| Summary: | Exogenous and endogenous DNA damaging agents such as UV light cause lesions in DNA, which halt the progress of DNA replication. Microbes such as E. coli have a response mechanism known as the SOS response which is initiated upon DNA damage. The SOS response ultimately results in the expression of the Y-family DNA polymerases which catalyze translesion synthesis to copy damaged DNA (Walsh 2011). Y-family polymerases specifically bypass certain types of DNA lesions.
Understanding DNA damage specificity of Y-family polymerases is necessary due to their mutagenic nature. They lack proofreading ability which removes incorrectly inserted nucleotides, and can therefore be error-prone (Jarosz 2007). Eventually, the insertion of the incorrect base can lead to mutations that confer a selective advantage in the cell, such as antibiotic resistance. In E. coli, there are two Y-family polymerases known as polymerase IV and polymerase V. Polymerase V, also
known as UmuD'2C , is capable of bypassing lesions caused by UV light exposure namely thymine-thymine cyclobutane pyrimidine dimers and T-T (6-4) photoproducts (Tang 2000). Polymerase IV, otherwise known as DinB, is capable of bypassing N2-dG adducts4 (among others), but not T-T CPDs or (6-4) photoproducts (Tang 2000; Jarosz 2006). The goal of this study is to understand how DinB is able to bypass certain types of lesions, but not others. In this study, directed evolution methods are
used to mutate the DinB gene and acquire variants that confer UV resistance. Characterization of these variants will help to understand which regions on the DinB protein allow for the specificity of lesion bypass. In addition, primer extension assays are used to study the lesion bypass efficiency of DinB variants in the Loop 1 region of the protein.
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