Spies Maria, Bianco Piero R, Dillingham Mark S, Handa Naofumi, Baskin Ronald J, Kowalczykowski Stephen C
Section of Microbiology, Center for Genetics and Development, University of California, Davis 9561, USA.
Cell. 2003 Sep 5;114(5):647-54. doi: 10.1016/s0092-8674(03)00681-0.
RecBCD enzyme is a heterotrimeric helicase/nuclease that initiates homologous recombination at double-stranded DNA breaks. Several of its activities are regulated by the DNA sequence chi (5'-GCTGGTGG-3'), which is recognized in cis by the translocating enzyme. When RecBCD enzyme encounters chi, the intensity and polarity of its nuclease activity are changed, and the enzyme gains the ability to load RecA protein onto the chi-containing, unwound single-stranded DNA. Here, we show that interaction with chi also affects translocation by RecBCD enzyme. By observing translocation of individual enzymes along single molecules of DNA, we could see RecBCD enzyme pause precisely at chi. Furthermore, and more unexpectedly, after pausing at chi, the enzyme continues translocating but at approximately one-half the initial rate. We propose that interaction with chi results in an enzyme in which one of the two motor subunits, likely the RecD motor, is uncoupled from the holoenzyme to produce the slower translocase.
RecBCD酶是一种异源三聚体解旋酶/核酸酶,它在双链DNA断裂处启动同源重组。其多种活性受DNA序列chi(5'-GCTGGTGG-3')调控,移位酶可顺式识别该序列。当RecBCD酶遇到chi时,其核酸酶活性的强度和极性会发生变化,并且该酶获得了将RecA蛋白加载到含chi的解旋单链DNA上的能力。在此,我们表明与chi的相互作用也会影响RecBCD酶的移位。通过观察单个酶沿单分子DNA的移位,我们可以看到RecBCD酶在chi处精确暂停。此外,更出乎意料的是,在chi处暂停后,该酶继续移位,但速度约为初始速度的一半。我们提出与chi的相互作用导致一种酶,其中两个运动亚基之一,可能是RecD运动亚基,与全酶解偶联,从而产生较慢的转位酶。