Molecular basis for PrimPol recruitment to replication forks by RPA

Thomas A.  Guilliam; Nigel Brissett; Aaron  Ehlinger; Benjamin A.  Keen; Peter  Kolesar; Elaine M.  Taylor; Laura J.  Bailey; Howard D.  Lindsay; Walter J.  Chazin; Aidan J.  Doherty

doi:10.1038/ncomms15222

Molecular basis for PrimPol recruitment to replication forks by RPA

Thomas A. Guilliam, Nigel Brissett, Aaron Ehlinger, Benjamin A. Keen, Peter Kolesar, Elaine M. Taylor, Laura J. Bailey, Howard D. Lindsay, Walter J. Chazin, Aidan J. Doherty

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

Abstract

DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase-polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.

Original language	English
Article number	15222
Journal	Nature Communications
Volume	8
DOIs	https://doi.org/10.1038/ncomms15222
Publication status	Published - 23 May 2017

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title = "Molecular basis for PrimPol recruitment to replication forks by RPA",

abstract = "DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase-polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.",

author = "Guilliam, {Thomas A.} and Nigel Brissett and Aaron Ehlinger and Keen, {Benjamin A.} and Peter Kolesar and Taylor, {Elaine M.} and Bailey, {Laura J.} and Lindsay, {Howard D.} and Chazin, {Walter J.} and Doherty, {Aidan J.}",

note = "This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article{\textquoteright}s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/",

year = "2017",

month = may,

day = "23",

doi = "10.1038/ncomms15222",

language = "English",

volume = "8",

journal = "Nature Communications",

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T1 - Molecular basis for PrimPol recruitment to replication forks by RPA

AU - Guilliam, Thomas A.

AU - Brissett, Nigel

AU - Ehlinger, Aaron

AU - Keen, Benjamin A.

AU - Kolesar, Peter

AU - Taylor, Elaine M.

AU - Bailey, Laura J.

AU - Lindsay, Howard D.

AU - Chazin, Walter J.

AU - Doherty, Aidan J.

N1 - This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

PY - 2017/5/23

Y1 - 2017/5/23

N2 - DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase-polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.

AB - DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase-polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.

UR - https://europepmc.org/articles/PMC5457501

U2 - 10.1038/ncomms15222

DO - 10.1038/ncomms15222

M3 - Article

C2 - 28534480

SN - 2041-1723

VL - 8

JO - Nature Communications

JF - Nature Communications

M1 - 15222

ER -

Molecular basis for PrimPol recruitment to replication forks by RPA

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