Background

The RecG DNA helicase plays a crucial role in stalled replication fork rescue. We have recently discovered that interaction of RecG with single-strand DNA binding protein (SSB) remodels RecG, allowing it to spontaneously translocate upstream of the fork. Based on these findings, we hypothesized that mispairing of DNA could limit such translocation of RecG.

Methods

Here, we used atomic force microscopy (AFM) to directly test this hypothesis and investigate how sensitive RecG translocation is to different types of mispairing.

Results

We found that a CC mispairing, at a distance of 30 bp from the fork position, prevents translocation of RecG over this mispairing. A G-bulge, placed at the same distance, also has a similar blocking efficiency. However, a CC mispairing, 10 bp away from the fork, does not prevent RecG translocation beyond 10 bp distance, but decreases complex yield. Modeling of RecG-DNA complexes show that 10 bp distance from the fork is within the binding footprint of RecG on DNA.

Conclusions

Our results suggest that the RecG translocation upstream of the replication fork is limited by mispairings in the parental arm of the replication fork.

General significance

These findings led us to propose dual functions for RecG, in which the thermally driven translocation of RecG can be a mechanism for the additional control of the DNA paring in which RecG can detect the lesions in front of the replication fork, adding to the fidelity of the DNA replication machinery.

中文翻译：

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DNA 错配对 RecG 易位的限制

背景

RecG DNA 解旋酶在停滞的复制叉救援中起着至关重要的作用。我们最近发现 RecG 与单链 DNA 结合蛋白 (SSB) 的相互作用重塑了 RecG，使其能够自发地转移到叉子的上游。基于这些发现，我们假设 DNA 错配可能会限制 RecG 的这种易位。

方法

在这里，我们使用原子力显微镜 (AFM) 直接检验这一假设并研究 RecG 易位对不同类型的错配的敏感程度。

结果

我们发现 C C 错配，距离叉位置 30 bp，防止 RecG 在这个错配上易位。放置在相同距离处的 G 型凸起也具有相似的阻挡效率。然而，距离叉子 10 bp 的 C C 错配不会阻止 RecG 易位超过 10 bp 距离，但会降低复合物产量。RecG-DNA 复合物的建模表明，距叉子 10 bp 的距离在 RecG 对 DNA 的结合足迹内。

结论

我们的结果表明，复制叉上游的 RecG 易位受到复制叉父臂错配的限制。

一般意义

这些发现使我们提出了 RecG 的双重功能，其中 RecG 的热驱动易位可以作为额外控制 DNA 配对的机制，其中 RecG 可以检测复制叉前的损伤，增加了保真度DNA复制机制。