Prasad R, Beard W A, Chyan J Y, Maciejewski M W, Mullen G P, Wilson S H
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
J Biol Chem. 1998 May 1;273(18):11121-6. doi: 10.1074/jbc.273.18.11121.
The amino-terminal 8-kDa domain of DNA polymerase beta functions in binding single-stranded DNA (ssDNA), recognition of a 5'-phosphate in gapped DNA structures, and as a 5'-deoxyribose phosphate (dRP) lyase. NMR and x-ray crystal structures of this domain have suggested several residues that may interact with ssDNA or play a role in the dRP lyase reaction. Nine of these residues were altered by site-directed mutagenesis. Each mutant was expressed in Escherichia coli, and the recombinant protein was purified to near homogeneity. CD spectra of these mutant proteins indicated that the alteration did not adversely affect the global protein structure. Single-stranded DNA binding was probed by photochemical cross-linking to oligo(dT)16. Several mutants (F25W, K35A, K60A, and K68A) were impaired in ssDNA binding activity, whereas other mutants (H34G, E71Q, K72A, E75A, and K84A) retained near wild-type binding activity. The 5'-phosphate recognition activity of these mutants was examined by UV cross-linking to a 5-nucleotide gap DNA where the 5' terminus in the gap was either phosphorylated or unphosphorylated. The results indicate that Lys35 is involved in 5'-phosphate recognition of DNA polymerase beta. Finally, the dRP lyase activity of these mutants was evaluated using a preincised apurinic/apyrimidinic DNA. Alanine mutants of Lys35 and Lys60 are significantly reduced in dRP lyase activity, consistent with the lower ssDNA binding activity. More importantly, alanine substitution for Lys72 resulted in a greater than 90% loss of dRP lyase activity, without affecting DNA binding. Alanine mutants of Lys68 and Lys84 had wild-type dRP lyase activity. The triple alanine mutant, K35A/K68A/K72A, was devoid of dRP lyase activity, suggesting that the effects of the alanine substitution at Lys72 and Lys35 were additive. The results suggest that Lys72 is directly involved in formation of a covalent imino intermediate and are consistent with Lys72 as the predominant Schiff base nucleophile in the dRP lyase beta-elimination catalytic reaction.
DNA聚合酶β的氨基末端8 kDa结构域具有结合单链DNA（ssDNA）、识别缺口DNA结构中的5'-磷酸以及作为5'-脱氧核糖磷酸（dRP）裂解酶的功能。该结构域的核磁共振（NMR）和X射线晶体结构表明，有几个残基可能与ssDNA相互作用或在dRP裂解酶反应中发挥作用。通过定点诱变改变了其中9个残基。每个突变体都在大肠杆菌中表达，重组蛋白被纯化至接近均一性。这些突变蛋白的圆二色光谱（CD）表明，这种改变并未对蛋白质的整体结构产生不利影响。通过与寡聚（dT）16进行光化学交联来探测单链DNA结合情况。几个突变体（F25W、K35A、K60A和K68A）的ssDNA结合活性受损，而其他突变体（H34G、E71Q、K72A、E75A和K84A）保留了接近野生型的结合活性。通过与一个5核苷酸缺口DNA进行紫外交联来检测这些突变体的5'-磷酸识别活性，该缺口DNA的5'末端要么被磷酸化，要么未被磷酸化。结果表明，赖氨酸35参与了DNA聚合酶β的5'-磷酸识别。最后，使用预先切割的无嘌呤/无嘧啶DNA评估这些突变体的dRP裂解酶活性。赖氨酸35和赖氨酸60的丙氨酸突变体的dRP裂解酶活性显著降低，这与较低的ssDNA结合活性一致。更重要的是，用丙氨酸取代赖氨酸72导致dRP裂解酶活性丧失超过90%，而不影响DNA结合。赖氨酸68和赖氨酸84的丙氨酸突变体具有野生型dRP裂解酶活性。三重丙氨酸突变体K35A/K68A/K72A没有dRP裂解酶活性，这表明赖氨酸72和赖氨酸35处丙氨酸取代的影响是累加的。结果表明，赖氨酸72直接参与共价亚氨基中间体的形成，并且与赖氨酸72作为dRP裂解酶β-消除催化反应中主要的席夫碱亲核试剂一致。
