Prasad R, Beard W A, Wilson S H
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-1068.
J Biol Chem. 1994 Jul 8;269(27):18096-101.
Purified mammalian DNA polymerase beta (beta-pol) fills short gaps of up to 6 nucleotides by a processive mechanism, and this gap-filling activity requires a PO4 group on the 5'-side of the gap (Singhal, R. K., and Wilson, S. H. (1993) J. Biol. Chem. 268, 15906-15911). To assess details of bimolecular binding between beta-pol and a 5-nucleotide (nt) gapped radiolabeled heteropolymeric DNA substrate, beta-pol.DNA complexes were formed, photochemically cross-linked using UV light, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. A 39-nt template was annealed with two 17-mer oligonucleotides, generating a 5-nt gap. The results indicate that beta-pol binds to both the template and primer strands, and binding is strongly enhanced by a 5'-PO4 on the downstream oligonucleotide, even though little cross-linking is observed to this oligonucleotide. The results suggest that beta-pol recognizes the 5'-side of a long single-stranded gap in DNA, provided it contains a 5'-PO4. Additional beta-pol.DNA binding measurements were performed using a competition assay to assess the ability of heteropolymeric DNA to inhibit synthesis on a homopolymeric template-primer system. The results indicate that in addition to the 5'-PO4, the length of the single-stranded template nucleic acid adjacent to the 5'-PO4 is also important for tight binding. Proteolysis of the cross-linked beta-pol.DNA complex with trypsin resulted in a single radiolabeled tryptic product corresponding to nucleic acid cross-linked to the 8-kDa domain. The results demonstrate that the role of the 8-kDa domain is to direct beta-pol binding to the phosphorylated 5'-position in gapped DNA substrates.
纯化的哺乳动物DNA聚合酶β（β-pol）通过一种持续合成机制填补长达6个核苷酸的短缺口，这种缺口填补活性需要在缺口的5'端有一个磷酸基团（辛哈尔，R.K.，和威尔逊，S.H.（1993年）《生物化学杂志》268卷，15906 - 15911页）。为了评估β-pol与一个5核苷酸（nt）缺口的放射性标记杂聚DNA底物之间双分子结合的细节，形成了β-pol.DNA复合物，用紫外光进行光化学交联，并通过SDS - 聚丙烯酰胺凝胶电泳和放射自显影进行分析。将一个39 nt的模板与两条17聚体寡核苷酸退火，产生一个5 nt的缺口。结果表明，β-pol与模板链和引物链都结合，并且下游寡核苷酸上的5'-磷酸基团强烈增强了结合，尽管对该寡核苷酸几乎没有观察到交联。结果表明，β-pol识别DNA中长单链缺口的5'端，前提是它含有一个5'-磷酸基团。使用竞争测定法进行了额外的β-pol.DNA结合测量，以评估杂聚DNA抑制同聚模板 - 引物系统上合成的能力。结果表明，除了5'-磷酸基团外，与5'-磷酸基团相邻的单链模板核酸的长度对于紧密结合也很重要。用胰蛋白酶对交联的β-pol.DNA复合物进行蛋白酶解产生了一个单一的放射性标记胰蛋白酶产物，对应于与8 kDa结构域交联的核酸。结果表明，8 kDa结构域的作用是将β-pol引导至缺口DNA底物中磷酸化的5'位置进行结合。
