Aronshtam A, Marinus M G
Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Nucleic Acids Res. 1996 Jul 1;24(13):2498-504. doi: 10.1093/nar/24.13.2498.
The mutL gene product is part of the dam-directed mismatch repair system of Escherichia coli but has no known enzymatic function. It forms a complex on heteroduplex DNA with the mismatch recognition MutS protein and with MutH, which has latent endonuclease activity. An N-terminal hexahistidine-tagged MutL was constructed which was active in vivo. As a first stop to determine the functional domains of MutL, we have isolated 72 hydroxylamine-induced plasmid-borne mutations which impart a dominant-negative phenotype to the wild-type strain for increased spontaneous mutagenesis. None of the mutations complement a mutL deletion mutant, indicating that the mutant proteins by themselves are inactive. All the dominant mutations but one could be complemented by the wild-type mutL at about the same gene dosage. DNA sequencing indicated that the mutations affected 22 amino acid residues located between positions 16 and 549 of the 615 amino acid protein. In the N-terminal half of the protein, 12 out of 15 amino acid replacements occur at positions conserved in various eukaryotic MutL homologs. All but one of the sequence changes affecting the C-terminal end of the protein are nonsense mutations.
MutL基因产物是大肠杆菌中由dam指导的错配修复系统的一部分，但目前尚无已知的酶促功能。它能与错配识别蛋白MutS以及具有潜在内切核酸酶活性的MutH在异源双链DNA上形成复合物。构建了一种在体内具有活性的N端带有六组氨酸标签的MutL。作为确定MutL功能结构域的第一步，我们分离出了72个由羟胺诱导的质粒携带突变，这些突变赋予野生型菌株显性负性表型，导致自发诱变增加。没有一个突变能互补mutL缺失突变体，这表明突变蛋白自身是无活性的。除了一个突变外，所有显性突变都能在大约相同的基因剂量下被野生型mutL互补。DNA测序表明，这些突变影响了615个氨基酸的蛋白质中位于第16至549位之间的22个氨基酸残基。在该蛋白质的N端一半区域，15个氨基酸替换中有12个发生在各种真核生物MutL同源物中保守的位置。影响该蛋白质C端的序列变化中，除了一个以外，其余都是无义突变。
