DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts

Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effec...

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Main Authors:	Gaggioli, Vincent, Le Viet, Barbara, Germe, Thomas, Hyrien, Olivier
Format:	Online
Language:	English
Published:	Oxford University Press 2013
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753627/

id	pubmed-3753627
recordtype	oai_dc
spelling	pubmed-37536272013-08-27 DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts Gaggioli, Vincent Le Viet, Barbara Germe, Thomas Hyrien, Olivier Genome Integrity, Repair and Replication Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes. ICRF-193 slowed down replication origin cluster activation and fork progression in a checkpoint-independent manner, without altering replicon size. In contrast, topo IIα depletion accelerated origin cluster activation, and topo IIα add-back negated overinitiation. Therefore, topo IIα is not required for DNA replication, but topo IIα clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks. Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo IIα restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters. Oxford University Press 2013-08 2013-06-11 /pmc/articles/PMC3753627/ /pubmed/23757188 http://dx.doi.org/10.1093/nar/gkt494 Text en © The Author(s) 2013. Published by Oxford University Press. http://creativecommons.org/licenses/by/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Gaggioli, Vincent Le Viet, Barbara Germe, Thomas Hyrien, Olivier
spellingShingle	Gaggioli, Vincent Le Viet, Barbara Germe, Thomas Hyrien, Olivier DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts
author_facet	Gaggioli, Vincent Le Viet, Barbara Germe, Thomas Hyrien, Olivier
author_sort	Gaggioli, Vincent
title	DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts
title_short	DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts
title_full	DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts
title_fullStr	DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts
title_full_unstemmed	DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts
title_sort	dna topoisomerase iiα controls replication origin cluster licensing and firing time in xenopus egg extracts
description	Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes. ICRF-193 slowed down replication origin cluster activation and fork progression in a checkpoint-independent manner, without altering replicon size. In contrast, topo IIα depletion accelerated origin cluster activation, and topo IIα add-back negated overinitiation. Therefore, topo IIα is not required for DNA replication, but topo IIα clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks. Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo IIα restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters.
publisher	Oxford University Press
publishDate	2013
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753627/
_version_	1612006165420441600

DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts

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