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The Pol β variant containing exon α is deficient in DNA polymerase but has full dRP lyase activity.
Scientific Reports ( IF 3.8 ) Pub Date : 2019-07-09 , DOI: 10.1038/s41598-019-45846-0 Da-Peng Dai 1, 2 , Rajendra Prasad 1 , Phyllis R Strauss 1, 3 , Samuel H Wilson 1
Scientific Reports ( IF 3.8 ) Pub Date : 2019-07-09 , DOI: 10.1038/s41598-019-45846-0 Da-Peng Dai 1, 2 , Rajendra Prasad 1 , Phyllis R Strauss 1, 3 , Samuel H Wilson 1
Affiliation
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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.
中文翻译：
含有外显子α的Polβ变异体缺乏DNA聚合酶，但具有完整的dRP裂解酶活性。
DNA聚合酶（Pol）β是碱基切除修复（BER）中的关键酶，BER是维持基因组完整性的重要修复系统。我们先前报道了在正常和结肠癌细胞系中含有外显子α（105个核苷酸）的Polβ转录物的存在。转录本在外显子VI和VII之间插入，并被预测为编码野生型39 kDa酶的〜42 kDa变异体。然而，关于含外显子α的Polβ（外显子αPolβ）变体的生化特性知之甚少。在这里，我们首先获得证据表明42kDa外显子αPolβ变异体在小鼠胚胎成纤维细胞中表达。然后将外显子αPolβ变体在大肠杆菌中过表达，纯化并对其生化特性进行表征。对外显子αPolβ的动力学研究表明，它缺乏与缺口DNA的DNA结合，在填补缺口期间，dNTP的聚合酶活性大大降低，而dNTP的Km更高。另一方面，外显子αPolβ变体的5'-dRP裂解酶活性与野生型Polβ相似。这些结果表明外显子αPolβ变异体是碱基切除修复缺陷，但确实在间隙边缘对dRP组进行了5'修饰。了解此Polβ变异体的生物学含义值得进一步研究。
更新日期：2019-07-09
中文翻译：
含有外显子α的Polβ变异体缺乏DNA聚合酶，但具有完整的dRP裂解酶活性。
DNA聚合酶（Pol）β是碱基切除修复（BER）中的关键酶，BER是维持基因组完整性的重要修复系统。我们先前报道了在正常和结肠癌细胞系中含有外显子α（105个核苷酸）的Polβ转录物的存在。转录本在外显子VI和VII之间插入，并被预测为编码野生型39 kDa酶的〜42 kDa变异体。然而，关于含外显子α的Polβ（外显子αPolβ）变体的生化特性知之甚少。在这里，我们首先获得证据表明42kDa外显子αPolβ变异体在小鼠胚胎成纤维细胞中表达。然后将外显子αPolβ变体在大肠杆菌中过表达，纯化并对其生化特性进行表征。对外显子αPolβ的动力学研究表明，它缺乏与缺口DNA的DNA结合，在填补缺口期间，dNTP的聚合酶活性大大降低，而dNTP的Km更高。另一方面，外显子αPolβ变体的5'-dRP裂解酶活性与野生型Polβ相似。这些结果表明外显子αPolβ变异体是碱基切除修复缺陷，但确实在间隙边缘对dRP组进行了5'修饰。了解此Polβ变异体的生物学含义值得进一步研究。
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