Human HELB is a processive motor protein that catalyzes RPA clearance from single-stranded DNA.
Human HELB is a processive motor protein that catalyzes RPA clearance from single-stranded DNA.
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DOI：
10.1073/pnas.2112376119
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发表时间：
2022-04-12
影响因子：
11.1
通讯作者：
中科院分区：
文献类型：
--
作者：
单链DNA （ssDNA）是许多细胞DNA交易的关键中间体，包括DNA复制、修复和重组。新生的ssDNA被复制蛋白A （RPA）复合体快速结合，形成核蛋白丝，稳定ssDNA并介导下游加工事件。然而，矛盾的是，RPA对ssDNA的高亲和力可能会阻止进一步因子的募集。在这项工作中，我们表明RPA-ssDNA核蛋白丝是人类HELB解旋酶的特异性靶向。RPA - ssdna募集HELB激活HELB易位活性，导致上游RPA复合物的过程去除。这种RPA清除活性可能支持HELB在复制和重组中的多种作用。人DNA解旋酶B （Human DNA helicase B， HELB）是一种特性较差的解旋酶，在DNA复制和重组过程中发挥正调控和负调控作用。在这项工作中，我们使用散装和单分子方法来表征HELB蛋白的生化活性，特别关注其与复制蛋白a （RPA）和RPA -单链DNA （ssDNA）细丝的相互作用。HELB是一种与ssDNA紧密结合的单体蛋白，位点大小约为20个核苷酸。它将ATP水解与ssDNA沿5 ‘至3 ’方向的易位结合在一起，并形成DNA环。HELB也表现出经典的解旋酶活性，但在没有辅助力的情况下，这种活性非常弱。HELB特异性结合人RPA，增强其atp酶和ssDNA转位酶活性，但抑制DNA解绕。在RPA核蛋白细丝上直接观察HELB表明，转移HELB会同时清除RPA中的ssDNA。这种活性允许其他蛋白质进入ssDNA中间体，尽管它们被RPA屏蔽，可能支持HELB在细胞DNA交易中的多种作用。
Single-stranded DNA (ssDNA) is a key intermediate in many cellular DNA transactions, including DNA replication, repair, and recombination. Nascent ssDNA is rapidly bound by the Replication Protein A (RPA) complex, forming a nucleoprotein filament that both stabilizes ssDNA and mediates downstream processing events. Paradoxically, however, the very high affinity of RPA for ssDNA may block the recruitment of further factors. In this work, we show that RPA–ssDNA nucleoprotein filaments are specifically targeted by the human HELB helicase. Recruitment of HELB by RPA–ssDNA activates HELB translocation activity, leading to processive removal of upstream RPA complexes. This RPA clearance activity may underpin the diverse roles of HELB in replication and recombination. Human DNA helicase B (HELB) is a poorly characterized helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single-molecule approaches to characterize the biochemical activities of HELB protein with a particular focus on its interactions with Replication Protein A (RPA) and RPA–single-stranded DNA (ssDNA) filaments. HELB is a monomeric protein that binds tightly to ssDNA with a site size of ∼20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5′ to 3′ direction accompanied by the formation of DNA loops. HELB also displays classical helicase activity, but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA, which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from ssDNA. This activity, which can allow other proteins access to ssDNA intermediates despite their shielding by RPA, may underpin the diverse roles of HELB in cellular DNA transactions.
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影响因子：
3.3
作者：
Bell SP;Labib K
通讯作者：
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影响因子：
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作者：
Luzzietti N;Brutzer H;Klaue D;Schwarz FW;Staroske W;Clausing S;Seidel R
通讯作者：
Seidel R
DOI：
10.1073/pnas.2020185118
发表时间：
2021-02-23
影响因子：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
Brosh RM Jr