title: Identification of critical residues for the tight binding of both correct and incorrect nucleotides to human DNA polymerase λ.
abstract: DNA polymerase λ (Pol λ) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase β. Pre-steady-state kinetic studies have shown that the Pol λ-DNA complex binds both correct and incorrect nucleotides 130-fold tighter, on average, than the DNA polymerase β-DNA complex, although the base substitution fidelity of both polymerases is 10(-)(4) to 10(-5). To better understand Pol λ's tight nucleotide binding affinity, we created single-substitution and double-substitution mutants of Pol λ to disrupt the interactions between active-site residues and an incoming nucleotide or a template base. Single-turnover kinetic assays showed that Pol λ binds to an incoming nucleotide via cooperative interactions with active-site residues (R386, R420, K422, Y505, F506, A510, and R514). Disrupting protein interactions with an incoming correct or incorrect nucleotide impacted binding to each of the common structural moieties in the following order: triphosphate≫base>ribose. In addition, the loss of Watson-Crick hydrogen bonding between the nucleotide and the template base led to a moderate increase in K(d). The fidelity of Pol λ was maintained predominantly by a single residue, R517, which has minor groove interactions with the DNA template.
authors.affiliation: Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Brown
authors.forename: Lindsey R
authors.initials: LR
authors.lastname: Pack
authors.forename: Shanen M
authors.initials: SM
authors.lastname: Sherrer
authors.forename: Ajay K
authors.initials: AK
authors.lastname: Kshetry
authors.forename: Sean A
authors.initials: SA
authors.lastname: Newmister
authors.forename: Jason D
authors.initials: JD
authors.lastname: Fowler
authors.forename: John-Stephen
authors.initials: JS
authors.lastname: Taylor
authors.forename: Zucai
authors.initials: Z
authors.lastname: Suo
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D020029:Base Pairing; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D003854:Deoxyribonucleotides; D050505:Mutant Proteins; D011994:Recombinant Proteins; D004247:DNA; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta