Influence of DNA Structure on DNA Polymerase β Active Site Function

Abstract

In the ternary substrate complex of DNA polymerase (pol) ␤, the nascent base pair (templating and incoming nucleotides) is sandwiched between the duplex DNA terminus and polymerase. To probe molecular interactions in the dNTP-binding pocket, we analyzed the kinetic behavior of wild-type pol ␤ on modified DNA substrates that alter the structure of the DNA terminus and represent mutagenic intermediates. The DNA substrates were modified to 1) alter the sequence of the duplex terminus (matched and mismatched), 2) introduce abasic sites near the nascent base pair, and 3) insert extra bases in the primer or template strands to mimic frameshift intermediates. The results indicate that the nucleotide insertion efficiency (k cat /K m , dGTP-dC) is highly dependent on the sequence identity of the matched (i.e. WatsonCrick base pair) DNA terminus (template/primer, G/C ϳ A/T > T/A ϳ C/G). Mismatches at the primer terminus strongly diminish correct nucleotide insertion efficiency but do not affect DNA binding affinity. Transition intermediates are generally extended more easily than transversions. Most mismatched primer termini decrease the rate of insertion and binding affinity of the incoming nucleotide. In contrast, the loss of catalytic efficiency with homopurine mismatches at the duplex DNA terminus is entirely due to the inability to insert the incoming nucleotide, since K d(dGTP) is not affected. Abasic sites and extra nucleotides in and around the duplex terminus decrease catalytic efficiency and are more detrimental to the nascent base pair binding pocket when situated in the primer strand than the equivalent position in the template strand.DNA polymerases select (bind and incorporate) a nucleoside triphosphate (dNTP) from a pool of structurally similar molecules to preserve Watson-Crick base pairing rules. DNA polymerase (pol) 1 ␤ is a model polymerase to study mechanisms utilized to assure efficient and faithful DNA synthesis. Its small size, lack of essential accessory proteins, and absence of a proofreading exonuclease have facilitated its biochemical, kinetic, and structural characterization. More importantly, it shares many general structural and mechanistic features exhibited by other DNA polymerases (1).DNA polymerase ␤ contributes two important enzymatic activities during single-nucleotide base excision DNA repair. A deoxyribose phosphate lyase activity is associated with the amino-terminal 8-kDa lyase domain. This activity excises a deoxyribose phosphate intermediate during repair of abasic sites and generates a 5Ј-phosphate in a single-nucleotide gap.The nucleotidyl transferase activity of pol ␤ is associated with the 31-kDa polymerase domain that fills the single-nucleotide gap. In addition, the polymerase activity of pol ␤ is necessary for several alternate repair pathways that require longer gapfilling DNA synthesis (e.g. long patch base excision repair) (2, 3). A general feature observed in the structures of all DNA polymerases that include a template overhang (i.e. singlestranded DNA) is that the tra...

Structure of DNA polymerase β with a benzo[ c ]phenanthrene diol epoxide-adducted template exhibits mutagenic features

“…The inability to obtain a ternary substrate complex with ddCTP strongly suggests that this is partly due to a dramatically reduced binding affinity for ddCTP resulting from phenanthrene incursion into the dNTP binding site and the inability of the N subdomain to close. Whereas correct nucleotide insertion is dramatically decreased, dGTP misinsertion is hardly affected relative to that observed with an unadducted templating guanine (42). Accordingly, dGMP misinsertion is greatly preferred over dCMP incorporation translating to a misinsertion frequency approaching 1.…”

“…The reaction mixture contained 50 mM Tris⅐HCl (pH 7.4), 100 mM KCl, 10 mM MgCl 2 , 20 nM Pol ␤, 200 nM DNA, and 200 M each dNTP. After various time intervals, reactions were quenched and the products were analyzed as described previously (42).…”

DNA Sequence Context Effects on the Glycosylase Activity of Human 8-Oxoguanine DNA Glycosylase

“…6, a reconstituted BER system using purified OGG1, pol ␤, APE1, and Lig I was strongly inhibited by a 5Ј-neighboring T:G, but not by a 5Ј-neighboring 5-meC:G. The inhibition of BER by an adjacent T:G mismatch should reflect not only the decreased excision rate of OGG1, but also the poor primer extension activity of pol ␤ with a mismatch terminus (66). The strand joining activity of Lig I could also be decreased by a mismatched base pair upstream of a matched primer terminus.…”

Conformational Transition Pathway of Polymerase β/DNA upon Binding Correct Incoming Substrate

“…Given the role of pol in maintaining genome integrity and the relation between pol 's malfunction and various diseases (e.g., cancer, premature aging), [50][51][52] pol has attracted many experimental (see ref 53 and references therein) and theoretical investigations. 8,54,[55][56][57] Our prior modeling work of the pol dynamics before and after the chemical step of nucleotidyl transfer reaction using MD 48,[58][59][60][61] and transition path sampling (TPS) 22 employed the CHARMM force field.…”