Fork-Remodeling Helicase Rad5 Preferentially Reverses Replication Forks with Gaps in the Leading Strand.
Fork-Remodeling Helicase Rad5 Preferentially Reverses Replication Forks with Gaps in the Leading Strand.
复制标题
分叉重塑解旋酶 Rad5 优先逆转前导链中存在缺口的复制叉。
DOI：
10.1016/j.jmb.2023.167946
复制
发表时间：
2023
影响因子：
5.6
通讯作者：
Washington,MTodd
中科院分区：
文献类型：
--
作者：
Ling,JustinA;Gildenberg,MelissaS;Honda,Masayoshi;Kondratick,ChristineM;Spies,Maria;Washington,MTodd
当复制叉在DNA损伤位点停止时，DNA损伤旁路通路促进受损DNA的复制。模板转换是一种DNA损伤旁路通路，其中叉形反转解旋酶将停滞的复制叉转化为四向DNA连接，称为鸡爪中间体，随后由复制DNA聚合酶延长。在酵母中，Rad5解旋酶以一种未知的机制进行叉型逆转。为了更好地了解Rad5的机制及其对不同分叉DNA底物的特异性，我们使用基于fret的检测方法实时观察分叉逆转。我们检测了Rad5在前导链或滞后链中存在短间隙以及滞后链中存在或不存在RPA和RNA引物的情况下结合和催化各种叉DNA底物逆转的能力。我们发现Rad5优先逆转分叉DNA底物，在前导链上有短间隙（10到30 nt.）。因此，Rad5优先逆转叉子DNA底物，形成具有5 '悬垂的鸡爪中间体，可以在模板切换的后续步骤中被复制DNA聚合酶延长。
DNA damage bypass pathways promote the replication of damaged DNA when replication forks stall at sites of DNA damage. Template switching is a DNA damage bypass pathway in which fork-reversal helicases convert stalled replication forks into four-way DNA junctions called chicken foot intermediates, which are subsequently extended by replicative DNA polymerases. In yeast, fork-reversal is carried out by the Rad5 helicase using an unknown mechanism. To better understand the mechanism of Rad5 and its specificity for different fork DNA substrates, we used a FRET-based assay to observe fork reversal in real time. We examined the ability of Rad5 to bind and catalyze the reversal of various fork DNA substrates in the presence of short gaps in the leading or lagging strand as well as in the presence or absence of RPA and RNA primers in the lagging strand. We found that Rad5 preferentially reverses fork DNA substrates with short gaps (10 to 30 nt.) in the leading strand. Thus, Rad5 preferentially reverses fork DNA substrates that form chicken foot intermediates with 5′ overhangs that can be extended by replicative DNA polymerases during the subsequent steps of template switching.