Genome Integrity and Structural Biology Laboratory, National Institutes of Health, NIEHS, 111 T.W. Alexander Drive, P.O. Box 12233, Research Triangle Park, North Carolina, NC, 27709, USA.
The MOH Key Laboratory of Geriatrics, Beijing Hospital, National Center of Gerontology, Beijing, 100730, P.R. China.
Sci Rep. 2019 Jul 9;9(1):9928. doi: 10.1038/s41598-019-45846-0.
DNA polymerase (Pol) β is a key enzyme in base excision repair (BER), an important repair system for maintaining genomic integrity. We previously reported the presence of a Pol β transcript containing exon α (105-nucleotide) in normal and colon cancer cell lines. The transcript carried an insertion between exons VI and VII and was predicted to encode a ~42 kDa variant of the wild-type 39 kDa enzyme. However, little is known about the biochemical properties of the exon α-containing Pol β (exon α Pol β) variant. Here, we first obtained evidence indicating expression of the 42 kDa exon α Pol β variant in mouse embryonic fibroblasts. The exon α Pol β variant was then overexpressed in E. coli, purified, and characterized for its biochemical properties. Kinetic studies of exon α Pol β revealed that it is deficient in DNA binding to gapped DNA, has strongly reduced polymerase activity and higher Km for dNTP during gap-filling. On the other hand, the 5'-dRP lyase activity of the exon α Pol β variant is similar to that of wild-type Pol β. These results indicate the exon α Pol β variant is base excision repair deficient, but does conduct 5'-trimming of a dRP group at the gap margin. Understanding the biological implications of this Pol β variant warrants further investigation.
DNA 聚合酶（Pol）β是碱基切除修复（BER）中的关键酶，是维持基因组完整性的重要修复系统。我们之前曾报道过在正常和结肠癌细胞系中存在含有外显子α（105 个核苷酸）的 Pol β 转录本。该转录本在第六和第七外显子之间有一个插入，预测编码野生型 39kDa 酶的~42kDa 变体。然而，对于含有外显子α的 Pol β（外显子α Pol β）变体的生化特性知之甚少。在这里，我们首先获得了在小鼠胚胎成纤维细胞中表达 42kDa 外显子α Pol β 变体的证据。然后，在大肠杆菌中外源过表达外显子α Pol β 变体，并对其生化特性进行了纯化和表征。外显子α Pol β 的动力学研究表明，它在结合缺口 DNA 方面存在缺陷，在缺口填充过程中聚合酶活性和对 dNTP 的 Km 值明显降低。另一方面，外显子α Pol β 变体的 5'-dRP 裂解酶活性与野生型 Pol β 相似。这些结果表明外显子α Pol β 变体碱基切除修复功能缺失，但确实可以在缺口边缘进行 dRP 基团的 5'-修剪。进一步研究这种 Pol β 变体的生物学意义是必要的。
