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

Published: 4 February 2021| Version 1 | DOI: 10.17632/35ksks3k3n.1
Contributors:
Sumedha Agashe,

Description

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 resolvase Sgs1-Top3-Rmi1 (STR) complex 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 resolution 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 synergizes with STR and Mus81-Mms4 resolvases for recovery, but not with 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 activity of other DNA resolvases at NPSs.

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Steps to reproduce

As per materials and methods in Agashe et al., 2020 ChIP-qPCR bar graphs were prepared with Excel (plotting mean and standard error bars, showing each datapoint on the graph). The signals for 2D gel images are adjusted with ImageJ, background for spot assays are adjusted with Adobe Photoshop.

Institutions

Fondazione Istituto FIRC di Oncologia Molecolare

Categories

DNA Analysis, Two-Dimensional Gel Electrophoresis, Western Blot, DNA Damage Response

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