Our objective is to understand the molecular mechanisms responsible for misrepair mutagenesis in Escherichia coli. These processes are elicited in response to DNA damage inflicted by a variety of mutagens, both physical and chemical, that occur in our environment naturally or are produced by human activity. These agents not only cause mutations, but, by altering certain regulatory genes, may also play a role in carcinogenesis. Such mutagens are a potential health hazard, therefore, though the extent to which this potential for harm is realized is not yet clear in many cases. Direct measurement of hazard at the low levels of damage usually encountered is rarely feasible, and extrapolation from experimental results with high doses of mutagen administered to non-human organisms is hampered by lack of information about the relevant mechanisms. Work in this project should help to provide such information. Our specific goals are to determine the types and frequencies of mutations induced by specific DNA lesions, namely, cyclobutane bipyrimidines and 6,4 Pyo adducts; to obtain evidence in vivo and in vitro for bypass replication, the process thought to cause mutations, and to see if the mechanisms for bypass replication differ when single- as opposed to double-stranded DNA is replicated; to investigate the properties of the umuDC gene, which is thought to play an important role in bypass replication; and to investigate the phenomenon of untargeted mutagenesis as a means of gaining insight into changes in the replication complex that result from UV irradiation. We will use a combination of genetical and molecular biological techniques to achieve these aims, including the sequencing of induced mutations, the construction of synthetic oligonucleotide hybrid phages containing a single defined lesion at a specific site, and the development of an in vitro system with which to study bypass replication.
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