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This molecular epidemiology study examines the DNA-repair capacities (DRCs) of basal cell carcinoma (BCC) skin cancer patients (88) and their controls (135) by using a plasmid/host-cell reactivation assay. In this assay UV-damaged expression vector plasmid is transfected into peripheral blood T lymphocytes from the subjects. The host-cellular repair enzymes repair the photochemical damage in the plasmid, and 40 hr later the plasmid-encoded reporter chloramphenicol acetyltransferase is measured. An age-related decline in this DRC, amounting to approximately 0.61% per yr occurred in the controls from 20 to 60 yr of age. Reduced DRC was a particularly important risk factor for young individuals with BCC and for those individuals with a family history of skin cancer. Young individuals with BCC repaired DNA damage poorly when compared with controls. As the BCC patients aged, however, differences between cases and controls gradually disappeared. The normal decline in DNA repair with increased age may account for the increased risk of skin cancer that begins in middle age, suggesting that the occurrence of skin cancer in the young may represent precocious aging. Patients with reduced DRCs and overexposure to sunlight had an estimated risk of BCC > 5-fold greater than the control group. Such a risk was even greater (10-fold) in female subjects.

The helicase action of the Escherichia coli UvrAB complex on a covalently closed circular DNA template was monitored using bacterial DNA topoisomerase I, which specifically removes negative supercoils. In the presence of E. coli DNA topoisomerase I and ATP, the UvrAB complex gradually introduced positive supercoils into the input relaxed plasmid DNA template. Positive supercoils were not produced when E. coli DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I or when both E. coli and eukaryotic DNA topoisomerases I were added simultaneously. These results suggest that like other DNA helix-tracking processes, the ATP-dependent action of the UvrAB complex on duplex DNA simultaneously generates both positive and negative supercoils, which are not constrained by protein binding but are torsionally strained. The supercoiling activity of UvrAB on UV-damaged DNA was also studied using UV-damaged plasmid DNA and a mutant UvrA protein that lacks the 40 C-terminal amino acids and is defective in preferential binding to UV-damaged DNA. UvrAB was found to preferentially supercoil the UV-damaged DNA template, whereas the mutant protein supercoiled UV-damaged and undamaged DNA with equal efficiency. Our results therefore suggest that the DNA helix-tracking activity of UvrAB may be involved in searching and/or prepriming the damaged DNA for UvrC incision. A possible role of supercoiled domains in the incision process is discussed.

The cloned uvrA and uvrB genes of Escherichia coli K-12 were amplified by linkage to the PL promoter of plasmid pKC30. The uvrC gene was amplified in the high-copy-number plasmid pRLM 24. The three gene products (purified in each case to greater than 95% purity) and ATP are required to effectively incise UV-damaged DNAs. The uvrABC proteins bind tightly to damaged sites in DNA, requiring the initial attachment of the uvrA protein in the presence of ATP before productive binding of the uvrB and uvrC proteins. Using a cloned tandem double insert of the lac p-o region as a damaged DNA substrate for the uvrABC complex and analyzing the incision both 5' and 3' to each pyrimidine dimer, we found that one break occurs 7 nucleotides 5' to a pyrimidine dimer and a second break is made 3-4 nucleotides 3' from the same pair of pyrimidines in the dimer. No such breaks are found in the strand complementary to the dimer. The size of the incised fragment in the DNA suggests that incision may be coordinated with excision reactions in repair processes.

We have constructed a hybrid pKC30-uvrA plasmid (pGHY5003) in which transcription of the uvrA gene can be induced under pL control to amplify the uvrA gene product to 7% of cellular protein. To construct pGHY5003, we developed a genetic selection using the basal level of expression (30 degrees C) from pL in thermosensitive cI857 lysogens to isolate appropriately tailored repair genes inserted at the Hpa I site of pKC30 from recombinant DNA mixtures with a variety of products. In addition, a post-UV-irradiation radiolabeling method was adapted to screen inserts for temperature-inducible polypeptide synthesis directed by transcription under pL control rapidly. This should prove generally useful for isolating genes inserted at the Hpa I site of plasmid pKC30 with the following characteristics: (i) genetically functional hybrid plasmids selected from a large population of exonucleolytically tailored fragments ligated into Hpa I of pKC30 and (ii) production of high-level amplification for the gene product of interest by screening for post-UV-irradiation temperature inducibility of polypeptides synthesized from hybrid plasmids. The level of amplification obtained for the uvrA gene product from pGHY5003 is approximately 10,000-fold higher than estimates of the level of uvrA protein in logarithmic phase Escherichia coli.

The bimodal-incision nature of the reaction of UV-irradiated DNA catalyzed by the Escherichia coli uvrABC protein complex potentially leads to excision of a 12- to 13-nucleotide-long damaged fragment. However, the oligonucleotide fragment containing the UV-induced pyrimidine dimer is not released under nondenaturing in vitro reaction conditions. Also, the uvrABC proteins are stably bound to the incised DNA and do not turn over after the incision event. In this communication it is shown that release of the damaged fragment from the parental uvrABC-incised DNA is dependent upon either chelating conditions or the simultaneous addition of the uvrD gene product (helicase II) and the polA gene product (DNA polymerase I) when polymerization of deoxynucleoside triphosphate substrates is concomitantly catalyzed. The product of this multiprotein-catalyzed series of reactions serves as a substrate for polynucleotide ligase, resulting in the restoration of the integrity of the strands of DNA. The addition of the uvrD protein to the incised DNA-uvrABC complex also results in turnover of the uvrC protein. It is suggested that the repair processes of incision, excision, resynthesis, and ligation are coordinately catalyzed by a complex of proteins in a "repairosome" configuration.

The Escherichia coli UvrB protein possesses an amino acid sequence motif common to many ATPases. The role of this motif in UvrB has been investigated by site-directed mutagenesis. Three UvrB mutants, with amino acid replacements at lysine-45, failed to confer UV resistance when tested in the UV-sensitive strain N364 (delta uvrB), while five other mutants constructed near this region of UvrB confer wild-type levels of UV resistance. Because even the conservative substitution of arginine for lysine-45 in UvrB results in failure to confer UV resistance, we believe we have identified an amino acid side chain in UvrB essential to nucleotide excision repair in E. coli. The properties of two purified mutant UvrB proteins, lysine-45 to alanine (K45A) and asparagine-51 to alanine (N51A), were analyzed in vitro. While the K45A mutant is fully defective in incision of UV-irradiated DNA, K45A is capable of interaction with UvrA in forming an ATP-dependent nucleoprotein complex. The K45A mutant, however, fails to activate the characteristic increase in ATPase activity observed with the wild-type UvrB in the presence of UvrA and DNA. From these results we conclude that there is a second nucleotide-dependent step in incision following initial complex formation, which is defective in the K45A mutant. This experimental approach may prove of general applicability in the study of function and mechanism of other ATPase motif proteins.

Two nucleases that catalyze the excision of photoproducts from UV-irradiated DNA have been extensively purified from M. luteus ( M. lysodeikticus ). The first enzyme, an endonuclease, has been purified 5000-fold and is free of conflicting nuclease activities. It introduces single-strand breaks into irradiated DNA but does not act on native or single-stranded DNA. The purified enzyme is activated but not dependent on Mg ++ and has an approximate molecular weight of 15,000. Photoproduct excision is absolutely dependent on the second enzyme, a magnesium requiring exonuclease. This enzyme, which has been purified 1000-fold, is devoid of conflicting nucleases. It hydrolyzes irradiated and unirradiated denatured DNA at the same rate, but has no activity on RNA. It only acts on double-stranded DNA which has been both irradiated and pretreated with the endonuclease. The combined action of the endo- and exonuclease results in the quantitative removal of photoproduct regions from UV-irradiated DNA. Approximately five nucleotides are released for every single-strand incision.

The irradiation of DNA in solution with ultraviolet light with doses approximating those used for the inactivation of transforming activity causes the irreversible hydrogenbond breakage, most likely in internal regions of the DNA molecule and the formation of formamide- and heat-stable linkages between the two complementary DNA strands. Repair of the latter damage is catalyzed by the photoreactivating enzyme from bakers' yeast. The cross-links are in keeping with the observations of others on the irradiation of thymine in the frozen state and may be due to the formation of dimers of thymine. (auth)