Repair of abasic sites by mammalian cell extracts

Abstract

Hamster cell extracts that perform repair synthesis on covalently closed circular DNA containing pyrimidine dimers, were used to study the repair of apurinic/apyrimidinic (AP) sites and methoxyamine (MX)-modified AP sites. Plasmid molecules were heat-treated at pH 5 and incubated with MX when required. The amount of damage introduced ranged from 0.2 to 0.9 AP sites/kb. Extracts were prepared from the Chinese hamster ovary CHO-9 cell line and from its derivative, 43-3B clone which is mutated in the nucleotide excision repair (NER) ERCC1 gene. AP and MX-AP sites stimulated repair synthesis by CHO-9 cell extracts. The level of synthesis correlated with the number of lesions and was of similar magnitude to the repair stimulated by 4.3 u.v. photoproducts/kb. Repair of AP and MX-AP sites was faster than the repair of u.v. damage and was independent of ERCC1 gene product. The high level of repair replication was due to a very efficient and rapid incision of plasmids carrying AP or MX-AP sites, performed by abundant AP endonucleases present in the extract. The calculated average repair patch sizes were: 7 nucleotides per AP site; 10 nucleotides per MX-AP site; 28 nucleotides per (6-4) u.v. photoproduct or cyclobutane pyrimidine dimer. The data indicate that AP and MX-AP sites are very efficiently repaired by base-excision repair in mammalian cells and suggest that MX-AP sites may also be processed via alternative repair mechanisms.

XRCC1 Is Specifically Associated with Poly(ADP-Ribose) Polymerase and Negatively Regulates Its Activity following DNA Damage

“…For mammalian cells, two distinct branches of the BER pathway that differ in the type of DNA polymerase required have been reported (10,15,16). The short patch pathway involves DNA polymerase ␤, XRCC1, and DNA ligase III (23), while the long patch pathway requires DNA polymerase ␤ or ␦, PCNA, DNase IV (FEN1), and DNA ligase I (25).…”

“…The short patch pathway involves DNA polymerase ␤, XRCC1, and DNA ligase III (23), while the long patch pathway requires DNA polymerase ␤ or ␦, PCNA, DNase IV (FEN1), and DNA ligase I (25). Although the entire process for uracil residues has been reconstituted in vitro with cell extracts (15,16) or purified proteins (25), the recent disruption of genes involved in BER has contributed to establishing the importance of XRCC1 (56), which apparently is not absolutely required in in vitro BER assays but has turned out to be a factor as crucial (49) as DNA polymerase ␤ (48) or human AP endonuclease (HAP1) (57) for animal viability. Similarly, PARP, which was found to negatively regulate BER in cell extracts (41,42), recently appeared to be essential for the survival of mice under genotoxic stress (35).…”

Novel Role of Base Excision Repair in Mediating Cisplatin Cytotoxicity

“…Differential Induction of Abasic Sites-Abasic sites are intermediates of the BER pathway, which are formed either spontaneously by hydrolysis of the N-glycosylic bond or following chemical modification of the bases by DNA-damaging agents and subsequent base removal by DNA glycosylases (29). To assess the involvement of BER in cisplatin cytotoxicity, we tested the ability of cisplatin, oxaliplatin, mitomycin C, and transplatin to induce AP sites.…”

Hypersensitivity phenotypes associated with genetic and synthetic inhibitor-induced base excision repair deficiency

“…In addition, the MX-adducted AP site is relatively resistant to enzymatic removal by APE [3]. Thus, MX effectively blocks single-nucleotide BER, however, repair may occur through alternate long-patch pathways [88]. Exposure to MX sensitizes pol β wild-type, but not pol β null cells, to the cytotoxic effects of MMS and MNU [3] (Fig.…”