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RPA-coated single-stranded DNA promotes the ETAA1-dependent activation of ATR

Lyu, Ke and Kumagai, Akiko and Dunphy, William G. (2019) RPA-coated single-stranded DNA promotes the ETAA1-dependent activation of ATR. Cell Cycle, 18 (8). pp. 898-913. ISSN 1538-4101. https://resolver.caltech.edu/CaltechAUTHORS:20190415-103113214

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Abstract

Besides TopBP1, ETAA1 has been identified more recently as an activator of the ATR-ATRIP complex in human cells. We have examined the role of ETAA1 in the Xenopus egg-extract system, which has been instrumental in the study of ATR-ATRIP. Depletion of ETAA1 from egg extracts did not noticeably reduce the activation of ATR-ATRIP in response to replication stress, as monitored by the ATR-dependent phosphorylation of Chk1 and RPA. Moreover, lack of ETAA1 did not appear to affect DNA replication during an unperturbed S-phase. Significantly, we find that TopBP1 is considerably more abundant than ETAA1 in egg extracts. We proceeded to show that ETAA1 could support the activation of ATR-ATRIP in response to replication stress if we increased its concentration in egg extracts by adding extra full-length recombinant ETAA1. Thus, TopBP1 appears to be the predominant activator of ATR-ATRIP in response to replication stress in this system. We have also explored the biochemical mechanism by which ETAA1 activates ATR-ATRIP. We have developed an in vitro system in which full-length recombinant ETAA1 supports activation of ATR-ATRIP in the presence of defined components. We find that binding of ETAA1 to RPA associated with single-stranded DNA (ssDNA) greatly stimulates its ability to activate ATR-ATRIP. Thus, RPA-coated ssDNA serves as a direct positive effector in the ETAA1-mediated activation of ATR-ATRIP.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1080/15384101.2019.1598728DOIArticle
ORCID:
AuthorORCID
Lyu, Ke0000-0002-2483-2253
Kumagai, Akiko0000-0003-2422-8053
Dunphy, William G.0000-0001-7598-8939
Additional Information:© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. Received 14 Jan 2019, Accepted 12 Mar 2019, Published online: 12 Apr 2019. We are grateful to Kanomi Sasaki-Capela for technical assistance. Dr. Marc Wold (University of Iowa) kindly provided the construct for expression of human RPA in bacteria. We also thank Dr. Oscar Fernandez-Capetillo (Karolinska Institute) for providing ETP46464. Anti-Xenopus RPA32 serum was the kind gift of Dr. Vincenzo Costanzo (FIRC Institute of Molecular Oncology). This work was supported by NIH grants GM070891 and GM043974 to W.G.D. No potential conflict of interest was reported by the authors.
Funders:
Funding AgencyGrant Number
NIHGM043974
Subject Keywords:ETAA1, ATR, ATRIP, TopBP1, Chk1, RPA, Xenopus egg extract
Issue or Number:8
Record Number:CaltechAUTHORS:20190415-103113214
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20190415-103113214
Official Citation:Ke Lyu, Akiko Kumagai & William G. Dunphy (2019) RPA-coated single-stranded DNA promotes the ETAA1-dependent activation of ATR, Cell Cycle, 18:8, 898-913, DOI: 10.1080/15384101.2019.1598728
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:94703
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:16 Apr 2019 21:41
Last Modified:03 Oct 2019 21:06

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