Jaremko, W. J. | bioRxiv Science

bioRxiv2022

Pre-Steady-State Kinetic Characterization of an Antibiotic-Resistant Mutation of Staphylococcus aureus DNA Polymerase PolC

The emergence and spread of antibiotic resistance in bacterial pathogens are serious and ongoing threats to public health. Since chromosome replication is essential to cell growth and pathogenesis, the essential DNA polymerases in bacteria have long been targets of antimicrobial development, although none have yet advanced to the market. Here we use transient-state kinetic methods to characterize the inhibition of the PolC replicative DNA polymerase from Staphylococcus aureus by ME-EMAU, a member of the 6-anilinouracil compounds that specifically target PolC enzymes, which are found in low-GC content Gram-positive bacteria. We find that ME-EMAU binds to S. aureus PolC with a dissociation constant of 14 nM, more than 200-fold tighter than the previously reported inhibition constant, which was determined using steady-state kinetic methods. This tight binding is driven by a very slow off rate, 0.006 s-1. We also characterized the kinetics of nucleotide incorporation by PolC containing a mutation of phenylalanine 1261 to leucine (F1261L). The F1261L mutation decreases ME-EMAU binding affinity by at least 3500-fold, but also decreases the maximal rate of nucleotide incorporation by 11.5-fold. This suggests that bacteria acquiring this mutation would be likely to replicate slowly and be unable to out-compete wild-type strains in the absence of inhibitor, reducing the likelihood of the resistant bacteria propagating and spreading resistance.

biochemistry↗

bioRxiv2020

Heterotrimeric PCNA Increases the Activity and Fidelity of Dbh, a Y-family Translesion DNA Polymerase Prone to Creating Single-Base Deletion Mutations

Dbh is a Y-family translesion DNA polymerase from Sulfolobus acidocaldarius, an archaeal species that grows in harsh environmental conditions. Biochemically, Dbh displays a distinctive mutational profile, creating single-base deletion mutations at extraordinarily high frequencies (up to 50%) in specific repeat sequences. In cells, however, Dbh does not appear to contribute significantly to spontaneous frameshifts in these same sequence contexts. This suggests that either the error-prone DNA synthesis activity of Dbh is reduced in vivo and/or Dbh is restricted from replicating these sequences. Here, we test the hypothesis that the propensity for Dbh to make single base deletion mutations is reduced through interaction with the S. acidocaldarius heterotrimeric sliding clamp processivity factor, PCNA-123. We first confirm that Dbh physically interacts with PCNA-123, with the interaction requiring both the PCNA-1 subunit and the C-terminal 10 amino acids of Dbh, which contain a predicted PCNA-interaction peptide (PIP) motif. This interaction stimulates the polymerase activity of Dbh, even on short, linear primer-template DNA by increasing the rate of nucleotide incorporation. This stimulation requires an intact PCNA-123 heterotrimer and a DNA duplex length of at least 18 basepairs, the minimal length predicted from structural data to bind to both the polymerase and the clamp. Finally, we find that PCNA-123 increases the fidelity of Dbh on a single-base deletion hotspot sequence 3-fold by promoting an increase in the rate of correct, but not incorrect, nucleotide addition and propose that PCNA-123 induces Dbh to adopt a more active conformation that is less prone to creating deletions during DNA synthesis. HighlightsO_LIPCNA increases the fidelity of Dbh polymerase on a deletion-hotspot sequence. C_LIO_LIThe interaction stimulates incorporation of the correct, but not incorrect, nucleotide. C_LIO_LIA minimal duplex length of 18 bp is required for PCNA to stimulate polymerase activity. C_LIO_LIStructural modeling suggests that PCNA induces a conformational change in Dbh. C_LI

biochemistry↗