Philip C. Hanawalt

DNA repair in an active gene: Removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall

DNA repair was measured in the dihydrofolate reductase gene in Chinese hamster ovary cells, amplified for the gene, by quantitating pyrimidine dimers with a specific UV-endonuclease. More than two thirds of the dimers had been removed from a 14.1 kb restriction fragment of the gene by 26 hr after irradiation (20 J/m2), while little removal was detected in fragments upstream of the gene and only 15% were removed from the genome overall. This suggests that damage processing can vary according to function or activity of affected sequences, which has general implications for correlations of DNA repair with survival and mutagenesis. Perhaps preferential repair of vital sequences facilitates UV-resistance of these cells despite low overall repair levels.

Transcription-coupled DNA repair: two decades of progress and surprises

Expressed genes are scanned by translocating RNA polymerases, which sensitively detect DNA damage and initiate transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes lesions from the template DNA strands of actively transcribed genes. Human hereditary diseases that present a deficiency only in TCR are characterized by sunlight sensitivity without enhanced skin cancer. Although multiple gene products are implicated in TCR, we still lack an understanding of the precise signals that can trigger this pathway. Futile cycles of TCR at naturally occurring non-canonical DNA structures might contribute to genomic instability and genetic disease.

Thymine deficiency and the normal DNA replication cycle. I.

In the absence of thymine, bacterial strains that require this compound for growth lose viability in a characteristic manner. If, at the time when thymine is removed from a culture, protein and RNA synthesis are inhibited a small fraction of the cells is found to be immune to thymineless death. Under conditions of in- hibited protein and RNA synthesis total DNA is found to increase by 40% or more. During this period of DNA synthesis the entire cell population gradually becomes immune; i.e. reaches a state in which subsequent removal of thymine does not cause death. To account for these observations we postulate that protein and/or RNA synthesis is necessary to initiate but not to sustain DNA replication. During renewed growth the acquired immunity to thymineless death is gradually lost and, in parallel, DNA synthesis is resumed. The steps by which a new DNA replication cycle is initiated require protein and/or RNA synthesis but take place as efficiently in the presence as in the absence of thymine. The chemical nature of these steps is unknown, but they are clearly independent of the replica- tion process proper.

EVIDENCE FOR REPAIR-REPLICATION OF ULTRAVIOLET DAMAGED DNA IN BACTERIA.

Density-labeling with the thymine analogue, 5-bromouracil, was used to follow DNA replication in ultraviolet irradiated bacteria. The partial degradation of the damaged DNA and simultaneous synthesis at random positions in the genome was demonstrated. This non-conservative mode of replication eventually resulted in density heterogeneity (due to differences in the 5-bromouracil-thymine ratio) among the isolated DNA fragments. Normal semi-conservative replication was observed if photoreactivating conditions followed the ultraviolet irradiation prior to density labeling. Molecular fragments containing these regions of random replication were thermally denatured and/or fragmented by sonication. Density-distribution analysis in the CsCl density-gradient indicated that the density label was incorporated into very short segments along single DNA strands. Our findings are consistent with the view that in ultraviolet-resistant organisms a mechanism for repair replication exists in which damaged single-strand regions of the chromosome can be excised and replaced, using the undamaged DNA strand as template.

Subpathways of nucleotide excision repair and their regulation

Nucleotide excision repair provides an important cellular defense against a large variety of structurally unrelated DNA alterations. Most of these alterations, if unrepaired, may contribute to mutagenesis, oncogenesis, and developmental abnormalities, as well as cellular lethality. There are two subpathways of nucleotide excision repair; global genomic repair (GGR) and transcription coupled repair (TCR), that is selective for the transcribed DNA strand in expressed genes. Some of the proteins involved in the recognition of DNA damage (including RNA polymerase) are also responsive to natural variations in the secondary structural features of DNA. Gratuitous repair events in undamaged DNA might then contribute to genomic instability. However, damage recognition enzymes for GGR are normally maintained at very low levels unless the cells are genomically stressed. GGR is controlled through the SOS stress response in E. coli and through the activated p53 tumor suppressor in human cells. These inducible responses in human cells are important, as they have been shown to operate upon chemical carcinogen DNA damage at levels to which humans are environmentally exposed. Interestingly, most rodent tissues are deficient in the p53-dependent GGR pathway. Since rodents are used as surrogates for environmental cancer risk assessment, it is essential that we understand how they differ from humans with respect to DNA repair and oncogenic responses to environmental genotoxins. In the case of terminally differentiated mammalian cells, a new paradigm has appeared in which GGR is attenuated but both strands of expressed genes are repaired efficiently.

p53-Mediated DNA Repair Responses to UV Radiation: Studies of Mouse Cells Lacking p53, p21, and/or gadd45 Genes

ABSTRACT Human cells lacking functional p53 exhibit a partial deficiency in nucleotide excision repair (NER), the pathway for repair of UV-induced DNA damage. The global genomic repair (GGR) subpathway of NER, but not transcription-coupled repair (TCR), is mainly affected by p53 loss or inactivation. We have utilized mouse embryo fibroblasts (MEFs) lacking p53 genes or downstream effector genes of the p53 pathway, gadd45 (Gadd45a) or p21(Cdkn1a), as well as MEFs lacking both gadd45and p21 genes to address the potential contribution of these downstream effectors to p53-associated DNA repair. Loss ofp53 or gadd45 had a pronounced effect on GGR, while p21 loss had only a marginal effect, determined by measurements of repair synthesis (unscheduled DNA synthesis), by immunoassays to detect removal of UV photoproducts from genomic DNA, and by assays determining strand-specific removal of CPDs from the mouse dhfr gene. Taken together, the evidence suggests a role for Gadd45, but relatively little role for p21, in DNA repair responses to UV radiation. Recent evidence suggests that Gadd45 binds to UV-damaged chromatin and may affect lesion accessibility. MEFs lacking p53 or gadd45 genes exhibited decreased colony-forming ability after UV radiation and cisplatin compared to wild-type MEFs, indicating their sensitivity to DNA damage. We provide evidence that Gadd45 affects chromatin remodelling of templates concurrent with DNA repair, thus indicating that Gadd45 may participate in the coupling between chromatin assembly and DNA repair.

A Phylogenomic Study of DNA Repair Genes, Proteins, and Processes

The ability to recognize and repair abnormal DNA structures is common to all forms of life. Studies in a variety of species have identified an incredible diversity of DNA repair pathways. Documenting and characterizing the similarities and differences in repair between species has important value for understanding the origin and evolution of repair pathways as well as for improving our understanding of phenotypes affected by repair (e.g., mutation rates, lifespan, tumorigenesis, survival in extreme environments). Unfortunately, while repair processes have been studied in quite a few species, the ecological and evolutionary diversity of such studies has been limited. Complete genome sequences can provide potential sources of new information about repair in different species. In this paper, we present a global comparative analysis of DNA repair proteins and processes based upon the analysis of available complete genome sequences. We use a new form of analysis that combines genome sequence information and phylogenetic studies into a composite analysis we refer to as phylogenomics. We use this phylogenomic analysis to study the evolution of repair proteins and processes and to predict the repair phenotypes of those species for which we now know the complete genome sequence.

EXPRESSION OF WILD-TYPE P53 IS REQUIRED FOR EFFICIENT GLOBAL GENOMIC NUCLEOTIDE EXCISION REPAIR IN UV-IRRADIATED HUMAN FIBROBLASTS

We have shown previously that Li-Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in the removal of UV-induced cyclobutane pyrimidine dimers from genomic DNA, but still proficient in the transcription-coupled repair pathway (Ford, J. M., and Hanawalt, P. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8876–8880). We have now utilized monoclonal antibodies specific for cyclobutane pyrimidine dimers or 6-4 photoproducts, respectively, to measure their repair in UV-irradiated human fibroblasts. Cells homozygous for p53 mutations were deficient in the repair of both photoproducts, whereas cells heterozygous for mutant p53 exhibited normal repair of 6-4 photoproducts, but decreased initial rates of removal of cyclobutane pyrimidine dimers, compared with normal cells. The specificity of the effect of wild-type p53 on nucleotide excision repair was demonstrated in a p53 homozygous mutant cell line containing a tetracycline-regulated wild-type p53 gene. Wild-type p53 expression and activity were suppressed in the presence of tetracycline, whereas withdrawal of tetracycline resulted in the induction of p53 expression, cell cycle checkpoint activation, and DNA damage-induced apoptosis. The regulated expression of wild-type p53 resulted in the recovery of normal levels of repair of both cyclobutane pyrimidine dimers and 6-4 photoproducts in genomic DNA, but did not alter the transcription-coupled repair of cyclobutane pyrimidine dimers. Therefore, the wild-type p53 gene product is an important determinant of nucleotide excision repair activity in human cells.

Xeroderma pigmentosum p48 gene enhances global genomic repair and suppresses UV-induced mutagenesis.

UV-damaged DNA-binding activity (UV-DDB) is deficient in some xeroderma pigmentosum group E individuals due to mutation of the p48 gene, but its role in DNA repair has been obscure. We found that UV-DDB is also deficient in cell lines and primary tissues from rodents. Transfection of p48 conferred UV-DDB to hamster cells, and enhanced removal of cyclobutane pyrimidine dimers (CPDs) from genomic DNA and from the nontranscribed strand of an expressed gene. Expression of p48 suppressed UV-induced mutations arising from the nontranscribed strand, but had no effect on cellular UV sensitivity. These results define the role of p48 in DNA repair, demonstrate the importance of CPDs in mutagenesis, and suggest how rodent models can be improved to better reflect cancer susceptibility in humans.

Molecular mechanisms for repair of DNA

Methylating agents may produce as many as nine alkylated purine and pyrimidine adducts in DNA, as well as forming phosphotriesters and inducing apurinic sites and strand breaks. Although some ofthese products are formed in proportionately small amounts, there are sufficient sites affected in the DNA of a mammalian cell to make even the most minor product of potential biological significance. It is not possible to specify the exact reaction sites resulting in biological damage, but it is possible to quantitate the excision-repair of such damage both in the bulk of the DNA and at DNA growing points. Excisionrepair can be measured in the bulk of the DNA by determining the specific activity of the NaCl eluate of a benzoylated naphthoylated DEAE-cellulose column of extracts of cells after treatment and incubation in the presence of hydroxyurea and labeled thymidine. The average number of nucleotides inserted per methyl methanesulfonate-induced methyl group is 0.1, per apurinic site is 9. Repair in growing-point regions after methyl methanesulfonate treatment occurs to approximately the same extent as in the bulk of the DNA. Alkylating agents are electrophilic reagents which can comhine with DNA and other cellular macromolecules. Different alkylating agents have specific biologi-