title: Evidence for a Watson-Crick hydrogen bonding requirement in DNA synthesis by human DNA polymerase kappa.
abstract: The efficiency and fidelity of nucleotide incorporation by high-fidelity replicative DNA polymerases (Pols) are governed by the geometric constraints imposed upon the nascent base pair by the active site. Consequently, these polymerases can efficiently and accurately replicate through the template bases which are isosteric to natural DNA bases but which lack the ability to engage in Watson-Crick (W-C) hydrogen bonding. DNA synthesis by Poleta, a low-fidelity polymerase able to replicate through DNA lesions, however, is inhibited in the presence of such an analog, suggesting a dependence of this polymerase upon W-C hydrogen bonding. Here we examine whether human Polkappa, which differs from Poleta in having a higher fidelity and which, unlike Poleta, is inhibited at inserting nucleotides opposite DNA lesions, shows less of a dependence upon W-C hydrogen bonding than does Poleta. We find that an isosteric thymidine analog is replicated with low efficiency by Polkappa, whereas a nucleobase analog lacking minor-groove H bonding potential is replicated with high efficiency. These observations suggest that both Poleta and Polkappa rely on W-C hydrogen bonding for localizing the nascent base pair in the active site for the polymerization reaction to occur, thus overcoming these enzymes' low geometric selectivity.
authors.affiliation: Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, TX 77555-1061, USA.
authors.forename: William T
authors.initials: WT
authors.lastname: Wolfle
authors.forename: M Todd
authors.initials: MT
authors.lastname: Washington
authors.forename: Eric T
authors.initials: ET
authors.lastname: Kool
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Spratt
authors.forename: Sandra A
authors.initials: SA
authors.lastname: Helquist
authors.forename: Louise
authors.initials: L
authors.lastname: Prakash
authors.forename: Satya
authors.initials: S
authors.lastname: Prakash
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006147:Guanine / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013936:Thymidine / Q000737:chemistry; D014050:Toluene / Q000031:analogs & derivatives / Q000737:chemistry; D000099270:Y-Family DNA Polymerases
chemical_list: D004247:DNA; D004259:DNA-Directed DNA Polymerase; D006147:Guanine; D013936:Thymidine; D014050:Toluene; C110527:difluorotoluene; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases