Localization of Cdc7 Protein Kinase During DNA Replication in Saccharomyces cerevisiae
DDK, a conserved serine-threonine protein kinase composed of a regulatory subunit, Dbf4, and a catalytic subunit, Cdc7, is essential for DNA replication initiation during S phase of the cell cycle through MCM2-7 helicase phosphorylation. The biological significance of DDK is well characterized, but...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Oxford University Press
2017-11-01
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| Series: | G3: Genes, Genomes, Genetics |
| Subjects: | |
| Online Access: | http://g3journal.org/lookup/doi/10.1534/g3.117.300223 |
| _version_ | 1819008978278940672 |
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| author | Daniel Rossbach D. Suzi Bryan Jay R. Hesselberth Robert Sclafani |
| author_facet | Daniel Rossbach D. Suzi Bryan Jay R. Hesselberth Robert Sclafani |
| author_sort | Daniel Rossbach |
| collection | DOAJ |
| description | DDK, a conserved serine-threonine protein kinase composed of a regulatory subunit, Dbf4, and a catalytic subunit, Cdc7, is essential for DNA replication initiation during S phase of the cell cycle through MCM2-7 helicase phosphorylation. The biological significance of DDK is well characterized, but the full mechanism of how DDK associates with substrates remains unclear. Cdc7 is bound to chromatin in the Saccharomyces cerevisiae genome throughout the cell cycle, but there is little empirical evidence as to specific Cdc7 binding locations. Using biochemical and genetic techniques, this study investigated the specific localization of Cdc7 on chromatin. The Calling Cards method, using Ty5 retrotransposons as a marker for DNA–protein binding, suggests Cdc7 kinase is preferentially bound to genomic DNA known to replicate early in S phase, including centromeres and origins of replication. We also discovered Cdc7 binding throughout the genome, which may be necessary to initiate other cellular processes, including meiotic recombination and translesion synthesis. A kinase dead Cdc7 point mutation increases the Ty5 retrotransposon integration efficiency and a 55-amino acid C-terminal truncation of Cdc7, unable to bind Dbf4, reduces Cdc7 binding suggesting a requirement for Dbf4 to stabilize Cdc7 on chromatin during S phase. Chromatin immunoprecipitation demonstrates that Cdc7 binding near specific origins changes during S phase. Our results suggest a model where Cdc7 is loosely bound to chromatin during G1. At the G1/S transition, Cdc7 binding to chromatin is increased and stabilized, preferentially at sites that may become origins, in order to carry out a variety of cellular processes. |
| first_indexed | 2024-12-21T00:49:03Z |
| format | Article |
| id | doaj.art-bd778d8bb12543ac8556f477dea0184c |
| institution | Directory Open Access Journal |
| issn | 2160-1836 |
| language | English |
| last_indexed | 2024-12-21T00:49:03Z |
| publishDate | 2017-11-01 |
| publisher | Oxford University Press |
| record_format | Article |
| series | G3: Genes, Genomes, Genetics |
| spelling | doaj.art-bd778d8bb12543ac8556f477dea0184c2022-12-21T19:21:27ZengOxford University PressG3: Genes, Genomes, Genetics2160-18362017-11-017113757377410.1534/g3.117.30022318Localization of Cdc7 Protein Kinase During DNA Replication in Saccharomyces cerevisiaeDaniel RossbachD. Suzi BryanJay R. HesselberthRobert SclafaniDDK, a conserved serine-threonine protein kinase composed of a regulatory subunit, Dbf4, and a catalytic subunit, Cdc7, is essential for DNA replication initiation during S phase of the cell cycle through MCM2-7 helicase phosphorylation. The biological significance of DDK is well characterized, but the full mechanism of how DDK associates with substrates remains unclear. Cdc7 is bound to chromatin in the Saccharomyces cerevisiae genome throughout the cell cycle, but there is little empirical evidence as to specific Cdc7 binding locations. Using biochemical and genetic techniques, this study investigated the specific localization of Cdc7 on chromatin. The Calling Cards method, using Ty5 retrotransposons as a marker for DNA–protein binding, suggests Cdc7 kinase is preferentially bound to genomic DNA known to replicate early in S phase, including centromeres and origins of replication. We also discovered Cdc7 binding throughout the genome, which may be necessary to initiate other cellular processes, including meiotic recombination and translesion synthesis. A kinase dead Cdc7 point mutation increases the Ty5 retrotransposon integration efficiency and a 55-amino acid C-terminal truncation of Cdc7, unable to bind Dbf4, reduces Cdc7 binding suggesting a requirement for Dbf4 to stabilize Cdc7 on chromatin during S phase. Chromatin immunoprecipitation demonstrates that Cdc7 binding near specific origins changes during S phase. Our results suggest a model where Cdc7 is loosely bound to chromatin during G1. At the G1/S transition, Cdc7 binding to chromatin is increased and stabilized, preferentially at sites that may become origins, in order to carry out a variety of cellular processes.http://g3journal.org/lookup/doi/10.1534/g3.117.300223replicationkinasecalling cardschromatinorigins |
| spellingShingle | Daniel Rossbach D. Suzi Bryan Jay R. Hesselberth Robert Sclafani Localization of Cdc7 Protein Kinase During DNA Replication in Saccharomyces cerevisiae G3: Genes, Genomes, Genetics replication kinase calling cards chromatin origins |
| title | Localization of Cdc7 Protein Kinase During DNA Replication in Saccharomyces cerevisiae |
| title_full | Localization of Cdc7 Protein Kinase During DNA Replication in Saccharomyces cerevisiae |
| title_fullStr | Localization of Cdc7 Protein Kinase During DNA Replication in Saccharomyces cerevisiae |
| title_full_unstemmed | Localization of Cdc7 Protein Kinase During DNA Replication in Saccharomyces cerevisiae |
| title_short | Localization of Cdc7 Protein Kinase During DNA Replication in Saccharomyces cerevisiae |
| title_sort | localization of cdc7 protein kinase during dna replication in saccharomyces cerevisiae |
| topic | replication kinase calling cards chromatin origins |
| url | http://g3journal.org/lookup/doi/10.1534/g3.117.300223 |
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