Department of Chemistry, University of California Davis, One Shields Avenue, Davis, CA 95616, USA.
DNA Repair (Amst). 2009 Dec 3;8(12):1400-10. doi: 10.1016/j.dnarep.2009.09.009.
MUTYH-associated polyposis (MAP) is the only inherited colorectal cancer syndrome that is associated with inherited biallelic mutations in a base excision repair gene. The MUTYH glycosylase plays an important role in preventing mutations associated with 8-oxoguanine (OG) by removing adenine residues that have been misincorporated opposite OG. MAP-associated mutations are present throughout MUTYH, with a large number coding for missense variations. To date the available information on the functional properties of MUTYH variants is conflicting. In this study, a kinetic analysis of the adenine glycosylase activity of MUTYH and several variants was undertaken using a correction for active fraction to control for differences due to overexpression and purification. Using these methods, the rate constants for steps involved in the adenine removal process were determined for the MAP variants Y165C, G382D, P391L and Q324R MUTYH. Under single-turnover conditions, the rate of adenine removal for these four variants was found to be 30-40% of WT MUTYH. In addition, the ability of MUTYH and the variants to suppress mutations and complement for the absence of MutY in Escherichia coli was assessed using rifampicin resistance assays. The presence of WT and Q324R MUTYH resulted in complete suppression of the mutation frequency, while G382D MUTYH showed reduced ability to suppress the mutation frequency. In contrast, the mutation frequency observed upon expression of P391L and Y165C MUTYH were similar to the controls, suggesting no activity toward preventing DNA mutations. Notably, though all variations studied herein resulted in similar reductions in adenine glycosylase activity, the effects in the bacterial complementation are quite different. This suggests that the consequences of a specific amino acid variation on overall repair in a cellular context may be magnified.
MUTYH 相关息肉病（MAP）是唯一一种与碱基切除修复基因中的双等位基因突变相关的遗传性结直肠癌综合征。MUTYH 糖苷酶在通过去除与 8-氧鸟嘌呤（OG）错配的腺嘌呤残基来防止与 OG 相关的突变方面发挥重要作用。MAP 相关突变存在于 MUTYH 全基因中，其中大量编码错义变异。迄今为止，有关 MUTYH 变体功能特性的可用信息存在冲突。在这项研究中，使用活性分数校正来控制过表达和纯化引起的差异，对 MUTYH 和几种变体的腺嘌呤糖苷酶活性进行了动力学分析。使用这些方法，确定了涉及腺嘌呤去除过程的步骤的速率常数，用于 MAP 变体 Y165C、G382D、P391L 和 Q324R MUTYH。在单轮条件下，发现这四个变体的腺嘌呤去除速率为 WT MUTYH 的 30-40%。此外，还使用利福平抗性测定评估了 MUTYH 及其变体抑制突变和弥补大肠杆菌中 MutY 缺失的能力。WT 和 Q324R MUTYH 的存在导致突变频率完全抑制，而 G382D MUTYH 显示出降低的抑制突变频率的能力。相比之下，在表达 P391L 和 Y165C MUTYH 时观察到的突变频率与对照相似，表明对防止 DNA 突变没有活性。值得注意的是，虽然本研究中研究的所有变异都导致腺嘌呤糖苷酶活性相似降低，但在细菌互补中的影响却大不相同。这表明在细胞环境中特定氨基酸变异对整体修复的后果可能会被放大。
