Rtt105 functions as a chaperone for replication protein A to preserve genome stability

Shuqi Li; Zhiyun Xu; Jiawei Xu; Linyu Zuo; Chuanhe Yu; Pu Zheng; Haiyun Gan; Xuezheng Wang; Longtu Li; Sushma Sharma; Andrei Chabes; Di Li; Sheng Wang; Sihao Zheng; Jinbao Li; Xuefeng Chen; Yujie Sun; Dongyi Xu; Junhong Han; Kuiming Chan; Zhi Qi; Jianxun Feng; Qing Li

doi:10.15252/embj.201899154

Rtt105 functions as a chaperone for replication protein A to preserve genome stability

Shuqi Li, Zhiyun Xu, Jiawei Xu, Linyu Zuo, Chuanhe Yu, Pu Zheng, Haiyun Gan, Xuezheng Wang, Longtu Li, Sushma Sharma, Andrei Chabes, Di Li, Sheng Wang, Sihao Zheng, Jinbao Li, Xuefeng Chen, Yujie Sun, Dongyi Xu, Junhong Han, Kuiming ChanZhi Qi, Jianxun Feng, Qing Li

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

20 Scopus citations

Abstract

Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA–ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA–importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA–ssDNA complex. Single-molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA. These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.

Original language	English (US)
Article number	e99154
Journal	EMBO Journal
Volume	37
Issue number	17
DOIs	https://doi.org/10.15252/embj.201899154
State	Published - Sep 3 2018
Externally published	Yes

Bibliographical note

Funding Information:
We thank Marc Wold for providing the yeast RPA expression constructs, Steven Brill for providing yeast Rfa1 and Rfa2 antibodies, Oscar Aparcio for the BrdU incorporation strains and plasmids, Chao Tang and Xiaojing Yang for providing GFP and mCherry plasmids, and Wenfeng Qian for providing various genetic background yeast strains. We thank Yun Zhang for assistance with the Illumina sequencing. We thank Hongxia Lv and Xiaochen Li for assistance with live-cell imaging. We thank Zhiguo Zhang, Lilin Du, and Hengyao Niu for critical reading of the manuscript. We thank Huiqiang Lou and Lee Zou for early discussion. This work was supported by grants from NSFC [31725015 (QL) and 31671332 (JF)]. Work in AC's laboratory was funded by the Swedish Research Council, the Swedish Cancer Society, and the Knut and Alice Wallenberg Foundation.

Publisher Copyright:
© 2018 The Authors

Keywords

RPA chaperone
Rtt105
replication fork
replication stress

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10.15252/embj.201899154

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@article{62d90915394d41e496868dac6638315d,

title = "Rtt105 functions as a chaperone for replication protein A to preserve genome stability",

abstract = "Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA–ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA–importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA–ssDNA complex. Single-molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA. These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.",

keywords = "RPA chaperone, Rtt105, replication fork, replication stress",

author = "Shuqi Li and Zhiyun Xu and Jiawei Xu and Linyu Zuo and Chuanhe Yu and Pu Zheng and Haiyun Gan and Xuezheng Wang and Longtu Li and Sushma Sharma and Andrei Chabes and Di Li and Sheng Wang and Sihao Zheng and Jinbao Li and Xuefeng Chen and Yujie Sun and Dongyi Xu and Junhong Han and Kuiming Chan and Zhi Qi and Jianxun Feng and Qing Li",

note = "Funding Information: We thank Marc Wold for providing the yeast RPA expression constructs, Steven Brill for providing yeast Rfa1 and Rfa2 antibodies, Oscar Aparcio for the BrdU incorporation strains and plasmids, Chao Tang and Xiaojing Yang for providing GFP and mCherry plasmids, and Wenfeng Qian for providing various genetic background yeast strains. We thank Yun Zhang for assistance with the Illumina sequencing. We thank Hongxia Lv and Xiaochen Li for assistance with live-cell imaging. We thank Zhiguo Zhang, Lilin Du, and Hengyao Niu for critical reading of the manuscript. We thank Huiqiang Lou and Lee Zou for early discussion. This work was supported by grants from NSFC [31725015 (QL) and 31671332 (JF)]. Work in AC's laboratory was funded by the Swedish Research Council, the Swedish Cancer Society, and the Knut and Alice Wallenberg Foundation. Publisher Copyright: {\textcopyright} 2018 The Authors",

year = "2018",

month = sep,

day = "3",

doi = "10.15252/embj.201899154",

language = "English (US)",

volume = "37",

journal = "EMBO Journal",

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T1 - Rtt105 functions as a chaperone for replication protein A to preserve genome stability

AU - Li, Shuqi

AU - Xu, Zhiyun

AU - Xu, Jiawei

AU - Zuo, Linyu

AU - Yu, Chuanhe

AU - Zheng, Pu

AU - Gan, Haiyun

AU - Wang, Xuezheng

AU - Li, Longtu

AU - Sharma, Sushma

AU - Chabes, Andrei

AU - Li, Di

AU - Wang, Sheng

AU - Zheng, Sihao

AU - Li, Jinbao

AU - Chen, Xuefeng

AU - Sun, Yujie

AU - Xu, Dongyi

AU - Han, Junhong

AU - Chan, Kuiming

AU - Qi, Zhi

AU - Feng, Jianxun

AU - Li, Qing

N1 - Funding Information: We thank Marc Wold for providing the yeast RPA expression constructs, Steven Brill for providing yeast Rfa1 and Rfa2 antibodies, Oscar Aparcio for the BrdU incorporation strains and plasmids, Chao Tang and Xiaojing Yang for providing GFP and mCherry plasmids, and Wenfeng Qian for providing various genetic background yeast strains. We thank Yun Zhang for assistance with the Illumina sequencing. We thank Hongxia Lv and Xiaochen Li for assistance with live-cell imaging. We thank Zhiguo Zhang, Lilin Du, and Hengyao Niu for critical reading of the manuscript. We thank Huiqiang Lou and Lee Zou for early discussion. This work was supported by grants from NSFC [31725015 (QL) and 31671332 (JF)]. Work in AC's laboratory was funded by the Swedish Research Council, the Swedish Cancer Society, and the Knut and Alice Wallenberg Foundation. Publisher Copyright: © 2018 The Authors

PY - 2018/9/3

Y1 - 2018/9/3

N2 - Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA–ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA–importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA–ssDNA complex. Single-molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA. These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.

AB - Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA–ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA–importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA–ssDNA complex. Single-molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA. These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.

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KW - replication fork

KW - replication stress

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DO - 10.15252/embj.201899154

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AN - SCOPUS:85052750558

SN - 0261-4189

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ER -

Rtt105 functions as a chaperone for replication protein A to preserve genome stability

Abstract

Bibliographical note

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