The LexA repressor of Escherichia coli represses a set of genes that are expressed in response to DNA damage. After inducing treatments, the repressor is inactivated in vivo by a specific cleavage reaction which requires RecA protein. Under physiological conditions in vitro, RecA-dependent cleavage also occurs. At alkaline pH, however, the specific cleavage reaction occurs spontaneously without RecA, a reaction which is termed autodigestion. The LexA repressor is, therefore, thought to cleave itself with RecA acting to stimulate autodigestion. A set of lexA (Ind⁻) mutants that are deficient in in vivo RecA-mediated cleavage but retain significant repressor function were isolated. These 20 mutations resulted in amino acid substitutions in 12 positions, most of which are conserved between LexA and four other cleavable proteins. All the mutations were located in the hinge region or C-terminal domain of the protein, portions of LexA previously implicated in the specific cleavage reactions. Furthermore, these mutations were clustered in three regions, around the cleavage site (Ala-84-Gly-85) and in blocks of conserved amino acids around two residues, Ser-119 and Lys-156, which are believed essential for the cleavage reactions. These three regions of the protein thus appear to play important roles in the cleavage reaction. Many of the mutant proteins were purified in order to further characterize their properties in both autodigestion and RecA-mediated cleavage. All of these mutant proteins are found to be deficient in both cleavage reactions. A mutant protein, replacing Lys-156 to Arg, requires a higher pH condition than the wild-type protein does for both cleavage reactions. The results suggest that deprotonation of Arg-156, and by inference Lys-156 in the wild-type protein, is required for both autodigestion and RecA-mediated cleavage; and that in the latter reaction RecA acts to reduce the pKa of Lys-156, allowing efficient cleavage of wild-type repressor under physiological conditions. Finally, several mutant proteins affecting amino acids around the cleavage site and the proposed nucleophile in the cleavage reaction (Ser-119) could not efficiently act as a competitive inhibitor in the RecA-mediated cleavage of wild-type repressor, presumably because they affect RecA binding.

Colon cancer is the second most common type of cancer in the United States. Its incidence is linked epidemiologically to high levels of bile acids in the feces. Bile acids have been implicated as promotors and cocarcinogens in the etiology of colon cancer and as comutagens and mutagens in bacteria. These observations suggest the hypothesis that bile acids may interact directly with DNA. Using agarose gel electrophoresis we showed that bile acids convert covalently closed circular plasmid DNA to the open circular form, indicating strand breakage. We next treated the single stranded circular DNA of phage M13 with bile acids and found that the transfection efficiency of this DNA declined up to a thousand-fold. The concentrations of bile acids used were of the same magnitude as the fecal bile acid concentrations found in colorectal cancer patients. This inactivation was largely prevented when the bile acids were pretreated with cellulose fiber.

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Infertility affects ~20% of couples in Europe and in 50% of cases the problem lies with the male. The development of assisted reproductive technologies (ART) such as in vitro fertilisation (IVF) and intra-cytoplasmic spermatozoa injection (ICSI) has allowed some couples to overcome male-factor infertility. However concerns remain over the increasing use of ART as elevated levels of DNA damage in sperm from infertile men have been reported and a link between DNA damage in sperm and early embryonic failure has been demonstrated. DNA damage in sperm, caused by oxidative stress may also be passed on from father to child resulting in an increased incidence of childhood cancer. This has led to fears that the use of damaged sperm in ART could contribute to early embryonic failure and/or birth defects. The studies described in this thesis used mouse models to investigate the relationship between DNA integrity in male germ cells and male fertility. This was achieved by studying both the effects of targeted ablation of genes involved in DNA repair and the impact of scrotal heat stress on testicular function and sperm DNA integrity. Three lines of transgenic mice with deletions in genes involved in genomic integrity (Ercc1, Msh2 and p53) were studied. All three genes are expressed in the testis. These studies confirmed and extended studies on Ercc1 knockout (-/-) mice showing reduced germ cell complement, increased apopotosis, an increased percentage of damaged sperm and demonstrated for the first time that depletion of Ercc1 results in an increased incidence of unrepaired double strand DNA breaks (DSB) in pachytene spermatocytes. The persistence of DSBs in spermatocytes and abnormal sperm chromatin structure confirmed that the repair functions of Ercc1 are essential for normal germ cell maturation. In the p53-/- mice these studies showed for the first time that there was an increase in DSBs in spermatocytes and an increase in numbers of sperm with damaged DNA. The level of apoptosis was also increased in the testes suggesting that caspase-3 mediated apoptosis is not entirely p53 dependent as been previously suggested. These studies demonstrated for the first time that targeted ablation of Msh2 compromises germ cell complement and as in the Ercc1-/- this resulted in gaps in the seminiferous epithelium consistent with clonal loss of germ cells. Consistent with a role for MSH2 in mismatch repair no DSBs were detected in spermatocytes from Msh2-/-. Testicular function is temperature dependant and due to their location in the scrotum testes are normally kept between 2ºC and 8ºC below core body temperature. In mice transient scrotal heat stress (30 minutes at 38°C, 40°C and 42°C) disrupted testicular function. Analysis of sperm and testis parameters revealed that stress at 38°C was sufficient to have subtle effects on epididymal function but the higher temperatures had additional consequences for testicular function which resulted in DNA damage in spermatocytes, germ cells loss and increased apoptosis. Further studies into the pathways of apoptosis demonstrated that the mitochondrial/intrinsic pathway plays a role in heat stress response. The fertility of males was altered in those heated to 42°C resulting in reduced pregnancy rate and litter size. Given that the paternal genome is reported to be required for the development of extraembryonic tissues and this will influence growth of the embryo, it was interesting to note an increase in resorption sites in pregnancies using 40°C males. IVF was used to demonstrate that embryos formed using sperm from males stressed at 42°C were compromised between the 4-cell and blastocyst stage suggesting that though sperm with DNA damage are still capable of fertilisation, the paternal DNA was introducing genomic instability to the embryo and having fatal effects on development. These studies have also shown that one possible underlying cause of the disturbance in testicular function is hypoxia, as a marked increase in Hif1 alpha (a marker of hypoxia) mRNA and relocalisation of the protein was observed in the testis. In conclusion, DNA damage in the male germ line caused either by induced stress, or by targeted ablation of DNA repair genes, can disrupt testicular architecture, function and therefore the fertility of mice. These data have demonstrated that deletion of Ercc1, Msh2 and p53 can have differential but overlapping affects on germ cell function and sperm production and that increased scrotal temperature can cause subfertility in male mice. This study has provided further confirmation of possible male-mediated effects on embryo survival and these findings should be taken into consideration when using sperm from infertile men in IVF/ICSI treatments where the normal quality control processes involved in fertilisation are bypassed.