Molecular cloning and biological characterization of the human excision repair gene ERCC-3

1990 ◽  
Vol 10 (6) ◽  
pp. 2570-2581
Author(s):  
G Weeda ◽  
R C van Ham ◽  
R Masurel ◽  
A Westerveld ◽  
H Odijk ◽  
...  
Keyword(s):  
High Efficiency ◽  
Excision Repair ◽  
Alkylating Agents ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Cdna Clones ◽  
Chromosomal Dna ◽  
Dominant Marker ◽  
Mammalian Expression ◽  
Group 3

In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion (L. H. Thompson, A. V. Carrano, K. Sato, E. P. Salazar, B. F. White, S. A. Stewart, J. L. Minkler, and M. J. Siciliano, Somat. Cell. Mol. Genet. 13:539-551, 1987).

scholarly journals Molecular cloning and biological characterization of the human excision repair gene ERCC-3.

1990 ◽  
Vol 10 (6) ◽  
pp. 2570-2581 ◽  
Author(s):  
G Weeda ◽  
R C van Ham ◽  
R Masurel ◽  
A Westerveld ◽  
H Odijk ◽  
...  
Keyword(s):  
High Efficiency ◽  
Excision Repair ◽  
Alkylating Agents ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Cdna Clones ◽  
Chromosomal Dna ◽  
Dominant Marker ◽  
Mammalian Expression ◽  
Group 3

In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion (L. H. Thompson, A. V. Carrano, K. Sato, E. P. Salazar, B. F. White, S. A. Stewart, J. L. Minkler, and M. J. Siciliano, Somat. Cell. Mol. Genet. 13:539-551, 1987).

scholarly journals An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

1994 ◽  
Vol 14 (1) ◽  
pp. 68-76 ◽  
Author(s):  
K W Caldecott ◽  
C K McKeown ◽  
J D Tucker ◽  
S Ljungquist ◽  
L H Thompson
Keyword(s):  
Dna Repair ◽  
Affinity Chromatography ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Affinity Purification ◽  
Alkylating Agents ◽  
Chinese Hamster ◽  
Dna Ligase ◽  
Base Excision ◽  
Dna Ligase Iii

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.

scholarly journals DNA Damage Induced by Alkylating Agents and Repair Pathways

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Natsuko Kondo ◽  
Akihisa Takahashi ◽  
Koji Ono ◽  
Takeo Ohnishi
Keyword(s):  
Dna Damage ◽  
Dna Methyltransferase ◽  
Clinical Medicine ◽  
Excision Repair ◽  
Alkylating Agents ◽  
Complementation Group ◽  
Clear Understanding ◽  
Repair System ◽  
Cellular Dna ◽  
Methylating Agents

The cytotoxic effects of alkylating agents are strongly attenuated by cellular DNA repair processes, necessitating a clear understanding of the repair mechanisms. Simple methylating agents form adducts atN- andO-atoms.N-methylations are removed by base excision repair, AlkB homologues, or nucleotide excision repair (NER).O6-methylguanine (MeG), which can eventually become cytotoxic and mutagenic, is repaired byO6-methylguanine-DNA methyltransferase, andO6MeG:T mispairs are recognized by the mismatch repair system (MMR). MMR cannot repair theO6MeG/T mispairs, which eventually lead to double-strand breaks. Bifunctional alkylating agents form interstrand cross-links (ICLs) which are more complex and highly cytotoxic. ICLs are repaired by complex of NER factors (e.g., endnuclease xeroderma pigmentosum complementation group F-excision repair cross-complementing rodent repair deficiency complementation group 1), Fanconi anemia repair, and homologous recombination. A detailed understanding of how cells cope with DNA damage caused by alkylating agents is therefore potentially useful in clinical medicine.

scholarly journals Expression and amplification of engineered mouse dihydrofolate reductase minigenes.

1983 ◽  
Vol 3 (2) ◽  
pp. 257-266 ◽  
Author(s):  
G F Crouse ◽  
R N McEwan ◽  
M L Pearson
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Specific Inhibitor ◽  
Cdna Clones ◽  
Coding Region ◽  
Chromosomal Dna ◽  
Lambda Phage ◽  
Ovary Cells

We constructed mouse dihydrofolate reductase (DHFR) minigenes (dhfr) that had 1.5 kilobases of 5' flanking sequences and contained either none or only one of the intervening sequences that are normally present in the coding region. They were greater than or equal to 3.2 kilobase long, about one-tenth the size of the corresponding chromosomal gene. Both of these minigenes complemented the DHFR deficiency in Chinese hamster ovary dhfr-1-cells at a high frequency after DNA-mediated gene transfer. The level of DHFR enzyme in various transfected clones varied over a 10-fold range but never was as high as in wild-type Chinese hamster ovary cells. In addition, the level of DHFR in primary transfectants did not vary directly with the copy number of the minigene, which ranged from fewer than five to several hundred per genome. The minigenes could be amplified to a level of over 2,000 copies per genome upon selection in methotrexate, a specific inhibitor of DHFR. In one case, the amplified minigenes were present in a tandem array; in two other cases, a rearranged minigene plasmid and its flanking chromosomal DNA sequence were amplified. Thus, the mouse dhfr minigenes could be transcribed, expressed, and amplified in Chinese hamster ovary cells, although the efficiency of expression was generally low. The key step in the construction of these minigenes was the generation in vivo of lambda phage recombinants by overlapping regions of homology between genomic and cDNA clones. The techniques used here for dhfr should be generally applicable to any gene, however large, and could be used to generate novel genes from members of multigene families.

Expression and amplification of engineered mouse dihydrofolate reductase minigenes

1983 ◽  
Vol 3 (2) ◽  
pp. 257-266
Author(s):  
G F Crouse ◽  
R N McEwan ◽  
M L Pearson
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Specific Inhibitor ◽  
Cdna Clones ◽  
Coding Region ◽  
Chromosomal Dna ◽  
Lambda Phage ◽  
Ovary Cells

We constructed mouse dihydrofolate reductase (DHFR) minigenes (dhfr) that had 1.5 kilobases of 5' flanking sequences and contained either none or only one of the intervening sequences that are normally present in the coding region. They were greater than or equal to 3.2 kilobase long, about one-tenth the size of the corresponding chromosomal gene. Both of these minigenes complemented the DHFR deficiency in Chinese hamster ovary dhfr-1-cells at a high frequency after DNA-mediated gene transfer. The level of DHFR enzyme in various transfected clones varied over a 10-fold range but never was as high as in wild-type Chinese hamster ovary cells. In addition, the level of DHFR in primary transfectants did not vary directly with the copy number of the minigene, which ranged from fewer than five to several hundred per genome. The minigenes could be amplified to a level of over 2,000 copies per genome upon selection in methotrexate, a specific inhibitor of DHFR. In one case, the amplified minigenes were present in a tandem array; in two other cases, a rearranged minigene plasmid and its flanking chromosomal DNA sequence were amplified. Thus, the mouse dhfr minigenes could be transcribed, expressed, and amplified in Chinese hamster ovary cells, although the efficiency of expression was generally low. The key step in the construction of these minigenes was the generation in vivo of lambda phage recombinants by overlapping regions of homology between genomic and cDNA clones. The techniques used here for dhfr should be generally applicable to any gene, however large, and could be used to generate novel genes from members of multigene families.

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.

scholarly journals Molecular cloning of the human DNA excision repair gene ERCC-6.

1990 ◽  
Vol 10 (11) ◽  
pp. 5806-5813 ◽  
Author(s):  
C Troelstra ◽  
H Odijk ◽  
J de Wit ◽  
A Westerveld ◽  
L H Thompson ◽  
...  
Keyword(s):  
Excision Repair ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Cyclobutane Pyrimidine Dimers ◽  
Repair Gene ◽  
Human Dna ◽  
Pyrimidine Dimers ◽  
Low Efficiency ◽  
Mutant Lines

The UV-sensitive, nucleotide excision repair-deficient Chinese hamster mutant cell line UV61 was used to identify and clone a correcting human gene, ERCC-6. UV61, belonging to rodent complementation group 6, is only moderately UV sensitive in comparison with mutant lines in groups 1 to 5. It harbors a deficiency in the repair of UV-induced cyclobutane pyrimidine dimers but permits apparently normal repair of (6-4) photoproducts. Genomic (HeLa) DNA transfections of UV61 resulted, with a very low efficiency, in six primary and four secondary UV-resistant transformants having regained wild-type UV survival. Southern blot analysis revealed that five primary and only one secondary transformant retained human sequences. The latter line was used to clone the entire 115-kb human insert. Coinheritance analysis demonstrated that five of the other transformants harbored a 100-kb segment of the cloned human insert. Since it is extremely unlikely that six transformants all retain the same stretch of human DNA by coincidence, we conclude that the ERCC-6 gene resides within this region and probably covers most of it. The large size of the gene explains the extremely low transfection frequency and makes the gene one of the largest cloned by genomic DNA transfection. Four transformants did not retain the correcting ERCC-6 gene and presumably have reverted to the UV-resistant phenotype. One of these appeared to have amplified an endogenous, mutated CHO ERCC-6 allele, indicating that the UV61 mutation is leaky and can be overcome by gene amplification.

Amplification and expression of heterologous ornithine decarboxylase in Chinese hamster cells

1988 ◽  
Vol 8 (2) ◽  
pp. 764-769
Author(s):  
T R Chiang ◽  
L McConlogue
Keyword(s):  
Ornithine Decarboxylase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Wild Type ◽  
Dominant Marker ◽  
Ovary Cells ◽  
Mammalian Expression ◽  
Mammalian Expression Vector

We have developed an amplifiable mammalian expression vector based on the enzyme ornithine decarboxylase (ODC). We show greater than 700-fold amplification of this vector in ODC-deficient Chinese hamster ovary cells. A passive coamplified marker, dihydrofolate reductase (dhfr), was amplified and overexpressed 1,000-fold. This ODC vector was a dominant marker in a variety of cell types and displayed at least 300-fold amplification in wild-type Chinese hamster ovary cells.

scholarly journals Amplification and expression of heterologous ornithine decarboxylase in Chinese hamster cells.

1988 ◽  
Vol 8 (2) ◽  
pp. 764-769 ◽  
Author(s):  
T R Chiang ◽  
L McConlogue
Keyword(s):  
Ornithine Decarboxylase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Wild Type ◽  
Dominant Marker ◽  
Ovary Cells ◽  
Mammalian Expression ◽  
Mammalian Expression Vector

We have developed an amplifiable mammalian expression vector based on the enzyme ornithine decarboxylase (ODC). We show greater than 700-fold amplification of this vector in ODC-deficient Chinese hamster ovary cells. A passive coamplified marker, dihydrofolate reductase (dhfr), was amplified and overexpressed 1,000-fold. This ODC vector was a dominant marker in a variety of cell types and displayed at least 300-fold amplification in wild-type Chinese hamster ovary cells.

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