Reactions of the UVRABC excision nuclease with DNA damaged by diamminedichloroplatinum(II)

Abstract

Mutants of Escherichia coli, which are blocked in excision repair (uvrA6, uvrB5, or uvrC34) are exceptionally sensitive to the antitumor drug cis-Pt(II)(NH3)2Cl2 (cis-DDP) but not the trans isomer. Plasmid DNA, damaged by either the cis or trans compound and treated with the UVRABC excision nuclease was cut as shown by conversion of supercoiled DNA to relaxed forms. All three protein products of the uvrA, uvrB, and uvrC genes were required for incision. End-labeled fragments damaged with cis-DDP and reacted with the UVRABC nuclease were cut at the 8th phosphodiester bond 5' and at the 4th phosphodiester bond 3' to adjacent GG's. DNA treated with trans-DDP was not cut appreciably at adjacent GG's by the repair enzyme as subsequent analysis of reaction products after enzyme digestion gave a pattern similar to those obtained with control untreated fragments. The results indicate that the UVRABC nuclease may promote cell survival by the removal of adjacent GG's which are crosslinked by cis-Pt(II)(NH3)2Cl2.

Dna Repair Enzymes

“…In the removal of (intrastrandl) diadducts there is some staggering on the 3' side: pyrimidine dimers as well as 6---4 photoproducts are removed by incising the 8th phosphodiester bond 5' and the 4th or 5th phosphodiester bond 3' to the photoprodUicts, producing fragments 12 or 13 nucleotides long (132,165). In contrast, Pt(NH3h[GG] diadduct is removed almost exclusively as a dodeca nucleotide by hydrolysis of the 8th (5 ') and the 4th (3 ') phosphodiester bonds (175). While the 5' incision for mono-and di-adduct is relatively fixed, sequence-dependent variations have been observed (34).…”

Repair synthesis by human cell extracts in DNA damaged bycis- andtrans-diamminedichloroplatinum(II)

“…The significance of repair of DNA lesions for cellular tolerance to cis-DDP is demonstrated by studies of repair deficient baceria (13,14,15,16), yeast cells (17), Chinese hamster cells (18) and human xeroderma pigInentosum cells (19,20,21). It has been shown in vitro that the E. coli UvrABC nuclease incises the 8th phosphodiester bond 5' and the 4th phosphodiester bond 3' to GG intrastrand cross-links, thus excising an oligomer containing the adduct (16).…”

“…It has been shown in vitro that the E. coli UvrABC nuclease incises the 8th phosphodiester bond 5' and the 4th phosphodiester bond 3' to GG intrastrand cross-links, thus excising an oligomer containing the adduct (16). Enhanced DNA repair may be a contributing factor to resistance of tumors to clinical therapy with cis-DDP.…”

The effect ofEscherichia coliUvr protein binding on the topology of supercoiled DNA

“…These findings suggest that the enzyme mechanisms may be similar and point to the significance of studying the E. coli uvr system as a model for a DNA repair deficiency amongst patients predisposed to skin cancer The incision components of the E. coli uvr repair system responsible for the initial recognition of damaged DNA consists of the UvrA protein which in the presence of ATP binds to the damaged DNA, as well as to undamaged DNA duplexes (2). The UvrA protein requires thesimultaneous presence of the UvrB and UvrC proteins to initiate a dual incision event in DNAs containing ultraviolet light induced pyrimidine dimers (3,4) and 6,4-pyrimidine-pyrimidone adducts (3), photoactivated psoralen mono-and diadducts (5), acetylaminofluorine-guanine adducts (5), cis-platinum-guanine adducts (6) and benzo [a]pyrene-guanine adducts (7). In almost all of those experiments carried out with DNA substrates of defined sequences, the dual incision events were shown to involve a 5'-incision event 7 nucleotides to a damaged site and 3-4 nucleotides 3'-to the same site.…”