UNWINDING OF HETEROLOGOUS DNA BY RECA PROTEIN DURING THE SEARCH FOR HOMOLOGOUS SEQUENCES
UNWINDING OF HETEROLOGOUS DNA BY RECA PROTEIN DURING THE SEARCH FOR HOMOLOGOUS SEQUENCES
复制标题
DOI：
10.1016/0022-2836(92)90129-8
复制
发表时间：
1992-07-05
影响因子：
5.6
通讯作者：
RADDING, CM
中科院分区：
文献类型：
--
作者：
ROULD, E;MUNIYAPPA, K;RADDING, CM
在体外寻找RecA蛋白启动的同源序列涉及突触前细丝和裸露的双链DNA，它们的多次接触产生核蛋白网络或共聚集体。然而，突触前细丝内的单链DNA的轴向间距是OFB形式DNA的1.5倍。为了研究RecA突触前丝和靶双链DNA中碱基间距之间的矛盾差异，我们探索了异种接触对DNA构象的影响，反之亦然。在小麦胚拓扑异构酶I存在的情况下，RecA突触前细丝诱导异源环状双链DNA连接数量的快速、有限的减少。这种异源双链DNA的有限解离，称为异源解离，在30秒内被检测到，并在几分钟内达到稳定状态。在γS存在下形成的突触前细丝，经凝胶过滤从游离RecA蛋白中分离出来，在与松弛的双链DNA和拓扑异构酶孵育时，也产生了一种拓扑异构体梯形。60 mM-NaC l或5 mM-ADP对异源接触的抑制导致异源解离相应减少。相反，与突触前纤维的异源接触的稳定性或数量与环状双链DNA的连接数量呈负相关。这些观察表明，与突触前细丝的异源接触导致双链DNA的有限解离，相反，DNA解离的能力稳定了瞬时的异源接触。
The search for homologous sequences promoted by RecA proteinin vitroinvolves a presynaptic filament and naked duplex DNA, the multiple contacts of which produce nucleoprotein networks or coaggregates. The single-stranded DNA within the presynaptic filaments, however, is extended to an axial spacing 1.5 times that ofB-form DNA. To investigate this paradoxical difference between the spacing of bases in the RecA presynaptic filamentversusthe target duplex DNA, we explored the effect of heterologous contacts on the conformation of DNA, andvice versa. In the presence of wheat germ topoisomerase I, RecA presynaptic filaments induced a rapid, limited reduction in the linking number of heterologous circular duplex DNA. This limited unwinding of heterologous duplex DNA, termed heterologous unwinding, was detected within 30 seconds and reached a steady state within a few minutes. Presynaptic filaments that were formed in the presence of ATPγS and separated from free RecA protein by gel filtration also generated a ladder of topoisomers upon incubation with relaxed duplex DNA and topoisomerase. The inhibition of heterologous contacts by 60 mm-NaCl or 5 mm-ADP resulted in a corresponding decrease in heterologous unwinding. In reciprocal fashion, the stability or number of heterologous contacts with presynaptic filaments was inversely related to the linking number of circular duplex DNA. These observations show that heterologous contacts with the presynaptic filament cause a limited unwinding of the duplex DNA, and conversely that the ability of the DNA to unwind stabilizes transient heterologous contacts.