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Comparative mutagenicity amongst c‐family bacterial DNA pol III’s (551.2)
Author(s) -
Morris Justin,
Mukherjee Purba,
Pata Janice
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.551.2
Subject(s) - dna polymerase , primer (cosmetics) , dna , polymerase , oligonucleotide , microbiology and biotechnology , dna polymerase ii , biology , processivity , chemistry , genetics , polymerase chain reaction , reverse transcriptase , gene , organic chemistry
As evidence points to the use of multiple DNA pol III’s by gram‐positive bacteria, a question remains: which biochemical factors (domain composition, DNA oligonucleotide (oligo) length, etc.) serve as the strongest determinants of fidelity? We have characterized various patterns of relative mutagenicity amongst the essential C‐family bacterial DNA polymerases found in Staphylococcus aureus. These include two PolC constructs: PolC‐Exomut and PolC‐ΔNΔExo, as well as DnaE. In vitro assays were performed using various primer‐template (p/t) DNA substrates and nucleotides. Additional assays paired the polymerase with the β clamp. DnaE showed the highest frequency of nucleotide misincorporation, especially on longer p/t DNA substrates, as well as in bypassing 8‐oxoguanine lesions. PolC‐Exomut, the construct with both N‐terminal and Exonuclease (Exo) domains, consistently showed the most fidelity. A direct relationship between fidelity and DNA substrate length was also observed. The presence of secondary proteins consistently increased overall addition, but not fidelity. Overall, these assays indicate DnaE as a more error‐prone polymerase compared to its PolC counterparts. Additionally, oligo length was shown to influence the accuracy of nucleotide addition. Future assays will further examine the effects of oligo length as well as fidelity patterns specifically attributable to the N‐terminal domain. Grant Funding Source : Supported by NIH Grant R01‐GM080573