Role of DDK in Replication Initiation

Rossbach, Daniel; Sclafani, Robert A.

doi:10.1007/978-3-319-24696-3_14

Cited by 7 publications

(7 citation statements)

References 80 publications

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“…Furthermore, ChIP-seq using a cdc7-7 strain did not reveal a significant enrichment of Cse4 to origins of DNA replication which are normally occupied by Cdc7 (Rossbach et al 2017). Similar to our observations, a previous study has shown that mcm5-bob1 cannot suppress the defect of cdc7-induced mutagenesis (Pessoa-Brandão and Sclafani 2004), indicating a different Cdc7 substrate in mutagenesis than the MCM2-7 complex (Rossbach and Sclafani 2016). Together, our results support a DNA replication-independent role of Cdc7 in regulating Cse4 proteolysis.…”

Section: Discussionsupporting

confidence: 89%

Dbf4-Dependent Kinase (DDK)-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A inSaccharomyces cerevisiae

Eisenstatt

Boeckmann

et al. 2020

G3 Genes|Genomes|Genetics

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The evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of CENP-A to non-centromeric chromatin contributes to chromosomal instability (CIN) in yeast, fly, and human cells and CENP-A is highly expressed and mislocalized in cancers. Defining mechanisms that prevent mislocalization of CENP-A is an area of active investigation. Ubiquitin-mediated proteolysis of overexpressed Cse4 (GALCSE4) by E3 ubiquitin ligases such as Psh1 prevents mislocalization of Cse4, and psh1Δ strains display synthetic dosage lethality (SDL) with GALCSE4. We previously performed a genome-wide screen and identified five alleles of CDC7 and DBF4 that encode the Dbf4-dependent kinase (DDK) complex, which regulates DNA replication initiation, among the top twelve hits that displayed SDL with GALCSE4. We determined that cdc7-7 strains exhibit defects in ubiquitin-mediated proteolysis of Cse4 and show mislocalization of Cse4. Mutation of MCM5 (mcm5-bob1) bypasses the requirement of Cdc7 for replication initiation and rescues replication defects in a cdc7-7 strain. We determined that mcm5-bob1 does not rescue the SDL and defects in proteolysis of GALCSE4 in a cdc7-7 strain, suggesting a DNA replication-independent role for Cdc7 in Cse4 proteolysis. The SDL phenotype, defects in ubiquitin-mediated proteolysis, and the mislocalization pattern of Cse4 in a cdc7-7 psh1Δ strain were similar to that of cdc7-7 and psh1Δ strains, suggesting that Cdc7 regulates Cse4 in a pathway that overlaps with Psh1. Our results define a DNA replication initiation-independent role of DDK as a regulator of Psh1-mediated proteolysis of Cse4 to prevent mislocalization of Cse4.

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Section: Discussionsupporting

confidence: 89%

Dbf4-Dependent Kinase (DDK)-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A inSaccharomyces cerevisiae

Eisenstatt

Boeckmann

et al. 2020

G3 Genes|Genomes|Genetics

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“…CDC7 is a conserved Ser/Thr kinase, which, in addition to its essential role in activation of DNA replication origins (reviewed in Rossbach and Sclafani, 2016 ), is implicated in intra S-phase checkpoint ( Costanzo et al., 2003 , Duncker et al., 2002 , Tsuji et al., 2008 , Weinreich and Stillman, 1999 , Yamada et al., 2013 ), DNA repair ( Princz et al., 2017 ), mitotic exit ( Miller et al., 2009 , Mishra et al., 2016 ), meiosis ( Buck et al., 1991 , Katis et al., 2010 , Lo et al., 2012 , Matos et al., 2008 , Murakami and Keeney, 2014 , Ogino et al., 2006 , Sasanuma et al., 2008 , Wan et al., 2008 ), and chromosome cohesion ( Natsume et al., 2013 , Takahashi et al., 2008 ). Catalytically inert in isolation, CDC7 becomes functional in complex with Dumbbell forming factor 4 (DBF4), the levels of which fluctuate throughout the cell cycle ( Ferreira et al., 2000 , Jackson et al., 1993 , Oshiro et al., 1999 ).…”

Section: Introductionmentioning

confidence: 99%

Structural Basis for the Activation and Target Site Specificity of CDC7 Kinase

et al. 2020

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Summary CDC7 is an essential Ser/Thr kinase that acts upon the replicative helicase throughout the S phase of the cell cycle and is activated by DBF4. Here, we present crystal structures of a highly active human CDC7-DBF4 construct. The structures reveal a zinc-finger domain at the end of the kinase insert 2 that pins the CDC7 activation loop to motif M of DBF4 and the C lobe of CDC7. These interactions lead to ordering of the substrate-binding platform and full opening of the kinase active site. In a co-crystal structure with a mimic of MCM2 Ser40 phosphorylation target, the invariant CDC7 residues Arg373 and Arg380 engage phospho-Ser41 at substrate P+1 position, explaining the selectivity of the S-phase kinase for Ser/Thr residues followed by a pre-phosphorylated or an acidic residue. Our results clarify the role of DBF4 in activation of CDC7 and elucidate the structural basis for recognition of its preferred substrates.

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“…DNA replication in eukaryotes is a tightly regulated process that ensures the genome is duplicated once and only once during the cell cycle. Failure to control replication mechanisms leads to chromosome instability, aneuploidy, and mutations, all hallmarks of many human diseases ( Rossbach and Sclafani 2016 ). DNA replication is initiated at specific segments of genomic DNA, termed origins of replication, during S phase of the cell cycle.…”

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confidence: 99%

Localization of Cdc7 Protein Kinase During DNA Replication in Saccharomyces cerevisiae

Rossbach

Bryan

Hesselberth

et al. 2017

G3 Genes|Genomes|Genetics

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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 G 1 . At the G 1 /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. KEYWORDSreplication kinase calling cards chromatin origins DNA replication in eukaryotes is a tightly regulated process that ensures the genome is duplicated once and only once during the cell cycle. Failure to control replication mechanisms leads to chromosome instability, aneuploidy, and mutations, all hallmarks of many human diseases (Rossbach and Sclafani 2016). DNA replication is initiated at specific segments of genomic DNA, termed origins of replication, during S phase of the cell cycle. In budding yeast, Saccharomyces cerevisiae, origins are known as ARSs (autonomous replicating sequences) and contain an 11 bp consensus sequence termed the ACS (ARS consensus sequence) that serves as a docking site for replisome proteins (Sclafani and Holzen 2007). Origins are classified by two key characteristics: efficiency and timing. Origin efficiency is determined by how frequently the origin initiates replication and whether it is used in a population of cells during every cellular division. Origin timing is a temporal reflection of when a given origin initiates replication during S phase. Origins can initiate replication throughout S phase and the time they initiate is quantified by the T rep (timing of replication), the time it takes to duplicate 50% of the DNA (Raghuraman et al. 2001;Yabuki et al. 2002).Initiation of DNA replic...

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Role of DDK in Replication Initiation

Cited by 7 publications

References 80 publications

Dbf4-Dependent Kinase (DDK)-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A inSaccharomyces cerevisiae

Dbf4-Dependent Kinase (DDK)-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A inSaccharomyces cerevisiae

Structural Basis for the Activation and Target Site Specificity of CDC7 Kinase

Localization of Cdc7 Protein Kinase During DNA Replication in Saccharomyces cerevisiae

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