Effects of non‐catalytic, distal amino acid residues on activity of E. coli DinB (DNA polymerase IV)

Abstract

DinB is one of two Y family polymerases in E. coli and is involved in copying damaged DNA. DinB is specialized to bypass deoxyguanosine adducts that occur at the N 2 position, with its cognate lesion being the furfuryl adduct. Active site residues have been identified that make contact with the substrate and carry out deoxynucleotide triphosphate (dNTP) addition to the growing DNA strand. In DNA polymerases, these include negatively charged aspartate and glutamate residues (D8, D103, and E104 in E. coli DNA polymerase IV DinB). These residues position the essential magnesium ions correctly to facilitate nucleophilic attack by the primer hydroxyl group on the a-phosphate group of the incoming dNTP. To study the contribution of DinB residues to lesion bypass, the computational methods THEMATICS and POOL were employed. These methods correctly predict the known active site residues, as well as other residues known to be important for activity. In addition, these methods predict other residues involved in substrate binding as well as more remote residues. DinB variants with mutations at the predicted positions were constructed and assayed for bypass of the N 2 -furfuryl-dG lesion. We find a wide range of effects of predicted residues, including some mutations that abolish damage bypass. Moreover, most of the DinB variants constructed are unable to carry out the extension step of lesion bypass. The use of computational prediction methods represents another tool that will lead to a more complete understanding of translesion DNA synthesis. Environ. Mol. Mutagen. 53:766-776, 2012. V V C 2012 Wiley Periodicals, Inc.

The DinB•RecA complex of Escherichia coli mediates an efficient and high‐fidelity response to ubiquitous alkylation lesions

“…Thus, while there is genetic evidence supporting the role of E. coli DinB and its homologs in the bypass of alkylation damage, the catalytic efficiency and fidelity of lesion bypass have not been explored in vitro. Kinetic analyses of E. coli DinB alone have been carried out previously; however, these have been limited to undamaged templates (Wagner et al, 1999;Sharma et al, 2013) or templates containing N 2 -dG adducts (Jarosz et al, 2006(Jarosz et al, , 2009Minko et al, 2008;Walsh et al, 2012), or oxidized bases (Hori et al, 2010). Nonetheless, analysis of N 2 -dG lesion bypass has allowed us to understand the preference and proficiency of DinB on this lesion, and to ultimately gain insights into the mechanism(s) regulating TLS DNA polymerases.…”

Increased Processivity, Misincorporation, and Nucleotide Incorporation Efficiency in Sulfolobus solfataricus Dpo4 Thumb Domain Mutants

“…Mutations in Dpo4 domains are likely to affect its properties. For Y-family DNA polymerases, mutations within the active site, palm, or finger domains affect translesion synthesis (37,38) and catalytic efficiency (24), whereas changes in the LF domain mainly influence processivity, fidelity, and lesion bypassing (39). In addition, thumb domain mutants of yeast mitochondrial RNA polymerase (40) and T7 DNA polymerase (41) showed altered processive synthesis.…”

“…The finger domain of Dpo4 contacts the template and the replicating base pair directly (6). Thus, key residues located in the four domains are likely to determine processivity, and mutating them can contribute to the activity of Y-family DNA polymerases (24,25). During catalysis, Dpo4 undergoes a conformational change that loosens binding to the DNA template and results in low processivity (26).…”

A rescue act: Translesion DNA synthesis past N2‐deoxyguanosine adducts

“…In addition, the furfuryl‐ N 2 ‐dG (fdG) adduct has been used as a model lesion to study the ability of PolIV to bypass N 2 ‐dG lesions . PolIV can mediate accurate replication past the fdG adduct . PolIV can incorporate the correct nucleotide dC opposite this replication blocking adducts more efficiently than the normal template dG .…”