title: 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 α-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.
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Walsh
authors.forename: Ramya
authors.initials: R
authors.lastname: Parasuram
authors.forename: Pradyumna R
authors.initials: PR
authors.lastname: Rajput
authors.forename: Eriks
authors.initials: E
authors.lastname: Rozners
authors.forename: Mary Jo
authors.initials: MJ
authors.lastname: Ondrechen
authors.forename: Penny J
authors.initials: PJ
authors.lastname: Beuning
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular
chemical_list: D000596:Amino Acids; D017931:DNA Primers; C094399:DinB protein, E coli; D029968:Escherichia coli Proteins