Smc5/6 functions with Sgs1-Top3-Rmi1 to complete chromosome replication at natural pause sites

Smc5/6 is essential for genome structural integrity by yet unknown mechanisms. Here we find that Smc5/6 co-localizes with the DNA crossed-strand processing complex Sgs1-Top3-Rmi1 (STR) at genomic regions known as natural pausing sites (NPSs) where it facilitates Top3 retention. Individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (JMs) composed of reversed forks, double Holliday Junctions and hemicatenanes, indicative of Smc5/6 regulating Sgs1 and Top3 DNA processing activities. We isolate an intra-allelic suppressor of smc6-56 proficient in Top3 retention but affected in pathways that act complementarily with Sgs1 and Top3 to resolve JMs arising at replication termination. Upon replication stress, the smc6-56 suppressor requires STR and Mus81-Mms4 functions for recovery, but not Srs2 and Mph1 helicases that prevent maturation of recombination intermediates. Thus, Smc5/6 functions jointly with Top3 and STR to mediate replication completion and influences the function of other DNA crossed-strand processing enzymes at NPSs.Smc5/6（Smc5/6）对于维持基因组结构完整性至关重要，但其具体作用机制迄今尚未阐明。本研究发现，Smc5/6可与DNA交叉链加工复合物Sgs1-Top3-Rmi1（STR）共定位于已知的自然暂停位点（natural pausing sites, NPSs），并在该区域促进Top3蛋白的驻留。单独缺失STR亚基或Smc5/6均会导致由反转叉、双Holliday联结体（double Holliday Junctions）与半连环（hemicatenanes）组成的联合分子（joint molecules, JMs）出现类似的异常积累，这提示Smc5/6可调控Sgs1与Top3的DNA加工活性。我们成功分离得到smc6-56的基因内抑制突变体，该突变体能够正常维持Top3的驻留，但在与Sgs1和Top3协同解决复制终止过程中产生的联合分子的通路中存在功能缺陷。在复制应激条件下，smc6-56抑制突变体的恢复过程需要STR与Mus81-Mms4复合物发挥功能，而无需Srs2与Mph1解旋酶（helicases）——这两类解旋酶可阻止重组中间体的成熟。综上，Smc5/6可与Top3及STR协同作用，介导复制过程的顺利完成，并影响自然暂停位点处其他DNA交叉链加工酶的功能。