Large-scale isolation of UV-sensitive clones of CHO cells

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187

The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined. Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro. The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide. Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography. Cell extracts of E. coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide. No significant repair replication on the 5' side of the lesion was observed. The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease. Gap-filling and ligation complete the repair reaction. Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase 13.DNA bases altered by hydrolytic deamination, oxygen radical-induced damage, and alkylation are often excised by DNA glycosylases (44,54). Certain mismatched residues, such as adenine or thymine opposite guanine, may also be removed by specific DNA glycosylases (1, 56, 57). Thus, a common intermediate in the repair of several different types of DNA damage is an apurinic-apyrimidinic (AP) site. Such sites are also generated by nonenzymatic hydrolysis of base-sugar bonds, in particular by depurination (26), and as a consequence of exposure to ionizing radiation (12,27,34). AP endonucleases, for example, Escherichia coli exonuclease III and endonuclease IV, rapidly incise DNA at the 5' side of an AP site (9). Subsequently, an excision step occurs to generate a small gap, which is filled in by repair replication. A DNA ligase completes the repair process. These consecutive events take place rapidly and efficiently in vivo. It has been shown that AP sites occur transiently in DNA after X-irradiation of mammalian cells but disappear in 2 to 4 min without any detectable accumulation of DNA containing single-strand interruptions (34).The excision of a depurinated-depyrimidinated site has remained the least understood of these events in the base excision repair process. Early work by Painter and Young (37) and Regan and Setlow (40) showed that repair replication of X-irradiated and alkylated DNA in vivo involved the filling-in of much smaller gaps than those observed after UV light-induced DNA damage. Subsequent studies have also indicated that patches only 1 to 4 nucleotides long may be generated during repair of AP sites (8,32). The excision of a 5'-terminal base-free sugar-phosphate residue, possibly accompanied by the removal of one or a few adjacent residues, has been ascribed to exonuclease acti...

Section: Materuils and Methodsmentioning

confidence: 99%

Generation of single-nucleotide repair patches following excision of uracil residues from DNA.

Dianov¹,

Price²,

Lindahl³

1992

274

187

“…Our results showed that AA8-4 DNA-treated cells gave 4 positives out of 14 total samples (in 11 experiments) versus 0 positives out of 12 samples (in 8 experiments) with UV-135 DNA-treated cells ( Table 2). The differences between the two DNA treatments in number and proportion of positive samples were very significant, either by the chi-square test (P = 0.044) or by the more appropriate binomial test for proportion of positives (P = 0.018).…”

mentioning

confidence: 65%

“…The molecular identification of a possible human ER gene complementing the XP-A genotype has been reported previously (23). However, several technical difficulties with the use of XP cell lines for this type of experiment have made another mammalian cell system attractive for repair gene transfer.The availability of ER-proficient CHO cells and the technical advantages of genetic manipulation in this system led to the isolation of a large series of recessive, UV-sensitive (UV') mutants which fall into five complementation groups (4,(25)(26)(27). These mutants were derived from CHO clone AA8-4, which is ER proficient by criteria of UV repair incision (4.…”

mentioning

confidence: 99%

See 1 more Smart Citation

DNA-mediated cotransfer of excision repair capacity and drug resistance into chinese hamster ovary mutant cell line UV-135.

MacInnes¹,

Bingham²,

Thompson³

et al. 1984

We have investigated DNA-mediated transfer of aminopterin resistance conferred by plasmid and UV resistance conferred by genomic DNA to the Chinese hamster ovary (CHO) cell line UV-135, a UVsensitive mutant defective in nucleotide excision repair. Plasmid pSV2gpt-CaPO4 coprecipitates induced aminopterin resistance with equal efficiency in the 6-thioguanine-resistant, aminopterin-sensitive, repairproficient parental line AA8-4(tg-1) and in . Genetic and molecular evidence for genomic DNAmediated transformation of UV-135(tg-2) cells with a putative excision repair gene were obtained by demonstrating that: (i) UV resistance transformation is dependent upon and specific for genomic DNA from excision repair-competent CHO cells: (ii) UV and drug coresistant colonies are bona fide transferants as verified by hybridization and Southern blotting analysis of pSV2gpt sequences in their genomic DNAs; (iii) confirmed transferants exhibit partial to near normal UV resistances for colony formation, and (iv) UVr transferants have near normal levels of excision repair capacity. The overall frequency of drug and UV resistance cotransformation was 8 x 10-8 per cell plated. This frequency was ca. 200-to 500-fold greater than that expected from coincident but independent UV' reversion and plasmid gene transfer events. DNA transfer techniques with this CHO system will be useful for further analysis of the essential structural DNA sequences, gene cloning, and expression of functional excision repair genes.Nucleotide excision repair (ER) is the major DNA metabolic pathway by which a variety of alkylation adducts, cross-links, and UV-induced photoproducts are eliminated from DNA (reviewed in reference 7). Genetic deficiency for repair of these damages is in part responsible for the propensity of most human xeroderma pigmentosum (XP) patients to develop skin cancers (21) and possibly for XPassociated degenerative changes in nonirradiated organ sites (10). Hence, XP and certain other genetic syndromes (17) correlate with altered ER capacity, alterations which can apparently increase cell susceptibility for certain initiation events in neoplasia. In none of these human syndromes has the normal products of these defective genes yet been identified.Molecular approaches are now available to identify. isolate, and manipulate expression of ER genes and polypeptides. In this work, we wanted to determine whether DNA transformation of repair-deficient cells would be useful for these purposes. The molecular identification of a possible human ER gene complementing the XP-A genotype has been reported previously (23). However, several technical difficulties with the use of XP cell lines for this type of experiment have made another mammalian cell system attractive for repair gene transfer.The availability of ER-proficient CHO cells and the technical advantages of genetic manipulation in this system led to the isolation of a large series of recessive, UV-sensitive (UV') mutants which fall into five complementation groups (4,(25)(26)(27). These mutan...

“…A number of different UV-sensitive mutants have been generated from established cell lines (5,15,23,25,27,30,34). One of these mutant lines, UV-20, isolated from a parental Chinese hamster ovary (CHO) cell line, is sensitive to a number of other DNA-damaging agents, including the DNA cross-linking agent mitomycin C (MM-C) (29).…”

mentioning

confidence: 99%

Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene.

Rubin¹,

Prideaux²,

Willard³

et al. 1985

The genes and gene products involved in the mammalian DNA repair processes have yet to be identified. Toward this end we made use of a number of DNA repair-proficient transformants that were generated after transfection of DNA from repair-proficient human cells into a mutant hamster line that is defective in the initial incision step of the excision repair process. In this report, biochemical evidence is presented that demonstrates that these transformants are repair proficient. In addition, we describe the molecular identification and cloning of unique DNA sequences closely associated with the transfected human DNA repair gene and demonstrate the presence of homologous DNA sequences in human cells and in the repair-proficient DNA transformants. The chromosomal location of these sequences was determined by using a panel of rodent-human somatic cell hybrids. Both unique DNA sequences were found to be on human chromosome 19.