M. Defais

Metal Antitumor Compounds: The Mechanism of Action of Platinum Complexes

Cisplatin (cis-diamminedichloroplatinum(II)) is widely used in the treatment of testicular and ovarian cancers. A number of biological and biochemical results indicate that the reaction of cisplatin with DNA is responsible for the cytotoxic action of this drug. The effect of platinum compounds on the conformation and stability of DNA has been investigated and several platinum-DNA adducts have been identified in vitro and in vivo. Preliminary experiments have quantified the effect of these different lesions on DNA replication, their capacity to induce mutations and their susceptibility to DNA repair processes. Additional DNA damage may be created by platinum(IV) compounds, perhaps during their reduction to platinum(II) compounds by the cell.

Mammalian Rad51 protein: a RecA homologue with pleiotropic functions.

During the last years, homologues of E coli RecA have been cloned in numerous species including man. These Rad51 proteins share sequence as well as functional homologies with the bacterial protein. Human Rad51 (HsRad51) is able to catalyze strand exchange in vitro between homologous DNAs, but with a lower efficiency compared to that of RecA. This suggests the requirement of additional factors. A very interesting feature of Rad51 is its essential role in mouse which could mean that it has gained an essential function in cell growth. The interaction of HsRad51 with several tumor suppressor genes namely p53, BRCA1 and BRCA2 implies possible role(s) of this protein in tumorigenesis. Thus, the continued study of Rad51 should bring important insights not only into homologous recombination mechanisms but also into cell proliferation regulation.

Modulation of the SOS response by truncated RecA proteins.

SummaryRecA protein plays several key roles in the SOS response. We have constructed truncated proteins and examined their capacity to accomplish Weigle reactivation and mutagenesis of bacteriophage lambda and recombination in Escherichia coli. Our data indicate that the 17 carboxyl terminal amino acids are not essential to RecA function. However in the presence of wild-type RecA protein, the truncated protein reduces the efficiency of recombination without affecting either mutagenesis or induction of an SOS gene or Weigle reactivation. The data presented here suggest that activation of RecA protein does not involve mixed multimers or is not affected by their presence.

Role of the E. coli umuC gene product in the repair of single-stranded DNA phage

SummaryThe umuC product of Escherichia coli has been suggested to have a central role in SOS induced error prone replication of DNA (Kato and Shinoura 1977). To investigate this possibility, we examined the effect of umuC mutations on error prone repair of singleand double-stranded DNA phages. No Weigle reactivation of M13 phage was detected in a umuC mutant. Reactivation of λ phage was reduced but still evident. However mutagenesis occurred in both cases. These results suggest that induced error prone replication of phage DNA can occur via umuC dependent (transdimer synthesis) and umuC independent mechanisms.

Overexpression of Metallothionein-II Sensitizes Rodent Cells to Apoptosis Induced by DNA Cross-linking Agent through Inhibition of NF-κB Activation

DNA cross-linking agents such as mitomycin C (MMC) and cisplatin are used as chemotherapeutic agents in cancer treatment. However, the molecular mechanism underlying their antitumor activity is not entirely clear. Critical steps in cytotoxicity toward cross-linking agents can involve DNA repair efficiency, inhibition of replication, cell-cycle checkpoints, regulation, and induction of apoptosis. The complexity of the mechanisms of the mammalian cell defense against cross-linking agents is reflected by the existence of many complementation groups identified in rodent cells that are specifically sensitive to MMC. We recently showed that increased induction of apoptosis contributes to the MMC sensitivity of the group represented by the V-H4 hamster mutant cell line. In this study, through the analyses of a substractive library, we discovered that sensitive V-H4 cells display a 40-fold increase of steady-state expression of metallothionein II (MT-II) mRNA compared with resistant parental V79 cells. Down-regulation of MT-II by antisense oligonucleotides partially restores MMC resistance in V-H4 cells, indicating that MT-II overexpression is directly involved in MMC hypersensitivity of these cells. MTs have been reported to regulate the activation of NF-κB, one of the key proteins that modulates the apoptotic response. Here we found that NF-κB activation by MMC is impaired in V-H4 cells and is partially restored following down-regulation of MT-II by antisense oligonucleotides. All these data suggest that the overexpression of MT-II in V-H4 cells impairs NF-κB activation by MMC, resulting in decreased cell survival and enhanced induction of apoptosis.

Association between the Herpes Simplex Virus-1 DNA Polymerase and Uracil DNA Glycosylase

Herpes simplex virus-1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery and other enzymes involved in DNA transactions. We recently reported that the HSV-1 DNA polymerase catalytic subunit (UL30) exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities. Moreover, UL30, in conjunction with the viral uracil DNA glycosylase (UL2), cellular apurinic/apyrimidinic endonuclease, and DNA ligase IIIα-XRCC1, performs uracil-initiated base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we show that the HSV-1 UL2 associates with the viral replisome. We identified UL2 as a protein that co-purifies with the DNA polymerase through numerous chromatographic steps, an interaction that was verified by co-immunoprecipitation and direct binding studies. The interaction between UL2 and the DNA polymerase is mediated through the UL30 subunit. Moreover, UL2 co-localizes with UL30 to nuclear viral prereplicative sites. The functional consequence of this interaction is that replication of uracil-containing templates stalls at positions −1 and −2 relative to the template uracil because of the fact that these are converted into non-instructional abasic sites. These findings support the existence of a viral repair complex that may be capable of replication-coupled base excision repair and further highlight the role of DNA repair in the maintenance of the HSV-1 genome.

Investigation of the Secondary DNA-binding Site of the Bacterial Recombinase RecA

The L2 loop is a DNA-binding site of RecA protein, a recombinase from Eschericha coli. Two DNA-binding sites have been functionally defined in this protein. To determine whether the L2 loop of RecA protein is part of the primary or secondary binding site, we have constructed proteins with site-specific mutations in the loop and investigated their biological, biochemical, and DNA binding properties. The mutation E207Q inhibits DNA repair and homologous recombination in vivo and prevents DNA strand exchange in vitro (Larminat, F., Cazaux, C., Germanier, M., and Defais, M. (1992) J. Bacteriol. 174, 6264–6269; Cazaux, C., Larminat, F., Villani, G., Johnson, N. P., Schnarr, M., and Defais, M. (1994) J. Biol. Chem. 269, 8246–8254). We have found that mutant protein RecAE207Qlacked one of the two single stranded DNA-binding sites of wild type RecA. The remaining site was functional, and biochemical activities of the mutant protein were the same as wild type RecA with ssDNA in the primary binding site. The second mutation, E207K, reduced but did not eliminate DNA repair, SOS induction, and homologous recombinationin vivo. In the presence of ATP, mutant protein RecAE207K catalyzed DNA strand exchange in vitro at a slower rate than wild type protein, and ssDNA binding at site I was competitively inhibited. These results show that the L2 loop is or is part of the functional secondary DNA-binding site of RecA protein.

UV induction of LexA independent proteins which could be involved in SOS repair

The SOS response is induced in E coli following treatments that interfere with DNA replication. The response is under the control of the recA and the lexA genes. Strains defective in LexA repressor constitutively express SOS proteins. However, SOS repair does not reach its maximum level in these strains. Instead, an activation of RecA protein and de novo protein synthesis are required for full repair. We have analyzed by 2-dimensional gel electrophoresis the induction of proteins after UV irradiation of lexA(Def) bacteria. Proteins which might participate in SOS repair are induced under these conditions.

Weigle reactivation and mutagenesis of bacteriophage λ in lexA(Def) mutants of E. coli K12

SummaryThe SOS response in UV-irradiated bacteria enhances the survival and mutagenesis of infecting damaged bacteriophage λ. In a lexA(Def) strain, SOS bacterial genes are fully derepressed by an inactivating mutation in the LexA repressor gene. We tested several lexA(Def) derivative strains for their capacity to constitutively promote high survival and mutagenesis of irradiated λ. We showed that UV irradiation of the lexA(Def) host bacteria is still necessary for optimal efficiency of both these SOS functions, which are dependent on the umuC gene product and an activated form of RecA protein.

Site-directed mutagenesis in the Escherichia coli recA gene

Escherichia coli RecA protein plays a fundamental role in genetic recombination and in regulation and expression of the SOS response. We have constructed 6 mutants in the recA gene by site-directed mutagenesis, 5 of which were located in the vicinity of the recA430 mutation responsible for a coprotease deficient phenotype and one which was at the Tyr 264 site. We have analysed the capacity of these mutants to accomplish recombination and to express SOS functions. Our results suggest that the region including amino acid 204 and at least 7 amino acids downstream interacts not only with LexA protein but also with ATP. In addition, the mutation at Tyr 264 shows that this amino acid is essential for RecA activities in vivo, probably because of its involvement in an ATP binding site, as previously shown in vitro.