (TRS) (CGG/CCG, CTG/CAG, and GAA/TTC) within or near specific genes. Mechanisms that mediate mutations of TRS include DNA replication, repair, and gene conversion and (or) recombination. The involvement of the repair systems in TRS instability was investigated in Escherichia coli on plasmid models, and the results showed that the deficiency of some nucleotide excision repair (NER) functions dramatically affects the stability of long CTG inserts. In such models in which there are tens or hundreds of plasmid molecules in each bacterial cell, repetitive sequences may interact between themselves and according to a recombination hypothesis, which may lead to expansions and deletions within such repeated tracts. Since one cannot control interaction between plasmids, it is also sometimes difficult to give precise interpretation of the results. Therefore, using modified lambda phage (λInCh), we have constructed a chromosomal model to study the instability of trinucleotide repeat sequences in E. coli. We have shown that the stability of (CTG/CAG)68 tracts in the bacterial chromosome is influenced by mutations in NER genes in E. coli. The absence of the uvrC or uvrD gene products greatly enhances the instability of the TRS in the chromosome, whereas the lack of the functional UvrA or UvrB proteins causes substantial stabilization of (CTG/CAG) tracts.

scholarly journals Functional nucleotide excision repair is required for the preferential removal of N-ethylpurines from the transcribed strand of the dihydrofolate reductase gene of Chinese hamster ovary cells.

1997 ◽  
Vol 17 (2) ◽  
pp. 564-570 ◽  
Author(s):  
A Sitaram ◽  
G Plitas ◽  
W Wang ◽  
D A Scicchitano
Keyword(s):  
Nucleotide Excision Repair ◽  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Excision Repair ◽  
Chinese Hamster Ovary Cells ◽  
Alkylating Agents ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Reductase Gene ◽  
Nucleotide Excision

Transcription-coupled repair of DNA adducts is an essential factor that must be considered when one is elucidating biological endpoints resulting from exposure to genotoxic agents. Alkylating agents comprise one group of chemical compounds which modify DNA by reacting with oxygen and nitrogen atoms in the bases of the double helix. To discern the role of transcription-coupled DNA repair of N-ethylpurines present in discrete genetic domains, Chinese hamster ovary cells were exposed to N-ethyl-N-nitrosourea, and the clearance of the damage from the dihydrofolate reductase gene was investigated. The results indicate that N-ethylpurines were removed from the dihydrofolate reductase gene of nucleotide excision repair-proficient Chinese hamster ovary cells; furthermore, when repair rates in the individual strands were determined, a statistically significant bias in the removal of ethyl-induced, alkali-labile sites was observed, with clearance occurring 30% faster from the transcribed strand than from its nontranscribed counterpart at early times after exposure. In contrast, removal of N-ethylpurines was observed in the dihydrofolate reductase locus in cells that lacked nucleotide excision repair, but both strands were repaired at the same rate, indicating that transcription-coupled clearance of these lesions requires the presence of active nucleotide excision repair.

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