Adenine removal activity and bacterial complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer.
Adenine removal activity and bacterial complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer.
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DOI：
10.1016/j.dnarep.2009.09.009
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发表时间：
2009-12-03
期刊：
影响因子：
3.8
通讯作者：
David SS
中科院分区：
文献类型：
--
作者：
Kundu S;Brinkmeyer MK;Livingston AL;David SS
MUTYH相关息肉病（MAP）是唯一一种与碱基切除修复基因中的遗传性双等位基因突变相关的遗传性结直肠癌综合征。MUTYH糖基化酶通过去除已被错误掺入相对于OG的腺嘌呤残基，在防止与8-氧代鸟嘌呤（OG）相关的突变中起重要作用。MAP相关突变存在于整个MUTYH中，其中大量编码错义变异。迄今为止，关于MUTYH变体的功能特性的可用信息是相互矛盾的。在这项研究中，MUTYH和几种变体的腺嘌呤糖基化酶活性的动力学分析进行了使用校正的活性级分，以控制由于过度表达和纯化的差异。使用这些方法，测定MAP变体Y165 C、G382 D、P391 L和Q324 R MUTYH的腺嘌呤去除过程中涉及的步骤的速率常数。在单转换条件下，发现这四种变体的腺嘌呤去除率为WT MUTYH的30-40%。此外，MUTYH和变体抑制突变和补充大肠杆菌中MutY缺失的能力也得到了证实。使用利福平抗性测定评估大肠杆菌。WT和Q324 R MUTYH的存在导致突变频率的完全抑制，而G382 D MUTYH显示抑制突变频率的能力降低。相反，在P391 L和Y165 C MUTYH表达时观察到的突变频率与对照相似，表明没有防止DNA突变的活性。值得注意的是，尽管本文研究的所有变化导致腺嘌呤糖基化酶活性的类似降低，但细菌互补中的效果是完全不同的。这表明，在细胞环境中，特定氨基酸变异对整体修复的影响可能会被放大。
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 E. 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.
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影响因子：
64.8
作者：
Fromme, JC;Banerjee, A;Verdine, GL
通讯作者：
Verdine, GL
影响因子：
9.7
作者：
Bai, Haibo;Grist, Scott;Lu, A-Lien
通讯作者：
Lu, A-Lien
影响因子：
2.9
作者：
Krishnamurthy, Nirmala;Zhao, Xiaobei;David, Sheila S.
通讯作者：
David, Sheila S.
影响因子：
14.8
作者：
Livingston, Alison L.;O'Shea, Valerie L.;David, Sheila S.
通讯作者：
David, Sheila S.
DOI：
10.1016/0027-5107(91)90157-j
发表时间：
1991-09-01
期刊：
MUTATION RESEARCH
影响因子：
--
作者：
AMES, BN;GOLD, LS
通讯作者：
GOLD, LS