We are studying the effect of DNA damage caused by DNA-protein cross links, the effect of these cross links on cell viability and DNA replication and the pathways that repair them.
The role of methylation reactions in the control of cellular processes especially by the methylation of DNA, the effect of nucleoside analog inhibitors of DNA methylases on cell growth, the mechanism of DNA repair as it relates to removal of DNA-protein cross links.
We have been studying the properties of DNA methylases and of drugs that inhibit their activity. Several of these proteins are autoregulated. In addition to binding to the DNA at the site that they methylate, they bind to their promoter inhibiting transcription of their respective genes. The properties of these binding reactions are very different. The recognition sequence that confers specificity for methylation consists of only 5 nucleotides. However, when they bind to the promoter they protect 50bp from DNase I digestion. One of our objectives is to determine the basis for the difference in binding specificity of these proteins at these sites.
We have demonstrated that DNA methylases can be inhibited by nucleoside analogs. The analog must be incorporated into DNA to be an effective inhibitor. These analogs cause the DNA methylase to bind tightly to the DNA containing them. Some of these analogs will kill the cells only when they contain DNA methylase; the cells are presumably killed by the binding of the enzyme to DNA. The cells can repair this DNA damage. They die when the repair system can no longer repair all the DNA-protein cross links that are formed. We have been studying the repair of these DNA-protein adducts by preparing DNA containing the analog in vitro and studying the survival of the DNA in vivo. The cells can replicate the DNA-protein complex with approximately 50% of the efficiency that they can replicate unbound DNA. Replication is not dependent on either the recA or uvrA gene product. Presently we are studying processes by which these DNA-protein adducts are repaired.