Investigating the role of the little finger domain of Y-family DNA polymerases in low fidelity synthesis and translesion replication.

Abstract:

Dpo4 and Dbh are Y-family polymerases that originate from two closely related strains of Sulfolobaceae. Quite surprisingly, however, the two polymerases exhibit different enzymatic properties in vitro. For example, Dpo4 can replicate past a variety of DNA lesions, yet Dbh does so with a much lower efficiency. When replicating undamaged DNA, Dpo4 is prone to make base pair substitutions, whereas Dbh predominantly makes single-base deletions. Overall, the two proteins are 54% identical, but the greatest divergence is found in their respective little finger (LF) domains, which are only 41% identical. To investigate the role of the LF domain in the fidelity and lesion-bypassing abilities of Y-family polymerases, we have generated chimeras of Dpo4 and Dbh in which their LF domains have been interchanged. Interestingly, by replacing the LF domain of Dbh with that of Dpo4, the enzymatic properties of the chimeric enzyme are more Dpo4-like in that the enzyme is more processive, can bypass an abasic site and a thymine-thymine cyclobutane pyrimidine dimer, and predominantly makes base pair substitutions when replicating undamaged DNA. The converse is true for the Dpo4-LF-Dbh chimera, which is more Dbh-like in its processivity and ability to bypass DNA adducts and generate single-base deletion errors. Our studies indicate that the unique but variable LF domain of Y-family polymerases plays a major role in determining the enzymatic and biological properties of each individual Y-family member.

Polymerases:

Topics:

Mutational Analysis, Historical Protein Properties (MW, pI, ...), Other Enzymatic Activities, Structure and Structure/Function

Status:

new topics/pols set partial results complete validated

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