Tseng Quincy, Orans Jillian, Hast Michael A, Iyer Ravi R, Changela Anita, Modrich Paul L, Beese Lorena S
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Aug 1;67(Pt 8):947-52. doi: 10.1107/S1744309111019300. Epub 2011 Jul 26.
MutSβ is a eukaryotic mismatch repair protein that preferentially targets extrahelical unpaired nucleotides and shares partial functional redundancy with MutSα (MSH2-MSH6). Although mismatch recognition by MutSα has been shown to involve a conserved Phe-X-Glu motif, little is known about the lesion-binding mechanism of MutSβ. Combined MSH3/MSH6 deficiency triggers a strong predisposition to cancer in mice and defects in msh2 and msh6 account for roughly half of hereditary nonpolyposis colorectal cancer mutations. These three MutS homologs are also believed to play a role in trinucleotide repeat instability, which is a hallmark of many neurodegenerative disorders. The baculovirus overexpression and purification of recombinant human MutSβ and three truncation mutants are presented here. Binding assays with heteroduplex DNA were carried out for biochemical characterization. Crystallization and preliminary X-ray diffraction analysis of the protein bound to a heteroduplex DNA substrate are also reported.
MutSβ是一种真核错配修复蛋白，它优先靶向螺旋外未配对核苷酸，并与MutSα（MSH2 - MSH6）存在部分功能冗余。尽管已表明MutSα识别错配涉及保守的Phe - X - Glu基序，但对MutSβ的损伤结合机制知之甚少。MSH3/MSH6联合缺陷会引发小鼠强烈的癌症易感性，而msh2和msh6的缺陷约占遗传性非息肉病性结直肠癌突变的一半。这三种MutS同源物也被认为在三核苷酸重复序列不稳定性中起作用，而三核苷酸重复序列不稳定性是许多神经退行性疾病的一个标志。本文介绍了重组人MutSβ及其三个截短突变体的杆状病毒过表达和纯化。进行了与异源双链DNA的结合测定以进行生化表征。还报道了与异源双链DNA底物结合的蛋白质的结晶和初步X射线衍射分析。
