Specificity of DNA damage inducible dna polymerase IV from Escherichia coli

Abstract

DNA polymerases are responsible for DNA replication during cell division. There are multiple families of polymerases (A, B, C, D, X) responsible for copying DNA during replication and repair. There is also a class of polymerases conserved throughout evolution, known as the Y family polymerases, that have reduced replication fidelity on undamaged DNA. However Y family DNA polymerases have the specialized property of replicating DNA by copying damaged DNA, a process known as translesion synthesis (TLS). Structural differences between Y family and replicative polymerases may account for the difference in enzymatic activity. However we demonstrate that the Klenow fragment (A family) can bypass a fluorescent cytosine analog known as 1, 3-diaza-2-oxophenothiazine (tC), that DinB, a Y family polymerase, cannot bypass. We show that DinB inserts dGTP faithfully, but cannot extend the DNA primer beyond that. Verifying which amino acid residues are responsible for both function and specificity of the Y family polymerases is accomplished by assessing the kinetic data of nucleotide incorporation events of DinB variants as compared to wild-type DinB. In order to determine candidates for residues to alter, we use a sequence alignment based approach as well as by using THEMATICS, a computational methodology developed by the Ondrechen group at Northeastern University that identifies ionizable active site residues. The variants are then tested for activity, lesion specificity and replication fidelity in vitro and in vivo.