Involvement of XRCC1 and DNA Ligase III Gene Products in DNA Base Excision Repair

Abstract

DNA ligase III and the essential protein XRCC1 are present at greatly reduced levels in the xrcc1 mutant CHO cell line EM-C11. Cell-free extracts prepared from these cells were used to examine the role of the XRCC1 gene product in DNA base excision repair in vitro. EM-C11 cell extract was partially defective in ligation of base excision repair patches, in comparison to wild type CHO-9 extracts. Of the two branches of the base excision repair pathway, only the single nucleotide insertion pathway was affected; no ligation defect was observed in the proliferating cell nuclear antigen-dependent pathway. Full complementation of the ligation defect in EM-C11 extracts was achieved by addition to the repair reaction of recombinant human DNA ligase III but not by XRCC1. This is consistent with the notion that XRCC1 acts as an important stabilizing factor of DNA ligase III. These data demonstrate for the first time that xrcc1 mutant cells are partially defective in ligation of base excision repair patches and that the defect is specific to the polymerase ␤-dependent single nucleotide insertion pathway. DNA base excision repair (BER)1 counteracts the mutagenic and cytotoxic effects of various kinds of base alterations that do not significantly distort the secondary structure of the double helix. A common intermediate of this pathway is the abasic (AP) site, that arises as a consequence of removal of altered bases by DNA-N-glycosylases or as spontaneous detachment of normal bases from the deoxyribose-phosphate backbone. It has been calculated that 2000 -10000 AP sites arise each day in a mammalian cell under physiological conditions (1). Therefore, the task of BER is engaging and important, and data obtained in Escherichia coli and transgenic mice show that this process is essential for survival (2-4). We have recently shown that, in addition to the polymerase ␤-dependent single nucleotide insertion pathway previously investigated in mammalian cells (5), a distinct proliferating cell nuclear antigen (PCNA)-dependent pathway is also present that incorporates a repair patch size of 7-14 nucleotides extending 3Ј to the site of the lesion (6). Our knowledge of the enzymology of the two pathways has several gaps. In particular, the enzymology of the ligation step is poorly defined. A role for the XRCC1 protein has been suggested on the basis of the sensitivity of xrcc1 mutant cell lines (the CHO derivatives EM9 and EM-C11) to agents that introduce DNA base damage (7, 8) and because of their reduced rate of single-strand break rejoining following exposure to ionizing radiation or alkylating agents (9, 10). Consistent with a role for XRCC1 in DNA ligation and BER is its observed interaction with DNA ligase III and DNA polymerase ␤ (7, 11, 12). Here, we have examined directly the role of XRCC1 and DNA ligase III in mammalian BER using a cellfree system. We report for the first time that (i) xrcc1 mutant cells are partially defective in ligation of BER patches and (ii) the defect involves only the polymerase ␤-dependent single nucle...

ATP for the DNA Ligation Step in Base Excision Repair Is Generated from Poly(ADP-ribose)

“…Heterodimerization of XRCC1 and Lig III leads to enhanced ligation activity (20). On the other hand, interaction of XRCC1 with either PARP-1 or Pol ␤ results in a down-regulation of the respective catalytic activity (13,18).…”

“…In subsequent steps, the adenylyl group is transferred to the 5Ј-phosphoryl donor, and eventually, AMP is released when the 5Ј-phosphoryl and the 3Ј-OH ends are joined. Owing to the fact that both PARP-1 and Lig III are constituents of the BER complex (17)(18)(19)(20), it appeared to be a likely possibility that Lig III (molecular weight, 103,000) is specifically using ATP generated from PAR as described above. To test this possibility, nuclear extracts were incubated in the presence of nicked DNA, dNTPs, and ␣-32 Plabeled NAD for several time intervals, similarly to reactions shown in Fig.…”

“…This protein is apparently essential and required for the BER pathway (16). Recent investigations demonstrated specific interactions of XRCC1 with Pol ␤, DNA ligase III (Lig III), and PARP-1 (13,(17)(18)(19)(20). Therefore, the complex of these proteins is supposed to control BER (1,9,19).…”

Terminally differentiated muscle cells are defective in base excision DNA repair and hypersensitive to oxygen injury

“…Long-patch repair synthesis is PCNA-dependent and involves FEN-1. The replicative DNA polymerases (2) and DNA ligase I have been shown to be indispensable for the resealing step (15,16). Therefore, changes in the gene expression of these key players of long-patch BER are likely to account for the impairment of this pathway in myotubes.…”

Reduced ligation during DNA base excision repair supported by BRCA2 mutant cells

“…DNA ligase III and DNA ligase I have been proposed as the major ligase activities involved in BER (Kubota et al, 1996;Prasad et al, 1996;Cappelli et al, 1997) and DNA ligase IV appears to participate in non homologous end joining (Grawunder et al, 1997;Teo and Jackson, 2000). Levels of the three DNA ligases were similar or greater in Capan-1 mutant cell extracts (lane 4) than in MCF7 (lane 1), T24 (lane 2) and BxPC-3 (lane 3) control cell extracts.…”