This Workshop focused on environmental and endogenous DNA damage and mutagenesis; specific genes, proteins and pathways involved in the recognition and repair of DNA lesions as a function of nucleotide sequence, chromatin structure and the transcriptional activity of target genes; the subversion of genomic stability, signal transduction pathways, cell-cycle control and apoptosis in oncogenesis; and genetic events in the process of carcinogenesis and tumor progression, with special emphasis on human colorectal and skin cancer and on radiation-induced thyroid carcinomas in children from Belarus after the Chernobyl reactor accident.
Aside from the germline mutations associated with rare hereditary cancer syndromes, most of the mutations in human cancer appear to result from unrepaired endogenous and exogenous DNA damage in the somatic cells of susceptible individuals. Dr. G.P. Pfeifer (City of Hope, Duarte, CA, USA) has compared DNA lesion spectra, repair rates and tumor-associated mutational spectra along sequences of the human TP53 gene. In non-melanoma skin tumors, the distribution of C = T transitions in TP53 is mirrored by UV-wavelength-dependent induction and lack of repair of cyclobutane-pyrimidine dimers (CPDs) at the same sequence positions. 5-methylcytosine becomes the preferred target for CPD formation when natural sunlight is used for irradiation. In lung cancer, the mutational spectrum of TP53 is characterized by a predominance of G = T transversions. Among the many components of cigarette smoke, polycyclic aromatic hydrocarbons are strongly implicated in carcinogenesis. The distribution of benzo[a]pyrenediol-epoxide(BPDE) DNA adducts along TP53 was mapped in bronchial epithelia. Strong and selective formation of these adducts occurred at the major mutational hotspots of TP53 in human lung cancers, i.e., at guanines in codons 157, 248 and 273. A majority of mutational hotspots in TP53 (and other genes) are located at CpG dinucleotides. Interestingly, there is a strong influence of 5-methylcytosine on the reactivity of BPDE with guanines at CpG sites, and a remarkably high number of somatic mutations of TP53 are found at methylated CpGs.
Packaging into nucleosomes and higher order chromatin structures restricts the accessibility and flexibility of DNA, and may thus affect gene regulation and transcription, the formation of DNA lesions, and DNA repair. The studies of Dr. F. Thoma (Zurich, Switzerland) have focused on CPDs induced by UV in yeast genes and minichromosomes with well-characterized chromatin structures (positioned nucleosomes), and on the processing of these lesions by photolyase in the presence of light (photoreactivation) and via the multi-enzyme-multi-step nucleotide excision repair (NER) pathway. Photoreactivation is tightly regulated by chromatin structure. Damaged open promoter regions of active genes are repaired within 15 minutes, nucleosomes are repaired in 2 hours. In contrast, repair of open promoters by NER is slow. Photolyase serves as a molecular tool to investigate chromatin structure and dynamics in vivo. Slow repair in nucleosomes is consistent with dynamic properties of nucleosomes that modulate the accessibility of CPDs. In active genes of NER-deficient strains (rad1β¬), photoreactivation was slow in the transcribed strand, but fast in the non-transcribed strand. In inactive genes, both strands were repaired at similar rates. These results suggest that RNA-polymerase II stalled at CPDs inhibits their accessibility to photolyase. In NER-proficient strains (RAD1), fast photoreactivation in the non-transcribed strand complements fast repair by NER in the transcribed strand (transcription-coupled repair). The combination of both repair pathways thus allows efficient restoration of damaged genes after UV irradiation. Analyses at nucleotide resolution showed that nucleosome structure and positioning modulate NER in the non-transcribed strand of an active gene.
One of the multiple modifications produced in DNA by endogenous reactive oxygen species, 8-oxo-7,8-dihydroguanine (8-OH-G), is supposed to play a critical role in mutagenesis, oncogenesis and aging. In E. coli, repair systems for 8-OH-G are known to involve the genes mutM, mutY and mutT. Reflecting the competition in the field of DNA repair, 5 groups have independently reported the cloning of the mammalian mutM homologue (MMH), a (8-OH-G)-specific DNA glycosylase/apurinic, apyrimidinic lyase (AP-lyase). Dr. S. Nishimura (Tsukuba, Japan) described the identification of the human gene (hMMH) by similarity search using a human expressed sequence tag (EST) database and comparison with the sequence of the yeast mutM homologue ogg1. hMMH is composed of 7 exons, and an 8th exon producing 4 alternative splicing isoforms. The hMMH protein (34% identical to yeast OGG1) was expressed in E. coli and purified to homogeneity. hMMH exhibits both glycosylase and AP-lyase activity on duplex DNA containing (8-OH-G)/C, but not on an oligonucleotide of intact G/C pairs. hMMH cleaves (8-OH-G)-containing DNA at 8-OH-G<-p-dN via β€-elimination. Duplex DNA containing (8-OH-G)/A is not cleaved, whereas (8-OH-G)/T and (8-OH-G)/G oligonucleotides are slightly cleaved. The enzymatic properties of hMMH are similar to those of yeast OGG1. hMMH was also able to rescue a spontaneous mutator strain of E. coli lacking mutM and mutY. In E. coli the mutT protein hydrolyzes 8-OH-dGTP to 8-OH-dGMP, thereby preventing mutagenesis by incorporation of 8-OH-dGTP opposite adenine or cytosine in DNA. As reported by Dr. H. Hayakawa (Fukuoka, Japan), an oxidized form of GTP, 8-OH-rGTP, can be incorporated into RNA opposite adenine, and thus potentially lead to the production of an abnormal protein. The hMTH1 protein, encoded by the human mutT homologue cloned and characterized by this group, hydrolyzes 8-OH-rGTP to 8-OH-rGMP, which is not re-phosphorylated by nucleoside diphosphatases. As evidenced by reconstitution experiments in vitro, hMTH1 thus prevents misincorporation of 8-OH-rGTP into RNA, strongly suggesting the maintenance not only of replicational but also of transcriptional fidelity to be the function of this protein.
Dr. M. Sekiguchi and Dr. K. Sakumi (Fukuoka, Japan) have found that mice defective in both alleles of the gene encoding the one-step DNA repair protein O 6 -methylguanine-DNA methyltransferase (MGMT) are highly sensitive to the cytotoxic and 1 Names and affiliations of participants are listed at the end of the report.