Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, Shropshire, SY10 7AG. UK.
J Cell Biochem. 2011 Jun;112(6):1612-21. doi: 10.1002/jcb.23075.
The mismatch repair protein, MSH3, together with MSH2, forms the MutSβ heterodimer which recognizes and repairs base pair mismatches and larger insertion/deletion loops in DNA. Lack of specific antibodies against mouse MSH3 has hampered studies of its expression and localization. Mouse MSH3 is not immunogenic in normal mice. This problem was overcome by immunizing msh3-knockout mice and generating a panel of ten monoclonal antibodies, two of which localize MSH3 specifically in cultured mouse cells and bind to an epitope containing amino-acids 33-37. The panel also includes two antibodies that recognise both mouse and human MSH3 and bind to a conserved epitope containing amino-acids 187-194. The mouse MSH3-specific antibodies show that MSH3 is a nuclear protein with a finely-granular nucleoplasmic distribution, largely absent from areas of condensed heterochromatin. Specificity of the localization was demonstrated by absence of immunostaining in a cell line from the msh3-knockout mouse. Furthermore, we show for the first time that stress treatment of mouse cells with ethanol or hydrogen peroxide caused the re-distribution of MSH3 into nuclear bodies containing the proliferating cell nuclear antigen (PCNA), a known binding partner of MutSβ.
错配修复蛋白 MSH3 与 MSH2 形成 MutSβ 异二聚体，识别和修复 DNA 中的碱基对错配和较大的插入/缺失环。缺乏针对小鼠 MSH3 的特异性抗体阻碍了其表达和定位的研究。在正常小鼠中，小鼠 MSH3 没有免疫原性。通过免疫 msh3 敲除小鼠并生成一组十个单克隆抗体克服了这个问题，其中两个抗体特异性地定位于培养的小鼠细胞中的 MSH3，并与包含氨基酸 33-37 的表位结合。该抗体组还包括两种识别小鼠和人 MSH3 的抗体，并与包含氨基酸 187-194 的保守表位结合。小鼠 MSH3 特异性抗体表明 MSH3 是一种核蛋白，具有精细颗粒状核质分布，主要不存在于浓缩异染色质区域。通过在 msh3 敲除小鼠的细胞系中缺乏免疫染色来证明定位的特异性。此外，我们首次表明，用乙醇或过氧化氢处理小鼠细胞会导致 MSH3 重新分布到含有增殖细胞核抗原 (PCNA) 的核体内，PCNA 是 MutSβ 的已知结合伴侣。
