UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Nucleic Acids Res. 2021 Aug 20;49(14):8177-8188. doi: 10.1093/nar/gkab591.
The oxidative base damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is a highly mutagenic lesion because replicative DNA polymerases insert adenine (A) opposite 8-oxoG. In mammalian cells, the removal of A incorporated across from 8-oxoG is mediated by the glycosylase MUTYH during base excision repair (BER). After A excision, MUTYH binds avidly to the abasic site and is thus product inhibited. We have previously reported that UV-DDB plays a non-canonical role in BER during the removal of 8-oxoG by 8-oxoG glycosylase, OGG1 and presented preliminary data that UV-DDB can also increase MUTYH activity. In this present study we examine the mechanism of how UV-DDB stimulates MUTYH. Bulk kinetic assays show that UV-DDB can stimulate the turnover rate of MUTYH excision of A across from 8-oxoG by 4-5-fold. Electrophoretic mobility shift assays and atomic force microscopy suggest transient complex formation between MUTYH and UV-DDB, which displaces MUTYH from abasic sites. Using single molecule fluorescence analysis of MUTYH bound to abasic sites, we show that UV-DDB interacts directly with MUTYH and increases the mobility and dissociation rate of MUTYH. UV-DDB decreases MUTYH half-life on abasic sites in DNA from 8800 to 590 seconds. Together these data suggest that UV-DDB facilitates productive turnover of MUTYH at abasic sites during 8-oxoG:A repair.
氧化碱基损伤,8-氧代-7,8-二氢鸟嘌呤(8-oxoG)是一种高度诱变的损伤,因为复制性 DNA 聚合酶会在 8-oxoG 对面插入腺嘌呤(A)。在哺乳动物细胞中,跨 8-oxoG 插入的 A 的去除是由碱基切除修复(BER)过程中的糖苷酶 MUTYH 介导的。在 A 切除后,MUTYH 与无碱基位点紧密结合,因此被产物抑制。我们之前报道过,在 8-氧代鸟嘌呤糖苷酶 OGG1 去除 8-氧代鸟嘌呤的过程中,UV-DDB 发挥了非典型的 BER 作用,并提出了初步数据表明,UV-DDB 还可以增加 MUTYH 的活性。在本研究中,我们研究了 UV-DDB 如何刺激 MUTYH 的机制。批量动力学测定表明,UV-DDB 可以使 MUTYH 切除 8-oxoG 对面 A 的周转率提高 4-5 倍。电泳迁移率变动分析和原子力显微镜表明,MUTYH 与 UV-DDB 之间存在短暂的复合物形成,从而将 MUTYH 从无碱基位点上置换下来。使用单分子荧光分析结合 MUTYH 的无碱基位点,我们表明 UV-DDB 与 MUTYH 直接相互作用,并增加了 MUTYH 的迁移率和离解速率。UV-DDB 将 MUTYH 在 DNA 无碱基位点上的半衰期从 8800 秒缩短至 590 秒。这些数据表明,UV-DDB 促进了 8-oxoG:A 修复过程中 MUTYH 在无碱基位点上的有效周转。
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