Rescue of collapsed replication forks is dependent on NSMCE2 to prevent mitotic DNA damage

Kelvin W. Pond; Christelle de Renty; Mary K. Yagle; Nathan A. Ellis

doi:10.1371/journal.pgen.1007942

Rescue of collapsed replication forks is dependent on NSMCE2 to prevent mitotic DNA damage

Kelvin W. Pond, Christelle de Renty, Mary K. Yagle, Nathan A. Ellis

Research output: Contribution to journal › Article › peer-review

24 Scopus citations

Abstract

NSMCE2 is an E3 SUMO ligase and a subunit of the SMC5/6 complex that associates with the replication fork and protects against genomic instability. Here, we study the fate of collapsed replication forks generated by prolonged hydroxyurea treatment in human NSMCE2-deficient cells. Double strand breaks accumulate during rescue by converging forks in normal cells but not in NSMCE2-deficient cells. Un-rescued forks persist into mitosis, leading to increased mitotic DNA damage. Excess RAD51 accumulates and persists at collapsed forks in NSMCE2-deficient cells, possibly due to lack of BLM recruitment to stalled forks. Despite failure of BLM to accumulate at stalled forks, NSMCE2-deficient cells exhibit lower levels of hydroxyurea-induced sister chromatid exchange. In cells deficient in both NSMCE2 and BLM, hydroxyurea-induced double strand breaks and sister chromatid exchange resembled levels found in NSCME2-deficient cells. We conclude that the rescue of collapsed forks by converging forks is dependent on NSMCE2.

Original language	English (US)
Article number	e1007942
Journal	PLoS genetics
Volume	15
Issue number	2
DOIs	https://doi.org/10.1371/journal.pgen.1007942
State	Published - Feb 2019

ASJC Scopus subject areas

Ecology, Evolution, Behavior and Systematics
Molecular Biology
Genetics
Genetics(clinical)
Cancer Research

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title = "Rescue of collapsed replication forks is dependent on NSMCE2 to prevent mitotic DNA damage",

abstract = "NSMCE2 is an E3 SUMO ligase and a subunit of the SMC5/6 complex that associates with the replication fork and protects against genomic instability. Here, we study the fate of collapsed replication forks generated by prolonged hydroxyurea treatment in human NSMCE2-deficient cells. Double strand breaks accumulate during rescue by converging forks in normal cells but not in NSMCE2-deficient cells. Un-rescued forks persist into mitosis, leading to increased mitotic DNA damage. Excess RAD51 accumulates and persists at collapsed forks in NSMCE2-deficient cells, possibly due to lack of BLM recruitment to stalled forks. Despite failure of BLM to accumulate at stalled forks, NSMCE2-deficient cells exhibit lower levels of hydroxyurea-induced sister chromatid exchange. In cells deficient in both NSMCE2 and BLM, hydroxyurea-induced double strand breaks and sister chromatid exchange resembled levels found in NSCME2-deficient cells. We conclude that the rescue of collapsed forks by converging forks is dependent on NSMCE2.",

author = "Pond, {Kelvin W.} and {de Renty}, Christelle and Yagle, {Mary K.} and Ellis, {Nathan A.}",

note = "Funding Information: Research reported in this publication was supported by the University of Arizona Cancer Center and the National Cancer Institute of the National Institutes of Health (https://www.cancer.gov) under award numbers R01CA140804 (NAE) and P30 CA023074 (NAE) and by funds from the University of Arizona. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank the University of Arizona Cancer Center Flow Cytometry Shared Resource for support. The authors thank Dr. Ghassan Mouneimne and Marco Padilla-Rodriguez for their help with image analysis, Dr. Gregory Rogers and Mengdie Wang for their help with microscopy, and Dr. Ted Weinert and Dr. Keith Maggert for their helpful comments on the manuscript. We also thank the University of Arizona Microscopy Alliance for their help with confocal imaging. Publisher Copyright: {\textcopyright} 2019 Pond et al. http://creativecommons.org/licenses/by/4.0/.",

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AU - Pond, Kelvin W.

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AU - Ellis, Nathan A.

N1 - Funding Information: Research reported in this publication was supported by the University of Arizona Cancer Center and the National Cancer Institute of the National Institutes of Health (https://www.cancer.gov) under award numbers R01CA140804 (NAE) and P30 CA023074 (NAE) and by funds from the University of Arizona. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank the University of Arizona Cancer Center Flow Cytometry Shared Resource for support. The authors thank Dr. Ghassan Mouneimne and Marco Padilla-Rodriguez for their help with image analysis, Dr. Gregory Rogers and Mengdie Wang for their help with microscopy, and Dr. Ted Weinert and Dr. Keith Maggert for their helpful comments on the manuscript. We also thank the University of Arizona Microscopy Alliance for their help with confocal imaging. Publisher Copyright: © 2019 Pond et al. http://creativecommons.org/licenses/by/4.0/.

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N2 - NSMCE2 is an E3 SUMO ligase and a subunit of the SMC5/6 complex that associates with the replication fork and protects against genomic instability. Here, we study the fate of collapsed replication forks generated by prolonged hydroxyurea treatment in human NSMCE2-deficient cells. Double strand breaks accumulate during rescue by converging forks in normal cells but not in NSMCE2-deficient cells. Un-rescued forks persist into mitosis, leading to increased mitotic DNA damage. Excess RAD51 accumulates and persists at collapsed forks in NSMCE2-deficient cells, possibly due to lack of BLM recruitment to stalled forks. Despite failure of BLM to accumulate at stalled forks, NSMCE2-deficient cells exhibit lower levels of hydroxyurea-induced sister chromatid exchange. In cells deficient in both NSMCE2 and BLM, hydroxyurea-induced double strand breaks and sister chromatid exchange resembled levels found in NSCME2-deficient cells. We conclude that the rescue of collapsed forks by converging forks is dependent on NSMCE2.

AB - NSMCE2 is an E3 SUMO ligase and a subunit of the SMC5/6 complex that associates with the replication fork and protects against genomic instability. Here, we study the fate of collapsed replication forks generated by prolonged hydroxyurea treatment in human NSMCE2-deficient cells. Double strand breaks accumulate during rescue by converging forks in normal cells but not in NSMCE2-deficient cells. Un-rescued forks persist into mitosis, leading to increased mitotic DNA damage. Excess RAD51 accumulates and persists at collapsed forks in NSMCE2-deficient cells, possibly due to lack of BLM recruitment to stalled forks. Despite failure of BLM to accumulate at stalled forks, NSMCE2-deficient cells exhibit lower levels of hydroxyurea-induced sister chromatid exchange. In cells deficient in both NSMCE2 and BLM, hydroxyurea-induced double strand breaks and sister chromatid exchange resembled levels found in NSCME2-deficient cells. We conclude that the rescue of collapsed forks by converging forks is dependent on NSMCE2.

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Rescue of collapsed replication forks is dependent on NSMCE2 to prevent mitotic DNA damage

Abstract

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