Abstract

The vast majority of the genome is transcribed by RNA polymerases. G+C-rich regions of the chromosomes and negative superhelicity can promote the invasion of the DNA by RNA to form R-loops, which have been shown to block DNA replication and promote genome instability. However, it is unclear whether the R-loops themselves are sufficient to cause this instability or if additional factors are required. We have investigated replisome collisions with transcription complexes and R-loops using a reconstituted bacterial DNA replication system. RNA polymerase transcription complexes co-directionally oriented with the replication fork were transient blockages, whereas those oriented head-on were severe, stable blockages. On the other hand, replisomes easily bypassed R-loops on either template strand. Replication encounters with R-loops on the leading-strand template (co-directional) resulted in gaps in the nascent leading strand, whereas lagging-strand template R-loops (head-on) had little impact on replication fork progression. We conclude that whereas R-loops alone can act as transient replication blocks, most genome-destabilizing replication fork stalling likely occurs because of proteins bound to the R-loops.

中文翻译：

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转录复合体和R环的复制体旁路。

摘要

RNA聚合酶可转录绝大多数基因组。富含G + C的染色体区域和负超螺旋现象可促进RNA入侵DNA形成R环，R环已被证明可阻止DNA复制并促进基因组不稳定。但是，不清楚R环本身是否足以引起这种不稳定性，或者是否需要其他因素。我们已经研究了使用重组细菌DNA复制系统的转录复合物与R环的复制体碰撞。与复制叉同向定向的RNA聚合酶转录复合物是暂时性阻滞，而正面逆转的是严重，稳定的阻滞。另一方面，复制体容易绕过任一模板链上的R环。前导链模板上的R环（同向）的复制遇到导致新生的前导链中的缺口，而滞后链模板R环（正面）对复制叉的进展影响很小。我们得出的结论是，尽管单独的R环可以充当瞬时复制块，但大多数基因组不稳定的复制叉停转很可能是由于与R环结合的蛋白质引起的。