Incision of DNA-protein crosslinks by UvrABC nuclease suggests a potential repair pathway involving nucleotide excision repair

Abstract

DNA-protein crosslinks (DPCs) arise in biological systems as a result of exposure to a variety of chemical and physical agents, many of which are known or suspected carcinogens. The biochemical pathways for the recognition and repair of these lesions are not well understood in part because of methodological difficulties in creating site-specific DPCs. Here, a strategy for obtaining site-specific DPCs is presented, and in vitro interactions of the Escherichia coli nucleotide excision repair (NER) UvrABC nuclease at sites of DPCs are investigated. To create site-specific DPCs, the catalytic chemistry of the T4 pyrimidine dimer glycosylase/apurinic/apyrimidinic site lyase (T4-pdg) has been exploited, namely, its ability to be covalently trapped to apurinic/apyrimidinic sites within duplex DNA under reducing conditions. Incubation of the DPCs with UvrABC proteins resulted in DNA incision at the 8th phosphate 5' and the 5th and 6th phosphates 3' to the protein-adducted site, generating as a major product of the reaction a 12-mer DNA fragment crosslinked with the protein. The incision occurred only in the presence of all three protein subunits, and no incisions were observed in the nondamaged complementary strand. The UvrABC nuclease incises DPCs with a moderate efficiency. The proper assembly and catalytic function of the NER complex on DNA containing a covalently attached 16-kDa protein suggest that the NER pathway may be involved in DPC repair and that at least some subset of DPCs can be removed by this mechanism without prior proteolytic degradation.

Figure 1

Preparation of site-specific DNA–protein crosslinks. (A) Sequence of the uracil-containing 60-mer oligodeoxynucleotide. (B) Urea-PAGE showing DNA substrate preparation. Lane 1, uracil-containing 60-mer; lane 2, uracil-containing 60-mer, digested with uracil DNA glycosylase and tested with T4-pdg (control of AP-site formation); reduced AP site-containing DNA before (lane 3) and after (lanes 4–6) purification; DPC-containing DNA before (lane 7) and after (lanes 8–10) purification. After purification, DNAs were subjected to the restriction endonuclease digestion with SnaBI (5, 9) or HaeIII (6, 10). (C) SDS/PAGE showing DPC-containing DNA substrates before (lane 1) and after (lane 2) HaeIII digestion.

Figure 2

Incision of DNA–protein crosslinks by UvrABC nuclease. (A) Urea-PAGE of 5′-terminally labeled DNAs incubated without (−) or with (+) UvrABC proteins. DNAs were AAF-guanine-containing 50-mer (AAF), uracil-containing 60-mer (U), reduced AP site-containing 60-mer (rAP), and DPC-containing 60-mer (DPC). (B) Urea-PAGE of 5′-terminally labeled DPC-containing 60-mer incubated without (−) or with (+) UvrA, UvrB, and UvrC proteins as indicated. (C) Urea-PAGE of 3′-terminally labeled DNAs incubated without (−) or with (+) UvrABC proteins. DNAs designated as in A. (D) SDS/PAGE of the internally labeled (at 5th position from 5′ side relative to the adducted site) DPC-containing 60-mer incubated without (−) and with (+) UvrABC nuclease. Left lane (U) represents uracil-containing 60-mer. Right lane (M, marker) contains 12-mer DNA with centrally positioned T4-pdg-DNA crosslink. (E) Positions of the 5′-side and 3′-side incisions by UvrABC nuclease.

Figure 3

Kinetics of DNA incision by UvrABC nuclease on the 5′-terminally labeled DNA substrates containing (+)-trans-benzo[a]pyrene diolepoxide or DPC. Data are the means ± SD of three independent experiments. The incision rate was calculated as the slope of the line obtained from unweighted linear regression.