Abstract
E. coli DNA Polymerase IV, also named DinB after its encoding gene, is a Y‐family DNA polymerase used by cells during stress‐induced mutagenesis. DinB functions as an error‐prone polymerase implicated in translesion synthesis. Kinetics studies have shown that DinB is more efficient at bypassing damaged DNA versus undamaged DNA, and that the polymerase is more effective at bypassing minor groove mutations, such as the N2‐furfuryl‐dG, than major groove mutations, such as the O6‐methylated‐dG. Recent hydrogen experiments suggest that DinB undergoes a conformational change only in the presence of select damages on the DNA template strand. These differentiations imply a possible mechanism of bypass discrimination, though discrete characteristic movements have not been identified. In this study, molecular dynamics (MD) simulations were used to explore these conformational movements through the generation and study of various systems of DNA/protein/incoming nucleotide combinations. A total of four systems were made: binary (DNA and protein), ternary‐CG (DNA, protein, correct incoming nucleotide), ternary O6‐methylated‐dG (deoxyguanosine with O6‐methylation on the template strand, protein, and correct incoming nucleotide), and binary O6‐methylated‐dG (deoxyguanosine with O6‐methylation on the template strand and protein). A ternary‐GG (DNA, protein, incorrect incoming nucleotide) system has been built, but has not yet been run through simulations. An N2‐furfuryl‐dG molecule was also built; however, the molecule requires further parameterization prior to dynamics run. The binary systems showed the highest levels of fluctuation, implying that, in lack of substrate, DinB has no conformational selectivity and is thus in a floppy and unrestrained state. Initially, the ternary O6‐methylated‐dG system seemed more similar to the ternary‐CG system than the binary system (the opposite of what was predicted). However, closer analysis of the ternary O6‐methylated‐dG system showed that residues key to stabilization of the incoming nucleotide were missing from the active site, suggesting an inability to reach catalytic competence without these residues to promote the appropriate conformational selection. Previously, it was believed that DinB adopted either an open‐inactive conformation (in the presence of a mutation or lack of substrate) or closed‐active conformation (no mutation and presence of proper substrate) in accordance with the induced‐fit theory of catalysis. However, our data suggest a less rigid breathing movement and mechanism of conformational selection as opposed to induced fit. Accordingly, data interpretation has moved from analysis of gross domain movements to analysis of the presence of key catalytic residues in the active site.Support or Funding InformationThis work was supported in part by the Rose M. Badgeley Residuary Charitable Trust Award and the Colette Mahoney Award to BSB.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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