Kim Seung-Hwan, Pytlos Małgorzata J, Sinden Richard R
Laboratory of DNA Structure and Mutagenesis, Center for Genome Research, Institute of Biosciences and Technology, Texas A&M University System Health Science Center, 2121 West Holcombe Blvd., Houston, TX 77030-3303, USA.
Mutat Res. 2006 Mar 20;595(1-2):5-22. doi: 10.1016/j.mrfmmm.2005.07.010.
(CTG)n.(CAG)n repeats undergo deletion at a high rate in plasmids in Escherichia coli in a process that involves RecA and RecB. In addition, DNA replication fork progression can be blocked during synthesis of (CTG)n.(CAG)n repeats. Replication forks stalled at (CTG)n.(CAG)n repeats may be rescued by replication restart that involves recombination as well as enzymes involved in replication and DNA repair, and this process may be responsible for the high rate of repeat deletion in E. coli. To test this hypothesis (CAG)n.(CTG)n deletion rates were measured in several E. coli strains carrying mutations involved in replication restart. (CAG)n.(CTG)n deletion rates were decreased, relative to the rates in wild type cells, in strains containing mutations in priA, recG, ruvAB, and recO. Mutations in priB and priC resulted in small reductions in deletion rates. In a recF strain, rates were decreased when (CAG)n comprised the leading template strand, but rates were increased when (CTG)n comprised the leading template. Deletion rates were increased slightly in a recJ strain. The mutational spectra for most mutant strains were altered relative to those in parental strains. In addition, purified PriA and RecG proteins showed unexpected binding to single-stranded, duplex, and forked DNAs containing (CAG)n and/or (CTG)n loop-outs in various positions. The results presented are consistent with an interpretation that the high rates of trinucleotide repeat instability observed in E. coli result from the attempted restart of replication forks stalled at (CAG)n.(CTG)n repeats.
(CTG)n.(CAG)n重复序列在大肠杆菌质粒中以较高频率发生缺失，该过程涉及RecA和RecB。此外，在(CTG)n.(CAG)n重复序列合成过程中，DNA复制叉的前进可能会受阻。停滞在(CTG)n.(CAG)n重复序列处的复制叉可能通过涉及重组以及复制和DNA修复相关酶的复制重启得以挽救，而这一过程可能是大肠杆菌中重复序列高缺失率的原因。为验证这一假设，在几种携带参与复制重启相关突变的大肠杆菌菌株中测量了(CAG)n.(CTG)n的缺失率。相对于野生型细胞中的缺失率，在priA、recG、ruvAB和recO发生突变的菌株中，(CAG)n.(CTG)n的缺失率降低。priB和priC的突变导致缺失率略有降低。在recF菌株中，当(CAG)n作为前导模板链时缺失率降低，但当(CTG)n作为前导模板时缺失率升高。在recJ菌株中缺失率略有升高。大多数突变菌株的突变谱相对于亲本菌株发生了改变。此外，纯化的PriA和RecG蛋白对含有位于不同位置的(CAG)n和/或(CTG)n环出结构的单链、双链和叉状DNA表现出意外的结合。所呈现的结果与一种解释一致，即在大肠杆菌中观察到的三核苷酸重复序列的高不稳定性是由于停滞在(CAG)n.(CTG)n重复序列处的复制叉试图重启所致。
