A new class of disordered elements controls DNA replication through initiator self-assembly

Matthew W. Parker; Maren Bell; Mustafa Mir; Jonchee A. Kao; Xavier Darzacq; Michael R. Botchan; James M. Berger

doi:10.7554/eLife.48562

A new class of disordered elements controls DNA replication through initiator self-assembly

Matthew W. Parker, Maren Bell, Mustafa Mir, Jonchee A. Kao, Xavier Darzacq, Michael R. Botchan, James M. Berger

School of Medicine

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

28 Scopus citations

Abstract

The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the McM2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and McM2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.

Original language	English (US)
Article number	e48562
Journal	eLife
Volume	8
DOIs	https://doi.org/10.7554/eLife.48562
State	Published - Sep 2019

ASJC Scopus subject areas

Neuroscience(all)
Immunology and Microbiology(all)
Biochemistry, Genetics and Molecular Biology(all)

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10.7554/eLife.48562

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title = "A new class of disordered elements controls DNA replication through initiator self-assembly",

abstract = "The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the McM2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and McM2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.",

author = "Parker, {Matthew W.} and Maren Bell and Mustafa Mir and Kao, {Jonchee A.} and Xavier Darzacq and Botchan, {Michael R.} and Berger, {James M.}",

note = "Funding Information: National Institute of General Medical Sciences, F32GM116393, National Cancer Institute, R01CA030490, The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Funding Information: We thank past and present members of the Berger and Botchan labs for helpful discussion and advice. We also thank Alison Killilea of the UC Berkeley Cell Culture facility for assistance with insect cell cultures. Parent pCopia and pCopia_mCherry-H2A vectors for work in Drosophila S2 tissue culture were a gift from the lab of Dr. Gary Karpen, UC Berkeley. Eric Greene (lab of Andy Martin, UC Berkeley) assisted in fluorescent anisotropy DNA-binding measurements. We thank Steve Bell for providing purified yeast ORC for analysis of phase separation behavior. This work was supported by an NIH NRSA postdoctoral fellowship (F32GM116393, to MWP) and by the NCI (R01CA030490, to JMB and MRB). Publisher Copyright: {\textcopyright} Parker et al.",

year = "2019",

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AU - Parker, Matthew W.

AU - Bell, Maren

AU - Mir, Mustafa

AU - Kao, Jonchee A.

AU - Darzacq, Xavier

AU - Botchan, Michael R.

AU - Berger, James M.

N1 - Funding Information: National Institute of General Medical Sciences, F32GM116393, National Cancer Institute, R01CA030490, The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Funding Information: We thank past and present members of the Berger and Botchan labs for helpful discussion and advice. We also thank Alison Killilea of the UC Berkeley Cell Culture facility for assistance with insect cell cultures. Parent pCopia and pCopia_mCherry-H2A vectors for work in Drosophila S2 tissue culture were a gift from the lab of Dr. Gary Karpen, UC Berkeley. Eric Greene (lab of Andy Martin, UC Berkeley) assisted in fluorescent anisotropy DNA-binding measurements. We thank Steve Bell for providing purified yeast ORC for analysis of phase separation behavior. This work was supported by an NIH NRSA postdoctoral fellowship (F32GM116393, to MWP) and by the NCI (R01CA030490, to JMB and MRB). Publisher Copyright: © Parker et al.

PY - 2019/9

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N2 - The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the McM2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and McM2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.

AB - The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the McM2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and McM2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.

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A new class of disordered elements controls DNA replication through initiator self-assembly

Abstract

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