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Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase
The Saccharomyces cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. We hypothesized that, as stimulators of e...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772018/ https://www.ncbi.nlm.nih.gov/pubmed/26728715 http://dx.doi.org/10.1101/gr.196857.115 |
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author | Peace, Jared M. Villwock, Sandra K. Zeytounian, John L. Gan, Yan Aparicio, Oscar M. |
author_facet | Peace, Jared M. Villwock, Sandra K. Zeytounian, John L. Gan, Yan Aparicio, Oscar M. |
author_sort | Peace, Jared M. |
collection | PubMed |
description | The Saccharomyces cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. We hypothesized that, as stimulators of early origin activation, Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide. Existing methods, however, are not well-suited to quantitative, genome-wide measurements of origin firing between strains and conditions. To overcome this limitation, we developed qBrdU-seq, a quantitative method for BrdU incorporation analysis of replication dynamics, and applied it to show that overexpression of Fkh1 and Fkh2 advances the initiation timing of many origins throughout the genome resulting in a higher total level of origin initiations in early S phase. The higher initiation rate is accompanied by slower replication fork progression, thereby maintaining a normal length of S phase without causing detectable Rad53 checkpoint kinase activation. The advancement of origin firing time, including that of origins in heterochromatic domains, was established in late G1 phase, indicating that origin timing can be reset subsequently to origin licensing. These results provide novel insights into the mechanisms of origin timing regulation by identifying Fkh1 and Fkh2 as rate-limiting factors for origin firing that determine the ability of replication origins to accrue limiting factors and have the potential to reprogram replication timing late in G1 phase. |
format | Online Article Text |
id | pubmed-4772018 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-47720182016-09-01 Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase Peace, Jared M. Villwock, Sandra K. Zeytounian, John L. Gan, Yan Aparicio, Oscar M. Genome Res Method The Saccharomyces cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. We hypothesized that, as stimulators of early origin activation, Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide. Existing methods, however, are not well-suited to quantitative, genome-wide measurements of origin firing between strains and conditions. To overcome this limitation, we developed qBrdU-seq, a quantitative method for BrdU incorporation analysis of replication dynamics, and applied it to show that overexpression of Fkh1 and Fkh2 advances the initiation timing of many origins throughout the genome resulting in a higher total level of origin initiations in early S phase. The higher initiation rate is accompanied by slower replication fork progression, thereby maintaining a normal length of S phase without causing detectable Rad53 checkpoint kinase activation. The advancement of origin firing time, including that of origins in heterochromatic domains, was established in late G1 phase, indicating that origin timing can be reset subsequently to origin licensing. These results provide novel insights into the mechanisms of origin timing regulation by identifying Fkh1 and Fkh2 as rate-limiting factors for origin firing that determine the ability of replication origins to accrue limiting factors and have the potential to reprogram replication timing late in G1 phase. Cold Spring Harbor Laboratory Press 2016-03 /pmc/articles/PMC4772018/ /pubmed/26728715 http://dx.doi.org/10.1101/gr.196857.115 Text en © 2016 Peace et al.; Published by Cold Spring Harbor Laboratory Press http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. |
spellingShingle | Method Peace, Jared M. Villwock, Sandra K. Zeytounian, John L. Gan, Yan Aparicio, Oscar M. Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase |
title | Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase |
title_full | Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase |
title_fullStr | Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase |
title_full_unstemmed | Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase |
title_short | Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase |
title_sort | quantitative brdu immunoprecipitation method demonstrates that fkh1 and fkh2 are rate-limiting activators of replication origins that reprogram replication timing in g1 phase |
topic | Method |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772018/ https://www.ncbi.nlm.nih.gov/pubmed/26728715 http://dx.doi.org/10.1101/gr.196857.115 |
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