RecA protein reinitiates strand exchange on isolated protein-free DNA intermediates. An ADP-resistant process.
RecA protein reinitiates strand exchange on isolated protein-free DNA intermediates. An ADP-resistant process.
复制标题
RecA 蛋白重新启动分离的无蛋白 DNA 中间体上的链交换。
DOI：
10.1016/s0022-2836(05)80264-5
复制
发表时间：
1990
影响因子：
5.6
通讯作者：
Radding,CM
中科院分区：
文献类型：
--
作者：
Rao,BJ;Jwang,B;Radding,CM
线性双链体DNA与包被有RecA蛋白的环状单链（正链）的有效同源配对需要单链被蛋白饱和和延伸。然而，链交换，从双链体DNA的链转移到核蛋白丝，这遵循同源配对，不需要RecA蛋白与单链DNA的稳定结合。当RecA蛋白被添加回分离的无蛋白质的DNA中间体中的存在下，足够的ADP强烈抑制RecA蛋白的单链DNA的结合，链交换仍然恢复在原来的速度，并完成。无蛋白质的DNA中间体的表征表明，它具有RecA蛋白结合的特殊位点或区域。脱蛋白中间体的部分新生取代正链无法作为RecA蛋白ATP酶活性的辅因子，约30%抵抗P1内切核酸酶的消化，P1内切核酸酶优先作用于单链DNA。在链交换完成时，当线性负链的远端5′端完全整合到异源双链DNA中时，核蛋白复合物仍然包含所有三条DNA链，新生的置换链仅在接下来的50至60分钟内解离。该中间产物经脱蛋白处理后得到一种复合物，其中也含有三条DNA链，其中新生的置换链对大肠杆菌核酸外切酶I和P1核酸内切酶都具有部分抗性。脱蛋白复合物在37°C和高到足以熔化双链体DNA的温度之间显示出宽的熔化转变。这些结果表明，链交换可以细分为两个阶段：（1）碱基对的交换，这会产生一个新的异源双链体对来代替亲本对;和（2）链分离，这是未配对链的物理位移来自核蛋白丝。在新的异源双链DNA的产生和第三条链的最终分离之间，存在着一种不寻常的DNA中间体，它可能含有天然DNA的三条链，长度为数千个碱基。
Efficient homologous pairingde novoof linear duplex DNA with a circular single strand (plus strand) coated with RecA protein requires saturation and extension of the single strand by the protein. However, strand exchange, the transfer of a strand from duplex DNA to the nucleoprotein filament, which follows homologous pairing, does not require the stable binding of RecA protein to single-stranded DNA. When RecA protein was added back to isolated protein-free DNA intermediates in the presence of sufficient ADP to inhibit strongly the binding of RecA protein to single-stranded DNA, strand exchange nonetheless resumed at the original rate and went to completion. Characterization of the protein-free DNA intermediate suggested that it has a special site or region to which RecA protein binds. Part of the nascent displaced plus strand of the deproteinized intermediate was unavailable as a cofactor for the ATPase activity of RecA protein, and about 30% resisted digestion by P1endonuclease, which acts preferentially on single-stranded DNA. At the completion of strand exchange, when the distal 5′ end of the linear minus strand had been fully incorporated into heteroduplex DNA, a nucleoprotein complex remained that contained all three strands of DNA from which the nascent displaced strand dissociated only over the next 50 to 60 minutes. Deproteinization of this intermediate yielded a complex that also contained three strands of DNA in which the nascent displaced strand was partially resistant to bothEscherichia coliexonuclease I and P1endonuclease. The deproteinized complex showed a broad melting transition between 37°C and temperatures high enough to melt duplex DNA. These results show that strand exchange can be subdivided into two stages: (1) the exchange of base-pairs, which creates a new heteroduplex pair in place of a parental pair; and (2) strand separation, which is the physical displacement of the unpaired strand from the nucleoprotein filament. Between the creation of new heteroduplex DNA and the eventual separation of a third strand, there exists an unusual DNA intermediate that may contain three-stranded regions of natural DNA that are several thousand bases in length.