MRC Laboratory of Molecular Biology, Cambridge, UK.
Sir William Dunn School of Pathology, Oxford, UK.
EMBO J. 2019 Feb 1;38(3). doi: 10.15252/embj.201899793. Epub 2018 Nov 26.
During DNA replication, conflicts with ongoing transcription are frequent and require careful management to avoid genetic instability. R-loops, three-stranded nucleic acid structures comprising a DNA:RNA hybrid and displaced single-stranded DNA, are important drivers of damage arising from such conflicts. How R-loops stall replication and the mechanisms that restrain their formation during S phase are incompletely understood. Here, we show how R-loop formation drives a short purine-rich repeat, (GAA), to become a replication impediment that engages the repriming activity of the primase-polymerase PrimPol. Further, the absence of PrimPol leads to significantly increased R-loop formation around this repeat during S phase. We extend this observation by showing that PrimPol suppresses R-loop formation in genes harbouring secondary structure-forming sequences, exemplified by G quadruplex and H-DNA motifs, across the genome in both avian and human cells. Thus, R-loops promote the creation of replication blocks at susceptible structure-forming sequences, while PrimPol-dependent repriming limits the extent of unscheduled R-loop formation at these sequences, mitigating their impact on replication.
在 DNA 复制过程中，与正在进行的转录相冲突的情况经常发生，需要谨慎处理以避免遗传不稳定性。R 环，由 DNA:RNA 杂交和取代的单链 DNA 组成的三链核酸结构，是由这些冲突引起的损伤的重要驱动因素。R 环如何导致复制停滞，以及在 S 期限制其形成的机制尚不完全清楚。在这里，我们展示了 R 环的形成如何驱动一个短的嘌呤丰富重复序列（GAA）成为复制障碍，从而使引发酶-聚合酶 PrimPol 的重新引发活性参与其中。此外，PrimPol 的缺失会导致在 S 期期间，这个重复序列周围的 R 环形成显著增加。我们通过展示 PrimPol 抑制基因组中形成二级结构的序列（如 G 四联体和 H-DNA 基序）中的 R 环形成，进一步扩展了这一观察结果，这在禽类和人类细胞中都得到了体现。因此，R 环促进了在易感结构形成序列处创建复制障碍，而 PrimPol 依赖性的重新引发限制了这些序列处未计划的 R 环形成的程度，从而减轻了它们对复制的影响。
