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A typical view of how DNA repair functions in chromatin usually depicts a struggle in which the DNA repair machinery battles to overcome the inhibitory effect of chromatin on the repair process. It may be that in this current interpretation the repair mechanisms are 'tilting at windmills', fighting an imaginary foe. An emerging picture suggests that we should not consider chromatin as an inhibitory force to be overcome like some quixotic giant by the DNA repair processes. Instead we should now recognize that DNA repair and chromatin metabolism are inextricably and mechanistically linked. Here we discuss the latest findings which are beginning to reveal how changes in chromatin dynamics integrate with the DNA repair process in response to UV induced DNA damage, with an emphasis on events in the yeast Saccharomyces cerevisiae.

This review assesses recent data on mutational risk to the germline after radiation exposure obtained by molecular analysis of tandemly repeated DNA loci (TRDLs): minisatellites in humans and expanded simple tandem repeats in mice. Some studies, particularly those including exposure to internal emitters, indicate that TRDL mutation can be used as a marker of human radiation exposure; most human studies, however, are negative. Although mouse studies have suggested that TRDL mutation analysis may be more widely applicable in biomonitoring, there are important differences between the structure of mouse and human TRDLs. Mutational mechanisms probably differ between the two species, and so care should be taken in predicting effects in humans from mouse data. In mice and humans, TRDL mutations are largely untargeted with only limited evidence of dose dependence. Transgenerational mutation has been observed in mice but not in humans, but the mechanisms driving such mutation transmission are unknown. Some minisatellite variants are associated with human diseases and may affect gene transcription, but causal relationships have not yet been established. It is concluded that at present the TRDL mutation data do not warrant a dramatic revision of germline or cancer risk estimates for radiation.

Nucleotide excision repair (NER) in eukaryotes removes DNA base damage as an oligonucleotide in a complex series of reactions. The nature of the dual incision reactions on either side of the damaged base has been extensively investigated. However, the precise mechanism of cleavage of the phosphodiester backbone of the DNA by the NER endonucleases and how this relates to removal of the damage-containing oligonucleotide during the excision process has not been determined. We previously isolated a stable heterotrimeric complex of Rad7/Rad16/Abf1 from yeast which functions in the conserved global genome repair (GGR) pathway. GGR removes lesions from DNA that is not actively transcribing. We have shown previously that the Rad7/Rad16/Abf1 heterotrimer is required to observe DNA repair synthesis and oligonucleotide excision during in vitro NER, but not needed to detect NER-dependent incision in such reactions. Here we report that this protein complex generates superhelicity in DNA through the catalytic activity of the Rad16 component. The torsion generated in the DNA by this complex is necessary to remove the damage-containing oligonucleotide during NER--a process referred to as excision. We conclude that in yeast the molecular mechanism of NER includes the generation of superhelical torsion in DNA., (Copyright 2003 Elsevier B.V.)

To date only a few studies have been undertaken on DNA adducts formed by epoxybutene (EB) and diepoxybutane (DEB), the two active metabolites of 1,3-butadiene. Our interests have focused on further investigating DNA alkylation by the two epoxides, especially in relation to the development of a method for human biomonitoring. Here, following the reaction of deoxyadenosine monophosphate and poly(dA-dT)(dA-dT) with DEB and subsequent HPLC, we have identified an adenine adduct. MS analyses indicate the structure of an adenine adducted by DEB at the N6 position. A HPLC/32P-postlabelling method was developed for its measurement in DNA samples and the adduct was detected in calf thymus DNA and DNA from Chinese hamster ovary cells exposed to DEB. The 100% labelling efficiency during postlabelling, the amount of the adduct and its elution before the normal nucleotides during HPLC suggest it could be a suitable indicator of BUT exposure.

The mutants irs1, irs2 and irs3 were previously isolated from the Chinese hamster line V79-4 on the basis of their hypersensitivity (2-3-fold) to cell inactivation by X-rays. Here the cross-sensitivities of the irs mutants to an array of chemical mutagens and topoisomerase inhibitors was determined in a differential cytotoxicity assay. Irs2 showed moderate hypersensitivity (2-3-fold) to simple alkylating agents and oxidative mutagens but was most sensitive (8-fold) to the topisomerase I inhibitor camptothecin. In contrast irs2 showed little or no increased sensitivity to four topoisomerase II inhibitors. Irs3 proved to be particularly hypersensitive to DNA crosslinking agents (5-15-fold) such as 1,3-butadiene diepoxide and mitomycin C. Irs1 was hypersensitive (3-fold or greater) to simple alkylating agents, oxidative mutagens and topoisomerase I and II inhibitors and exhibited extreme sensitivity (20-100-fold) to DNA crosslinking agents. The cellular hypersensitivities of irs2 and irs3 were reflected at the level of the chromosome. Camptothecin induced chromosomal aberrations in irs2 consisted almost exclusively of chromatid deletions and exchanges, whilst in irs3 1,3 butadiene diepoxide induced a 50-fold increase in chromatid exchanges compared with V79-4. The nature of irs2's camptothecin hypersensitivity was investigated. Analysis of the protein associated DNA single strand breaks produced by camptothecin indicated that there was no difference between V79-4 and irs2 in either the number of breaks induced or in the rate of their reversal following drug removal. In addition, levels of topoisomerase I activity in V79-4 and irs2 were indistinguishable. The data presented suggest that irs3 is likely to be defective in some aspect of DNA cross-link removal and irs2, whilst showing no gross defect in DNA strand break repair may fail to correctly respond to or repair certain types of strand breaks, possibly those associated with replicating DNA. The phenotypes of irs2 and irs3 respectively show similarities to those of cultured cells from the syndromes ataxia telangiectasia and Fanconi's anaemia.

Samples of clinically normal oral tissue were obtained from patients undergoing surgery for intraoral squamous cell carcinoma. DNA was extracted from samples obtained from 20 tobacco smokers, four exsmokers, and nine nonsmokers and analyzed for the presence of aromatic DNA adducts using two distinct modifications of the 32P postlabeling assay. 32P postlabeling following butanol extraction enhancement revealed a much wider range and substantially higher levels of DNA adducts than obtained following nuclease P1 enrichment. Adduct levels in smokers, exsmokers, and nonsmokers were 1133 +/- 354, 785 +/- 251, and 660 +/- 317 amol/microgram of DNA (+/- SD), respectively. The elevation of adduct levels in smokers compared with either nonsmokers or non- and exsmokers combined is statistically significant (P < 0.005). These observations are consistent with epidemiological evidence linking tobacco smoking with oral cancer. The differential enhancement of DNA adducts with the two 32P postlabeling protocols indicate that aromatic amines and nitroaromatics may be important sources of the DNA adducts detected in human oral tissue.

We have developed an HPLC-32P-postlabelling procedure to detect DNA adducts formed by epoxybutene and diepoxybutane. The method exploits the interaction of the two epoxides with deoxynucleotides and polydeoxynucleotides to optimize the HPLC enrichment of adducted nucleotides before 32P-postlabelling. Using this approach, a number of guanine adducts were identified after the exposure of dGMP, poly(dG-dC) or calf thymus DNA to epoxybutene and diepoxybutane, and a major adenine adduct was identified in poly(dA-dT) and calf thymus DNA exposed to diepoxybutane.

Samples of clinically normal oral tissue were obtained from 17 tobacco smokers and 7 non- or ex-smokers undergoing surgery for intra-oral squamous cell carcinoma. Isolated DNA was analysed for the presence of aromatic DNA adducts using the 32P-postlabelling technique with adduct enhancement by either butanol extraction or nuclease P1 enrichment. DNA adduction detected following butanol extraction was more diverse and at a higher level than obtained with the P1 method. Adduct levels in the smokers and non- or ex-smokers were 1163 +/- 375 and 774 +/- 318 amol/micrograms, respectively. This difference is statistically significant (p < 0.05). The differential enhancement of adducts with the two protocols suggested that arylamines may be the source of at least a proportion of the DNA adduction detected. These data indicate that oral tissues are likely to be a suitable source of material for human biomonitoring and furthermore they highlight the importance of utilizing more than one enhancement procedure when examining DNA adduction induced by complex mixtures such as tobacco smoke or those encountered at industrial plants.

UV sensitive Chinese hamster mutants belonging to ERCC groups 1, 2 and 6 together with one cross-link- and one X-ray-sensitive mutant have been examined for sensitivity to 4-nitroquinoline-1-oxide (4NQO) and the ability to repair 4NQO adducts at the N2 and C8 of guanosine. Despite the fact that all of the mutants examined were hyper-sensitive to 4NQO there was little difference between the mutants V-H1, V-H4, V-C4 and UV61 and the parental cell lines as regards the ability to remove these lesions from bulk DNA. The UV5 and UV20 mutants were both defective in the ability to remove N2 guanosine adducts yet repaired the C8 guanine adduct as normal. The fact that the mutants V-H1, V-H4, V-C4 and UV61 are 4NQO sensitive but repair the above adducts suggests that either some other lesion(s) is responsible for increased toxicity in these mutants, or that some regions of the genome may not be repaired as effectively as bulk DNA in these mutants, or that the quality of the repair is less than in the parental cells. Clearly the inability to remove UV induced pyrimidine dimers and the (6-4) photoproduct associated with the UV5 and UV20 mutants correlates with the inability to repair 4NQO-N2 guanosine adducts. However, mutants capable of (6-4) photoproduct repair but not dimer repair (VH-1 and UV61) can repair this lesion. Hence it is possible that the same domains in these repair proteins are required for the recognition of (6-4) photoproduct repair and 4NQO-N2 guanosine adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

The genotoxicity of tobacco particulate matter (TPM) derived from a low-tar, low-nicotine cigarette has been examined by measuring micronucleus induction in a primary pulmonary cell line, both in the absence and presence of an exogenous source of metabolic activation. In an attempt to correlate the cytogenetic damage observed with DNA adduct formation, DNA extracted from TPM-treated cells has been analysed with two different modifications of the 32P-postlabelling assay. The results from the 32P-postlabelling analysis taken together with the pattern of micronucleus induction provide strong evidence that bioreactivated aromatic carcinogens, such as benzo[a]pyrene, are unlikely to be responsible for the TPM-induced cytogenetic damage observed in cultured mammalian cells.

The yield of ultraviolet-induced dimers is similar for a fixed dose in both haploid and diploid Saccharomyces cerevisiae. The excision of these photo-products from the nuclear deoxyribonucleic acids of cells of both ploidies after ultraviolet incident doses of 2 times 10-3 to 4 times 10-3 ergs/mm2 decreased with the corresponding increasing dose. Postirradiation incubation in saline followed by a further incubation in nutrient medium increases the excision as compared to that seen in either nutrient medium or saline alone. Previous data regarding both pyrimidine dimer removal and the survival of haploid and diploid cells after ultraviolet irradiation and either immediate or delayed plating are discussed.

DNA replication and repair has been studied in human cells irradiated with single or split doses of UV at 254nm. A dose of 2 J.m(-2) followed by incubation in medium for periods of 12 to 48 h before a second irradiation with 5 or 10 J.m(-2) actually reduces the amount of DNA replication seen after the latter dose alone. Such a preirradiation did not perceptibly alter the rate of excision repair seen after the latter dose when measured by the incidence of UV endonuclease sensitive sites, 3H-bromodeoxyuridine incorporation, or the incidence of cytosine arabinoside induced breaks. Furthermore, the first two parameters were measured in molecules replicating or remaining unreplicated after the latter irradiation. No difference in repair rates was seen between these two classes of molecules.

We have compared the initial yield and subsequent enzymatic repair of DNA strand breaks and 1-beta-D-arabinofuranosylcytosine (araC)-detectable sites in cultured human cell lines after irradiation with soft (40-75 kVp) X rays and hard (60Co) gamma rays, two forms of low-LET radiation whose average ionization densities differ by 15-fold (i.e., approximately 4.5 keV/mu and approximately 0.3 keV/mu, respectively). Incubation of X- or gamma-irradiated cell cultures in the presence of araC resulted in the accumulation of DNA single-strand interruptions, as measured by conventional velocity sedimentation analysis in alkaline sucrose gradients. Since the drug is a selective inhibitor of DNA polymerase alpha, this observation implicates polymerase alpha in the repair of radiogenic damage. Specifically, DNA repair analysis in X-ray-treated cells suggested that polymerase alpha is a key participant in the repair of a distinct, albeit structurally undefined, class of radioproducts [presumably a combination of double-strand breaks and alkali-stable lesions (e.g., modified base/sugar residues and DNA crosslinks)], but takes little, if any, part in the restitution of single-strand breaks (whether frank scissions or alkali-labile bonds) induced by ionizing radiation. Accordingly, the drug did not significantly inhibit the repair of DNA damage in cultures exposed to less than or equal to 100 Gy of gamma radiation. A remarkable difference was evident with respect to the absolute yields of these two major classes of DNA injury--that is, araC-detectable sites and single-strand breaks--depending on the type of radiation administered. For the same dose, the level of araC-detectable sites found in cells treated with gamma rays (less than or equal to 150 Gy) was as much as 3.4 times lower than that observed in X-ray-exposed cells (i.e., one site per 1,155 and 335 eV, respectively). In contrast, the number of strand breaks induced by gamma rays was approximately 2.3-fold higher than that produced by X rays (one break per 27 and 61 eV, respectively). Assuming that, among other conditions, the relative biological effectiveness of the comparatively densely ionizing soft X rays is roughly twice that of the sparsely ionizing hard gamma rays, our data strongly imply that those radioproducts which are manifested as araC-detectable sites may be approximately 45 times more deleterious, on a per lesion basis, than those observed as single-strand breaks.

4-Nitroquinoline-1-oxide (4NQO) reacts with DNA primarily at the N2 and C8 of guanosine, with a small percent of reaction at the N6 of adenosine. In human cells it has been unclear whether or not all 4NQO-induced adducts are removed by a nucleotide excision repair mechanism. In this paper we demonstrate that the inhibitor of DNA polymerases alpha and delta, aphidicolin, blocks the repair of all 4NQO adducts. Hence excision repair must operate on all of these lesions. After 4NQO the residual excision repair seen in a xeroderma pigmentosum group A cell line virtually totally defective in UV repair was 40-60% of that in normal cells. Therefore there must be some differences between the excision repair operating on UV as opposed to 4NQO-induced DNA damage.

The photoreactivability of UV-induced pyrimidine dimers in the nuclear and mitochondrial DNAs of Saccharomyces cerevisiae has been investigated in conjunction with the fate of these photoproducts following postirradiation dark incubation in saline and nutrient media. In all instances, survival and "petite" induction were measured. An attempt has been made to relate these results to present ideas on the repair of UV damages in DNA.

One-third of all protein-coding genes from bacterial genomes cannot be annotated with a function. Here, to investigate the functions of these genes, we present genome-wide mutant fitness data from 32 diverse bacteria across dozens of growth conditions. We identified mutant phenotypes for 11,779 protein-coding genes that had not been annotated with a specific function. Many genes could be associated with a specific condition because the gene affected fitness only in that condition, or with another gene in the same bacterium because they had similar mutant phenotypes. Of the poorly annotated genes, 2,316 had associations that have high confidence because they are conserved in other bacteria. By combining these conserved associations with comparative genomics, we identified putative DNA repair proteins; in addition, we propose specific functions for poorly annotated enzymes and transporters and for uncharacterized protein families. Our study demonstrates the scalability of microbial genetics and its utility for improving gene annotations.A large-scale mutagenesis screen identifies mutant phenotypes for over 11,000 protein-coding genes in bacteria that had previously not been assigned a specific function., Author(s): Morgan N. Price [sup.1] , Kelly M. Wetmore [sup.1] , R. Jordan Waters [sup.2] , Mark Callaghan [sup.1] , Jayashree Ray [sup.1] , Hualan Liu [sup.1] , Jennifer V. [...]