Department of Biology, Tufts University, Medford, MA 02155, USA.
Genes (Basel). 2022 Mar 8;13(3):474. doi: 10.3390/genes13030474.
Repair of DNA double-strand breaks by homologous recombination (HR) requires a carefully orchestrated sequence of events involving many proteins. One type of HR, synthesis-dependent strand annealing (SDSA), proceeds via the formation of a displacement loop (D-loop) when RAD51-coated single-stranded DNA invades a homologous template. The 3' end of the single-stranded DNA is extended by DNA synthesis. In SDSA, the D-loop is then disassembled prior to strand annealing. While many helicases can unwind D-loops in vitro, how their action is choreographed in vivo remains to be determined. To clarify the roles of various DNA helicases during SDSA, we used a double-strand gap repair assay to study the outcomes of homologous recombination repair in lacking the BLM, HELQ, and FANCM helicases. We found that the absence of any of these three helicases impairs gap repair. In addition, flies lacking both BLM and HELQ or HELQ and FANCM had more severe SDSA defects than the corresponding single mutants. In the absence of BLM, a large percentage of repair events were accompanied by flanking deletions. Strikingly, these deletions were mostly abolished in the and double mutants. Our results suggest that the BLM, HELQ, and FANCM helicases play distinct roles during SDSA, with HELQ and FANCM acting early to promote the formation of recombination intermediates that are then processed by BLM to prevent repair by deletion-prone mechanisms.
同源重组 (HR) 修复 DNA 双链断裂需要一系列精心协调的事件，涉及许多蛋白质。HR 的一种类型，即依赖合成的链退火 (SDSA)，当 RAD51 包裹的单链 DNA 入侵同源模板时，通过形成置换环 (D-loop) 进行。单链 DNA 的 3' 端通过 DNA 合成延伸。在 SDSA 中，D-loop 先于链退火解组装。虽然许多解旋酶可以在体外解开 D-loop，但它们在体内的作用如何协调仍有待确定。为了阐明 SDSA 过程中各种 DNA 解旋酶的作用，我们使用双链缺口修复测定法研究了 BLM、HELQ 和 FANCM 解旋酶缺失的同源重组修复的结果。我们发现，这三种解旋酶中的任何一种缺失都会损害缺口修复。此外，BLM 和 HELQ 或 HELQ 和 FANCM 缺失的果蝇比相应的单突变体具有更严重的 SDSA 缺陷。在 BLM 缺失的情况下，很大一部分修复事件伴随着侧翼缺失。引人注目的是，这些缺失在 BLM 和 缺失的双突变体中大部分被消除。我们的结果表明，BLM、HELQ 和 FANCM 解旋酶在 SDSA 中发挥不同的作用，HELQ 和 FANCM 早期发挥作用，促进重组中间体的形成，然后由 BLM 处理，以防止通过易发生缺失的机制进行修复。
