The Y-family DNA polymerases help cells tolerate DNA damage by allowing replication to continue opposite lesions in the DNA template. This translesion DNA synthesis can be accurate, preserving the integrity of the genetic information, or it can be error-prone, producing a mutation in the genome even if the DNA damage in the template strand is repaired later. The Y-family polymerases that belong to the DinB subfamily are able to bypass damaged deoxyguanosine bases accurately by incorporating deoxycytidine nucleotides opposite the lesion. The DinB enzymes generally make fewer base-substitution errors than other types of Y-family polymerases, yet they make single-base deletion mutations, where a template base is skipped during replication, at a high rate. We are using the DinB homolog (Dbh) from Sulfolobus acidocaldarius as a model for the DinB class of DNA polymerases. Dbh has been demonstrated to accurately and efficiently bypass DNA damage at deoxyguanosine bases;it displays a strong preference for incorporating deoxycytidine nucleotides even on undamaged DNA;and it generates single-base deletion errors at an exceptionally high rate at specific sequences. The objective of this proposal is to provide a more complete understanding of how structural differences among the various Y-family DNA polymerases give rise to differing lesion-bypass activity and DNA replication fidelity. Our central hypothesis is that the exaggerated mutational specificity and lesion-bypass activity of Dbh will allow us to more easily identify the structural features that influence these activities.
The specific aims are (1) to determine how Dbh generates single-base deletion mutations, (2) to elucidate the mechanisms Dbh uses to replicate damaged DNA, and (3) to characterize how Dbh is regulated by interactions with other proteins. We will use a combination of X-ray crystallographic, computational and biochemical approaches to address these issues. These studies will contribute to our understanding of how the Y-family polymerases help cells tolerate DNA damage and also how they introduce mutations into the genome. Statement of Public Health Relevance An accumulation of multiple mutations in human cells can lead to cancerous cell growth, while mutations in bacteria can lead to antibiotic resistance. The Y-family DNA polymerases appear to be responsible for many of the mutations produced in both prokaryotic and eukaryotic cells. Inhibiting these polymerases, at appropriate times, could be a useful way to prevent cancers from progressing or to increase the efficacy of antibacterial drug treatments.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function C Study Section (MSFC)
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Preusch, Peter C
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Wadsworth Center
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Mukherjee, Purba; Wilson, Ryan C; Lahiri, Indrajit et al. (2014) Three residues of the interdomain linker determine the conformation and single-base deletion fidelity of Y-family translesion polymerases. J Biol Chem 289:6323-31
Manjari, Swati R; Pata, Janice D; Banavali, Nilesh K (2014) Cytosine unstacking and strand slippage at an insertion-deletion mutation sequence in an overhang-containing DNA duplex. Biochemistry 53:3807-16
Wilson, Ryan C; Jackson, Meghan A; Pata, Janice D (2013) Y-family polymerase conformation is a major determinant of fidelity and translesion specificity. Structure 21:20-31
Mukherjee, Purba; Lahiri, Indrajit; Pata, Janice D (2013) Human polymerase kappa uses a template-slippage deletion mechanism, but can realign the slipped strands to favour base substitution mutations over deletions. Nucleic Acids Res 41:5024-35
Lahiri, Indrajit; Mukherjee, Purba; Pata, Janice D (2013) Kinetic characterization of exonuclease-deficient Staphylococcus aureus PolC, a C-family replicative DNA polymerase. PLoS One 8:e63489
Wu, Yifeng; Wilson, Ryan C; Pata, Janice D (2011) The Y-family DNA polymerase Dpo4 uses a template slippage mechanism to create single-base deletions. J Bacteriol 193:2630-6
Pata, Janice D (2010) Structural diversity of the Y-family DNA polymerases. Biochim Biophys Acta 1804:1124-35