Sun Zhiqiang, Wang Yaqing, Bianco Piero R, Lyubchenko Yuri L
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198-6025, USA.
Center for Single Molecule Biophysics and Department of Microbiology and Immunology, University at Buffalo, SUNY, Buffalo, NY 14214, USA.
Nanoscale Adv. 2020 Mar 1;2(3):1318-1324. doi: 10.1039/c9na00712a. Epub 2020 Feb 11.
The RecG DNA helicase is a guardian of the bacterial genome where it dominates stalled DNA replication fork rescue. The single-stranded DNA binding protein (SSB) is involved in this process and promotes the binding of RecG to stalled replication forks. Atomic force microscopy (AFM) was used to investigate the interaction of RecG and SSB on a mobile fork substrate capable of being regressed. In the absence of proteins, the fork undergoes spontaneous dynamics between two states defined by the length of the DNA complementarity at the fork. Binding of SSB does not affect these dynamics as it binds to single-stranded regions as expected. In contrast, RecG interacts with the two states quite differently. We demonstrate that RecG has two modes of interaction with fork DNA in the presence of SSB and ATP. In the first mode, RecG translocates over the duplex region and this activity is defined by SSB-mediated remodeling of the helicase. In the second mode, RecG utilizes its helicase activity to regress the fork, in an ATP-dependent manner, displacing SSB on the ssDNA. Overall, our results highlight two functions of RecG that can be employed in the regulation of stalled DNA replication fork rescue.
RecG DNA解旋酶是细菌基因组的守护者，在其中主导停滞的DNA复制叉拯救过程。单链DNA结合蛋白（SSB）参与此过程，并促进RecG与停滞的复制叉结合。原子力显微镜（AFM）用于研究RecG和SSB在能够发生倒退的移动叉状底物上的相互作用。在没有蛋白质的情况下，叉在由叉处DNA互补长度定义的两种状态之间经历自发动态变化。SSB的结合不会影响这些动态变化，因为它如预期那样结合到单链区域。相比之下，RecG与这两种状态的相互作用截然不同。我们证明，在存在SSB和ATP的情况下，RecG与叉状DNA有两种相互作用模式。在第一种模式中，RecG在双链区域上移位，这种活性由SSB介导的解旋酶重塑所定义。在第二种模式中，RecG利用其解旋酶活性以ATP依赖的方式使叉倒退，取代单链DNA上的SSB。总体而言，我们的结果突出了RecG在调节停滞的DNA复制叉拯救中可发挥的两种功能。
