Effect of Dna Damage By Mitomycin C Or Ultraviolet Irradiation on Bacteriophage T4 Deoxyribonucleotide Synthetase.

In phage T(,4) infection deoxyribonucleoside triphosphates are formed by a channeled process from ribonucleotides by a system of enzymes, called the deoxyribonucleotide synthetase complex. By our concept the synthetase intimately interacts with the T4 DNA replication enzyme complex and transfers the deoxyribonucleotides directly to the 3' growing point. Agents altering the structure of DNA have been shown to inhibit the de novo synthesis of deoxyribonucleotides in T4-infection, not through direct action on the biosynthetic enzymes of the system but via DNA as an intermediary. As a result it had been proposed that T4 template DNA has a structural/regulatory role in the deoxyribonucleotide synthetase complex. This thesis has examined the inhibition of deoxyribonucleotide synthesis by mitomycin C, an antibiotic which forms covalent crosslinks between guanine residues on opposite str and s of DNA. To determine whether damage of DNA by mitomycin C preferentially inhibited one of the two pathways UDP (--->) dUDP (--->) dUMP (--->) dTMP and CDP (--->) dCDP (--->) dCMP (--->) HmdCMP T4 cd uvsX phage was employed. This double mutant carries, respectively, mutations in the structural gene for dCMP deaminase, to prevent conversion of dCMP to dTMP, and in a gene required for repair of DNA damage by mitomycin C or ultraviolet irradiation. In fact, mitomycin C inhibited the two pathways equally. However, in the presence of the uvsX('+) gene product the antibiotic did not inhibit deoxyribonucleotide synthesis, in keeping with the proposal that these agents function through their action on DNA itself. Together with other evidence the studies indicate that damage of template DNA inhibits phage-coded ribonucleoside diphosphate reductase, the limiting step in the pathway, but has no differential effects on the enzyme. While mitomycin C inhibited deoxyribonucleotide synthesis by about 30 to 35% in cultures infected by uvsX Dna('+) phage, the agent had little or no effect in infections with uvsX Dna('-) double mutants. Similarly, ultraviolet irradiation of a uvsX Dna(,ts) double mutant inhibited deoxyribonucleotide synthesis only if infection was at temperatures permissive for DNA synthesis. Since DNA damage by the level of ultraviolet irradiation or mitomycin C employed occurs at a limited number of sites, estimated to be in the order of 1 in several thous and base pairs, the probability is great that the deoxyribonucleotide synthetase complexes will assemble at undamaged replication origins on the template. A model is presented in which a phage deoxyribonucleotide synthetase complex, associated with the replication complex at the leading str and of the growing point, is inhibited only when it reaches the site of DNA damage; in the absence of DNA replication inhibition cannot occur. The model also predicts that the deoxyribonucleotide synthetase complexes associated with DNA replication complexes carrying out discontinuous DNA synthesis, i.e., forming Okazaki fragments on the lagging str and will not be inhibited. Mitomycin C crosslinking of DNA has been suggested by other workers to occur at the sequence pGpC:pGpC. In the alternating polymer poly(dGdC):poly(dGdC) 50% of the dinucleotide sequences are such sites. Reaction of this synthetic polymer with reduced mitomycin C resulted in antibiotic:polynucleotide complexes with binding ratios in a range of one drug molecule per 2 to 4 base pairs. At a ratio of 1:4 the mitomycin C-polynucleotide complex showed no thermal hyperchromic transition even at temperatures 18 degrees above the Tm of the untreated polynucleotide. This finding suggests that almost every mitomycin molecule bound to poly(dGdC):poly(dGdC) forms an interstr and crosslink, since the two str and s of the DNA do not unstack at all even under strongly denaturing conditions. The model reaction supports the proposal that pGpC:pGpC represents the site of mitomycin C-induced crosslinks.Ph.D.BiochemistryUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/158307/1/8116294.pd