Sen1 and Rrm3 ensure permissive topological conditions for replication termination [ChIP-seq]

Replication forks terminate at TERs and telomeres. Forks that converge or encounter transcription generate topological stress. Combining genetic, genomic and imaging approaches we found that Rrm3hPif1 and Sen1hSenataxin helicases assist termination at TERs, Sen1 at telomeres. rrm3 and sen1 are synthetic lethal, fail to terminate replication exhibiting lagging chromosomes and fragility at TERs and telomeres. sen1 rrm3 build up RNA-DNA hybrids at TERs, sen1 accumulates RNPII at TERs and telomeres. Double mutants exhibit X-shaped gapped or reversed converging forks. Rrm3 and Sen1 restrain Top1 and Top2 activities, preventing toxic accumulation of positive supercoil at TERs and telomeres. We suggest that Rrm3 and Sen1 coordinate the activities of fork-associated Top1 and Top2 with those of gene loop-associated Top1 and Top2 by preventing DNA and RNA polymerases slowing down when forks encounter transcription head-on or codirectionally, respectively. Hence Rrm3 and Sen1 are essential to generate permissive topological conditions for replication termination. Overall design: RNA Pol II_Rpb1-ChIP-seq (S-phase) and bTMP-seq

复制叉在复制终止序列（TER）与端粒处完成终止。相互汇聚或遭遇转录过程的复制叉会产生拓扑应力。本研究结合遗传学、基因组学与成像技术手段，发现Rrm3与Pif1解旋酶、Sen1及Senataxin解旋酶可协助复制终止序列（TER）处的复制终止，其中Sen1负责端粒处的复制终止辅助。rrm3与sen1基因存在合成致死效应，二者缺失会导致复制终止失败，并在TER与端粒处出现滞后染色体与染色体脆性位点。sen1与rrm3的双突变体可在TER处积累RNA-DNA杂交体，而单突变的sen1则会在TER与端粒处聚集RNA聚合酶II（RNPII）。双突变株会呈现出带有缺口的X型结构或反向汇聚的复制叉。Rrm3与Sen1可抑制拓扑异构酶I（Top1）与拓扑异构酶II（Top2）的活性，从而避免TER与端粒处出现有害的正超螺旋积累。我们提出，Rrm3与Sen1可分别通过在复制叉遭遇头对头转录或同向转录时，阻止DNA聚合酶与RNA聚合酶的减速，从而协调与复制叉相关的Top1、Top2与基因环相关的Top1、Top2的活性。因此，Rrm3与Sen1对于构建复制终止所需的宽松拓扑环境至关重要。实验设计概述：RNA聚合酶II Rpb1亚基染色质免疫沉淀测序（RNA Pol II_Rpb1-ChIP-seq，S期样本）与bTMP-seq