Cargando…
Mcm2 phosphorylation and the response to replicative stress
BACKGROUND: The replicative helicase in eukaryotic cells is comprised of minichromosome maintenance (Mcm) proteins 2 through 7 (Mcm2-7) and is a key target for regulation of cell proliferation. In addition, it is regulated in response to replicative stress. One of the protein kinases that targets Mc...
Autores principales: | , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2012
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517340/ https://www.ncbi.nlm.nih.gov/pubmed/22564307 http://dx.doi.org/10.1186/1471-2156-13-36 |
_version_ | 1782252388263919616 |
---|---|
author | Stead, Brent E Brandl, Christopher J Sandre, Matthew K Davey, Megan J |
author_facet | Stead, Brent E Brandl, Christopher J Sandre, Matthew K Davey, Megan J |
author_sort | Stead, Brent E |
collection | PubMed |
description | BACKGROUND: The replicative helicase in eukaryotic cells is comprised of minichromosome maintenance (Mcm) proteins 2 through 7 (Mcm2-7) and is a key target for regulation of cell proliferation. In addition, it is regulated in response to replicative stress. One of the protein kinases that targets Mcm2-7 is the Dbf4-dependent kinase Cdc7 (DDK). In a previous study, we showed that alanine mutations of the DDK phosphorylation sites at S164 and S170 in Saccharomyces cerevisiae Mcm2 result in sensitivity to caffeine and methyl methanesulfonate (MMS) leading us to suggest that DDK phosphorylation of Mcm2 is required in response to replicative stress. RESULTS: We show here that a strain with the mcm2 allele lacking DDK phosphorylation sites (mcm2(AA)) is also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU) and to the base analogue 5-fluorouracil (5-FU) but not the radiomimetic drug, phleomycin. We screened the budding yeast non-essential deletion collection for synthetic lethal interactions with mcm2(AA) and isolated deletions that include genes involved in the control of genome integrity and oxidative stress. In addition, the spontaneous mutation rate, as measured by mutations in CAN1, was increased in the mcm2(AA) strain compared to wild type, whereas with a phosphomimetic allele (mcm2(EE)) the mutation rate was decreased. These results led to the idea that the mcm2(AA) strain is unable to respond properly to DNA damage. We examined this by screening the deletion collection for suppressors of the caffeine sensitivity of mcm2(AA). Deletions that decrease spontaneous DNA damage, increase homologous recombination or slow replication forks were isolated. Many of the suppressors of caffeine sensitivity suppressed other phenotypes of mcm2(AA) including sensitivity to genotoxic drugs, the increased frequency of cells with RPA foci and the increased mutation rate. CONCLUSIONS: Together these observations point to a role for DDK-mediated phosphorylation of Mcm2 in the response to replicative stress, including some forms of DNA damage. We suggest that phosphorylation of Mcm2 modulates Mcm2-7 activity resulting in the stabilization of replication forks in response to replicative stress. |
format | Online Article Text |
id | pubmed-3517340 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-35173402012-12-08 Mcm2 phosphorylation and the response to replicative stress Stead, Brent E Brandl, Christopher J Sandre, Matthew K Davey, Megan J BMC Genet Research Article BACKGROUND: The replicative helicase in eukaryotic cells is comprised of minichromosome maintenance (Mcm) proteins 2 through 7 (Mcm2-7) and is a key target for regulation of cell proliferation. In addition, it is regulated in response to replicative stress. One of the protein kinases that targets Mcm2-7 is the Dbf4-dependent kinase Cdc7 (DDK). In a previous study, we showed that alanine mutations of the DDK phosphorylation sites at S164 and S170 in Saccharomyces cerevisiae Mcm2 result in sensitivity to caffeine and methyl methanesulfonate (MMS) leading us to suggest that DDK phosphorylation of Mcm2 is required in response to replicative stress. RESULTS: We show here that a strain with the mcm2 allele lacking DDK phosphorylation sites (mcm2(AA)) is also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU) and to the base analogue 5-fluorouracil (5-FU) but not the radiomimetic drug, phleomycin. We screened the budding yeast non-essential deletion collection for synthetic lethal interactions with mcm2(AA) and isolated deletions that include genes involved in the control of genome integrity and oxidative stress. In addition, the spontaneous mutation rate, as measured by mutations in CAN1, was increased in the mcm2(AA) strain compared to wild type, whereas with a phosphomimetic allele (mcm2(EE)) the mutation rate was decreased. These results led to the idea that the mcm2(AA) strain is unable to respond properly to DNA damage. We examined this by screening the deletion collection for suppressors of the caffeine sensitivity of mcm2(AA). Deletions that decrease spontaneous DNA damage, increase homologous recombination or slow replication forks were isolated. Many of the suppressors of caffeine sensitivity suppressed other phenotypes of mcm2(AA) including sensitivity to genotoxic drugs, the increased frequency of cells with RPA foci and the increased mutation rate. CONCLUSIONS: Together these observations point to a role for DDK-mediated phosphorylation of Mcm2 in the response to replicative stress, including some forms of DNA damage. We suggest that phosphorylation of Mcm2 modulates Mcm2-7 activity resulting in the stabilization of replication forks in response to replicative stress. BioMed Central 2012-05-07 /pmc/articles/PMC3517340/ /pubmed/22564307 http://dx.doi.org/10.1186/1471-2156-13-36 Text en Copyright ©2012 Stead et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Stead, Brent E Brandl, Christopher J Sandre, Matthew K Davey, Megan J Mcm2 phosphorylation and the response to replicative stress |
title | Mcm2 phosphorylation and the response to replicative stress |
title_full | Mcm2 phosphorylation and the response to replicative stress |
title_fullStr | Mcm2 phosphorylation and the response to replicative stress |
title_full_unstemmed | Mcm2 phosphorylation and the response to replicative stress |
title_short | Mcm2 phosphorylation and the response to replicative stress |
title_sort | mcm2 phosphorylation and the response to replicative stress |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517340/ https://www.ncbi.nlm.nih.gov/pubmed/22564307 http://dx.doi.org/10.1186/1471-2156-13-36 |
work_keys_str_mv | AT steadbrente mcm2phosphorylationandtheresponsetoreplicativestress AT brandlchristopherj mcm2phosphorylationandtheresponsetoreplicativestress AT sandrematthewk mcm2phosphorylationandtheresponsetoreplicativestress AT daveymeganj mcm2phosphorylationandtheresponsetoreplicativestress |