Action mechanism of ABC excision nuclease on a DNA substrate containing a psoralen crosslink at a defined position.

Abstract

Many carcinogenic as well as chemotherapeutic agents cause covalent linkages between complementary strands of DNA. If unrepaired, DNA crosslinks are blocks to DNA replication and transcription and therefore represent potentially lethal lesions to the cell. Genetic studies of Escherichia coli have demonstrated that the repair enzyme ABC excision nuclease, coded for by the three unlinked genes, uvrA, uvrB, and uvrC, plays a crucial role in DNA crosslink repair. To study the molecular events of ABC excision nudeasemediated crosslink repair, we have engineered a DNA fragment with a psoralen-DNA interstrand crosslink at a defined position, digested this substrate with pure enzyme, and analyzed the reaction products on DNA sequencing gels. We find that the excision nuclease (i) cuts only one of the two strands involved in the crosslink, (ii) incises the crosslink by hydrolyzing the ninth phosphodiester bond 5' and the third phosphodiester bond 3' to the furan-side thymine of the crosslink, and (iii) does not produce double-strand breaks at any significant level. Based on these data, we present a model by which ABC excision nuclease initiates crosslink repair in vivo.Several agents have been shown to produce DNA interstrand crosslinks (1) including mitomycin C (2), nitrous acid (3), nitrogen and sulfur mustards (4), formaldehyde (5), cisplatin (6), and psoralen plus light (7,8). Because oftheir predictable and highly characterized reactivity with DNA (9, 10), psoralens have been used most extensively to study the repair of DNA crosslinks in several organisms (11-17). These three-ring heterocyclic aromatic compounds (furocoumarins) contain two reactive double bonds that, upon absorption of near UV light (320-360 nm), photoreact with the 5,6 double bond in pyrimidines to form both monoadducts and interstrand crosslinked diadducts, primarily at 5' TpA 3' and to a lesser extent at 5' ApT 3' sequences (18).Genetic and biochemical studies of Escherichia coli have identified several proteins necessary for the removal of psoralen crosslinks, including products of the recA, uvrA, uvrB, uvrC, uvrD, and polA genes (12,(19)(20)(21)(22)(23)(24)(25)(26). Furthermore, it has been estimated that in the uvrA recA double mutant, one DNA crosslink per genome is lethal (24,25). Based on these data, several models of psoralen crosslink repair have been proposed that incorporate components of both the RecA-dependent recombination and the nucleotide excision repair pathways (12, 23).In E. coli, the incision and excision steps of nucleotide excision repair are mediated by a single enzyme, the ABC excision nuclease (see ref. 27). This enzyme is composed of three proteins UvrA (Mr, 103,874), UvrB (Mr, 76,118), and UvrC (Mr, 66,038), which act in concert to cleave both the eighth phosphodiester bond 5' and the fourth or fifth phosphodiester bond 3' to UV-induced cyclobutane pyrimidine dimers and 6-4 pyrimidine-pyrimidone intrastrand diadducts (28). The goal of this study was to characterize the molecular events of ABC excision nuclease-m...

Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA

“…The protein binds preferentially to damaged DNA, with a discrimination factor for UV lesions of -103. Interestingly, this is the same order of magnitude of damaged site discrimination as found for the E.coli UvrA protein (Seeberg and Steinum, 1982;Van Houten et al, 1987;Mazur and Grossman, 1991). Neither XP-A protein nor UvrA can introduce nicks into DNA on its own.…”

DNA Repair Defects Channel Interstrand DNA Cross-links into Alternate Recombinational and Error-prone Repair Pathways

“…Measurements of psoralen-induced DSBs by pulsed field gel electrophoresis showed a larger reduction in a triple rad2 rad6 rad52 mutant strain than in the single rad2 mutant, indicating a role for other repair epistasis groups in DSB formation (19). Alternatively, DSBs may not be the only recombinogenic intermediates; for instance, in bacteria recombination is initiated by single strand gaps opposite psoralen adducts (3,4).…”

Role of the human ERCC-1 gene in gene-specific repair of cisplatin-induced DNA damage

“…There are at least two possible processes by which a crosslink can be removed from the DNA. In one model, the crosslink is completely repaired by 1) incising one strand, 2) filing in the gap, and then 3) removing the resultant crosslinked oligomer from the other strand by homologous strand exchange (27) (25). Furthermore, it was shown that the most frequent DNA adduct induced by cisplatin, the intrastrand d(GpG) platinum crosslink is poorly repaired in human cell extracts (30,31).…”