"Helicase" Activity promoted through dynamic interactions between a ssDNA translocase and a diffusing SSB protein.
"Helicase" Activity promoted through dynamic interactions between a ssDNA translocase and a diffusing SSB protein.
复制标题
“解旋酶”活性通过ssDNA易位酶和扩散的SSB蛋白之间的动态相互作用促进。
DOI：
10.1073/pnas.2216777120
复制
发表时间：
2023-04-11
影响因子：
11.1
通讯作者：
Lohman, Timothy M.
中科院分区：
文献类型：
--
作者：
Mersch, Kacey N.;Sokoloski, Joshua E.;Nguyen, Binh;Galletto, Roberto;Lohman, Timothy M.
RPA是真核生物中一种单链（ss） DNA结合蛋白（SSB），它与基因组维持过程中短暂形成的ssDNA结合，并破坏ssDNA内的DNA二级结构。我们发现Pif1，一个ssDNA转位酶，可以化学机械地推动人类RPA沿着ssDNA进入双工DNA，导致双工DNA的定向解绕。结果证明了RPA获得定向解开DNA的能力的机制，这是一种迄今为止仅归因于解旋酶的活动。这些结果进一步表明，一个过程解旋酶的两个基本功能可以“分离”并由单独的蛋白质提供，一个携带DNA碱基对融化活性，另一个携带（atp酶驱动的）转位酶活性。这为更好地理解解旋酶机制提供了机会。复制蛋白A （RPA）是一种真核生物单链dna结合（SSB）蛋白，对基因组维持的各个方面都至关重要。RPA以高亲和力结合ssDNA，但也可沿ssDNA扩散。就其本身而言，RPA能够通过从双工DNA两侧的ssDNA扩散而短暂地破坏双工DNA的短区域。利用单分子全内反射荧光和光学捕获结合荧光方法，我们发现酿酒酵母Pif1可以利用其依赖于atp的5 ‘到3 ’转位酶活性，以化学力学的方式推动单个人类RPA （hRPA）异源三聚体沿ssDNA方向移动，其速度与Pif1单独转位的速度相当。我们进一步表明，利用其易位活性，Pif1可以将hRPA从ssDNA装载位点推入双工DNA，导致至少9bp的双工DNA稳定中断。这些结果强调了hRPA的动态特性，使其即使与ssDNA紧密结合也能很容易地重组，并证明了一种机制，通过ssDNA转位酶推动SSB蛋白的联合作用，可以实现定向DNA解绕。这些结果强调了任何过程DNA解旋酶的两个基本要求：瞬时DNA碱基对熔化（由hRPA提供）和atp依赖的定向ssDNA易位（由Pif1提供），并且这些功能可以通过使用两个单独的蛋白质来解除连接。
RPA is a single-stranded (ss) DNA-binding protein (SSB) in eukaryotes that binds to ssDNA formed transiently during genome maintenance and disrupts DNA secondary structures within ssDNA. We show that Pif1, a ssDNA translocase, can chemomechanically push human RPA along ssDNA and into duplex DNA, resulting in directional unwinding of duplex DNA. The results demonstrate a mechanism in which RPA gains the ability to unwind DNA directionally, an activity heretofore ascribed only to helicases. These results further show that the two basic functions of a processive helicase can be “unlinked” and provided by separate proteins, one carrying the DNA base pair melting activity and the other carrying the (ATPase-driven) translocase activity. This introduces opportunities to better understand helicase mechanisms. Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-binding (SSB) protein that is essential for all aspects of genome maintenance. RPA binds ssDNA with high affinity but can also diffuse along ssDNA. By itself, RPA is capable of transiently disrupting short regions of duplex DNA by diffusing from a ssDNA that flanks the duplex DNA. Using single-molecule total internal reflection fluorescence and optical trapping combined with fluorescence approaches, we show that S. cerevisiae Pif1 can use its ATP-dependent 5′ to 3′ translocase activity to chemomechanically push a single human RPA (hRPA) heterotrimer directionally along ssDNA at rates comparable to those of Pif1 translocation alone. We further show that using its translocation activity, Pif1 can push hRPA from a ssDNA loading site into a duplex DNA causing stable disruption of at least 9 bp of duplex DNA. These results highlight the dynamic nature of hRPA enabling it to be readily reorganized even when bound tightly to ssDNA and demonstrate a mechanism by which directional DNA unwinding can be achieved through the combined action of a ssDNA translocase that pushes an SSB protein. These results highlight the two basic requirements for any processive DNA helicase: transient DNA base pair melting (supplied by hRPA) and ATP-dependent directional ssDNA translocation (supplied by Pif1) and that these functions can be unlinked by using two separate proteins.
登录
查看更多内容
影响因子：
3.7
作者：
Gibb B;Ye LF;Gergoudis SC;Kwon Y;Niu H;Sung P;Greene EC
通讯作者：
Greene EC
DOI：
10.1073/pnas.2112376119
发表时间：
2022-04-12
影响因子：
11.1
作者：
通讯作者：
--
影响因子：
5.6
作者：
Awate S;Brosh RM Jr
通讯作者：
Brosh RM Jr
影响因子：
11.4
作者：
Baumann, P;West, SC
通讯作者：
West, SC
影响因子：
5.6
作者：
Cheng, W;Hsieh, J;Lohman, TM
通讯作者：
Lohman, TM