Dixon D A, Kowalczykowski S C
Section of Microbiology, University of California, Davis 95616, USA.
J Biol Chem. 1995 Jul 7;270(27):16360-70. doi: 10.1074/jbc.270.27.16360.
Genetic recombination occurring in wild type Escherichia coli is stimulated at DNA sequences known as chi sites, 5'-GCTGGTGG-3'. In vitro, homologous pairing between duplex DNA substrates dependent upon the RecA, RecBCD, and SSB proteins is stimulated by the presence of a chi sequence in the donor linear double-stranded DNA. We show that this stimulation is due to two factors: 1) the enhanced production of chi-specific single-stranded DNA fragments and 2) their preferential use in the RecA protein-promoted pairing step. Furthermore, under conditions of limiting Mg2+ concentration, joint molecule formation does not occur, even though DNA unwinding and chi-specific single-stranded DNA fragment production are observed. Also, under these conditions, chi-specific fragments derived from both the upstream and downstream regions of the DNA strand containing chi and from cleavage of the non-chi-containing DNA strand are detected. Finally, the behavior of mutant RecBCD enzymes (RecBC*D and RecBCD not equal to) in this in vitro reaction is shown to parallel their in vivo phenotypes with respect to chi stimulation of recombination. Thus we suggest that, in addition to its ability to regulate the degradative activities of RecBCD enzyme, chi itself may be a preferred site for initiation of homologous pairing in this concerted process.
野生型大肠杆菌中发生的基因重组在被称为chi位点（5'-GCTGGTGG-3'）的DNA序列处受到刺激。在体外，依赖于RecA、RecBCD和SSB蛋白的双链DNA底物之间的同源配对，会因供体线性双链DNA中chi序列的存在而受到刺激。我们发现这种刺激归因于两个因素：1）chi特异性单链DNA片段产量的增加；2）它们在RecA蛋白促进的配对步骤中的优先使用。此外，在镁离子浓度受限的条件下，即使观察到DNA解旋和chi特异性单链DNA片段的产生，也不会形成联合分子。同样，在这些条件下，可检测到来自含有chi的DNA链的上游和下游区域以及不含chi的DNA链切割产生的chi特异性片段。最后，在这个体外反应中，突变型RecBCD酶（RecBC*D和RecBCD不等）的行为在chi对重组的刺激方面与其体内表型平行。因此我们认为，除了其调节RecBCD酶降解活性的能力外，chi本身可能是这个协同过程中同源配对起始的优选位点。
