Role of key protein residues involved in DinB DNA damage specificity: an in silico study

Abstract

Y‐family DNA polymerases are a class of error‐prone polymerases able to perform DNA translesion synthesis. E. coli DNA Polymerase IV (also called DinB from the name of the gene) is a Y‐family DNA polymerase implicated in stress‐induced mutagenesis. As other DNA polymerases, DinB has the general architecture of a right hand, with palm, thumb, and fingers subdomains. In addition, Y‐family polymerases have an extra domain, namely the “little finger” or polymerase‐associated domain (PAD) and have a more solvent accessible active‐site than their high‐fidelity replicative counterparts. Kinetics studies have shown that DinB is particularly efficient at bypassing damaged guanosines, such as the N2‐furfuryl‐dG, and even more efficient in lesion bypass on damaged DNA than in replication of undamaged DNA. Hydrogen exchange experiments have suggested that the protein undergoes a conformational change in the presence of certain DNA damages on the template strand, but not others, implying a possible mechanism of bypass discrimination. However, the exact nature of such movement is not well characterized. Using molecular dynamics (MD) simulations we explored different DNA/protein/incoming nucleotides combinations to dissect the protein conformational change. Specifically, we analyzed a lesion that the protein can bypass (N2‐furfuryl‐dG) and one that instead the protein does not bypass well (O6‐Methylated dG). Our MD simulations revealed that there are some key residues in the fingers domain that are important switches for the conformational change. For example, R35 assumes a characteristic position when the protein is in a catalytically competent state, while it blocks the active site when the protein is not able to bypass the lesion. This result is in agreement with previous studies which showed that mutations of the R35 residue alter the protein specificity. Other areas of DinB which seem to undergo large movement reside in the PAD domain. This domain, which makes contact with the DNA major groove, has been implicated in DNA damage specificity, so it makes sense that residues on this domain assume different conformations, depending on the type of lesions present on the templating strand. A discussion of possible roles for some of the residues on the PAD will be presented.