scholarly journals Claspin recruits Cdc7 kinase for initiation of DNA replication in human cells

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Chi-Chun Yang ◽  
Masahiro Suzuki ◽  
Shiori Yamakawa ◽  
Syuzi Uno ◽  
Ai Ishii ◽  
...  
Keyword(s):  
Dna Replication ◽  
Human Cells ◽  
Cdc7 Kinase ◽  
Initiation Of Dna Replication

scholarly journals R-loops and initiation of DNA replication in human cells: a missing link?

2015 ◽  
Vol 6 ◽  
Author(s):  
Rodrigo Lombraña ◽  
Ricardo Almeida ◽  
Alba Álvarez ◽  
María Gómez
Keyword(s):  
Dna Replication ◽  
Human Cells ◽  
Missing Link ◽  
Initiation Of Dna Replication

scholarly journals Assembly of the Cdc45-Mcm2–7-GINS complex in human cells requires the Ctf4/And-1, RecQL4, and Mcm10 proteins

2009 ◽  
Vol 106 (37) ◽  
pp. 15628-15632 ◽  
Author(s):  
Jun-Sub Im ◽  
Sang-Hee Ki ◽  
Andrea Farina ◽  
Dong-Soo Jung ◽  
Jerard Hurwitz ◽  
...  
Keyword(s):  
Human Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Yeast Cells ◽  
Dna Unwinding ◽  
Cdk Inhibitor ◽  
Replication Forks ◽  
Cdc7 Kinase ◽  
Initiation Of Dna Replication ◽  
Stable Association ◽  
Prereplicative Complex

In eukaryotes, the activation of the prereplicative complex and assembly of an active DNA unwinding complex are critical but poorly understood steps required for the initiation of DNA replication. In this report, we have used bimolecular fluorescence complementation assays in HeLa cells to examine the interactions between Cdc45, Mcm2–7, and the GINS complex (collectively called the CMG complex), which seem to play a key role in the formation and progression of replication forks. Interactions between the CMG components were observed only after the G1/S transition of the cell cycle and were abolished by treatment of cells with either a CDK inhibitor or siRNA against the Cdc7 kinase. Stable association of CMG required all three components of the CMG complex as well as RecQL4, Ctf4/And-1, and Mcm10. Surprisingly, depletion of TopBP1, a homologue of Dpb11 that plays an essential role in the chromatin loading of Cdc45 and GINS in yeast cells, did not significantly affect CMG complex formation. These results suggest that the proteins involved in the assembly of initiation complexes in human cells may differ somewhat from those in yeast systems.

Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process

2000 ◽  
Vol 113 (11) ◽  
pp. 1929-1938 ◽  
Author(s):  
D. Coverley ◽  
C. Pelizon ◽  
S. Trewick ◽  
R.A. Laskey
Keyword(s):  
Dna Replication ◽  
Protein Kinase ◽  
Mammalian Cell ◽  
Cyclin A ◽  
S Phase ◽  
Human Cells ◽  
Cell Extracts ◽  
In Vitro Replication ◽  
Initiation Of Dna Replication

Cdc6 is essential for the initiation of DNA replication in all organisms in which it has been studied. In addition, recombinant Cdc6 can stimulate initiation in G(1) nuclei in vitro. We have analysed the behaviour of recombinant Cdc6 in mammalian cell extracts under in vitro replication conditions. We find that Cdc6 is imported into the nucleus in G(1)phase, where it binds to chromatin and remains relatively stable. In S phase, exogenous Cdc6 is destroyed in a process that requires import into the nucleus and phosphorylation by a chromatin-bound protein kinase. Recombinant cyclin A-cdk2 can completely substitute for the nucleus in promoting destruction of soluble Xenopus and human Cdc6. Despite this regulated destruction, endogenous Cdc6 persists in the nucleus after initiation, although the amount falls. Cdc6 levels remain constant in G(2) then fall again before mitosis. We propose that cyclin A-cdk2 phosphorylation results in destruction of any Cdc6 not assembled into replication complexes, but that assembled proteins remain, in the phosphorylated state, in the nucleus. This process could contribute to the prevention of reinitiation in human cells by making free Cdc6 unavailable for re-assembly into replication complexes after G(1) phase.

scholarly journals Regulation of the initiation of DNA replication in human cells

DNA Repair ◽  
2018 ◽  
Vol 72 ◽  
pp. 99-106 ◽  
Author(s):  
Tatiana N. Moiseeva ◽  
Christopher J. Bakkenist
Keyword(s):  
Dna Replication ◽  
Human Cells ◽  
Initiation Of Dna Replication

scholarly journals Novel Fission Yeast Cdc7-Dbf4-Like Kinase Complex Required for the Initiation and Progression of Meiotic Second Division

2002 ◽  
Vol 22 (1) ◽  
pp. 309-320 ◽  
Author(s):  
Taro Nakamura ◽  
Michiko Nakamura-Kubo ◽  
Tomohiro Nakamura ◽  
Chikashi Shimoda
Keyword(s):  
Dna Replication ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Structural Features ◽  
Regulatory Subunit ◽  
Primary Role ◽  
Biological Functions ◽  
Cdc7 Kinase ◽  
Initiation Of Dna Replication ◽  
Novel Protein

ABSTRACT Cdc7, a conserved serine/threonine protein kinase, controls initiation of DNA replication. A regulatory subunit, Dbf4, stimulates the kinase activity of Cdc7 and recruits it to the replication origins. Schizosaccharomyces pombe has a homologous kinase complex, composed of Hsk1 and Dfp1/Him1. Here, we report a novel protein kinase of S. pombe, Spo4, which shares common structural features with the Cdc7 kinases. In spite of the structural similarities, Spo4 is dispensable for mitotic growth and premeiotic DNA replication. Intriguingly, spo4 null mutants are defective in initiation and progression of the second meiotic division. Spindles for meiosis II are often fragmented. Spo4 kinase activity is markedly enhanced when the enzyme is associated with its regulatory subunit, Spo6, a Dbf4-like protein. Expression of Spo4 is specifically induced during meiosis. Spo4 is preferentially present in nuclei, but this nuclear localization does not require Spo6. These results suggest that Spo4 is a Cdc7 kinase whose primary role is in meiosis, not in DNA replication. This is the first report of an organism which has two Cdc7-related kinase complexes with different biological functions.

scholarly journals The Cdc6 Nucleotide–Binding Site Regulates Its Activity in DNA Replication in Human Cells

1999 ◽  
Vol 10 (8) ◽  
pp. 2631-2645 ◽  
Author(s):  
Utz Herbig ◽  
Clinton A. Marlar ◽  
Ellen Fanning
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Essential Role ◽  
Active Form ◽  
Human Cells ◽  
Amino Acid Substitutions ◽  
Human Homologue ◽  
Mutant Proteins ◽  
Initiation Of Dna Replication ◽  
Atp Binding And Hydrolysis

The Cdc6 protein of budding yeast and its homologues in other species play an essential role in the initiation of DNA replication. A cDNA encoding a human homologue of Cdc6 (HsCdc6) has been cloned and expressed as a fusion protein in a soluble and functionally active form. The purified protein bound specifically to ATP and slowly hydrolyzed it, whereas HsCdc6 mutants containing amino acid substitutions in the Walker A or B motifs were defective. The mutant proteins retained the ability to bind HsOrc1 and HsCdc6 but displayed aberrant conformations in the presence of nucleotides. Microinjection of either mutant protein into human cells in G1 inhibited DNA replication, suggesting that ATP binding and hydrolysis by HsCdc6 are essential for DNA replication.

scholarly journals Mutation of Cyclin/cdk Phosphorylation Sites in HsCdc6 Disrupts a Late Step in Initiation of DNA Replication in Human Cells

2000 ◽  
Vol 11 (12) ◽  
pp. 4117-4130 ◽  
Author(s):  
Utz Herbig ◽  
Jason W. Griffith ◽  
Ellen Fanning
Keyword(s):  
Dna Replication ◽  
Inhibitory Effect ◽  
Human Cells ◽  
Phosphorylation Sites ◽  
Cyclin Dependent Kinases ◽  
Late Step ◽  
Initiation Of Dna Replication ◽  
Cdk Phosphorylation

Cyclin-dependent kinases (Cdk) are essential for promoting the initiation of DNA replication, presumably by phosphorylating key regulatory proteins that are involved in triggering the G1/S transition. Human Cdc6 (HsCdc6), a protein required for initiation of DNA replication, is phosphorylated by Cdk in vitro and in vivo. Here we report that HsCdc6 with mutations at potential Cdk phosphorylation sites was poorly phosphorylated in vitro by Cdk, but retained all other biochemical activities of the wild-type protein tested. Microinjection of mutant HsCdc6 proteins into human cells blocked initiation of DNA replication or slowed S phase progression. The inhibitory effect of mutant HsCdc6 was lost at the G1/S transition, indicating that phosphorylation of HsCdc6 by Cdk is critical for a late step in initiation of DNA replication in human cells.

A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity

2008 ◽  
Vol 4 (6) ◽  
pp. 357-365 ◽  
Author(s):  
Alessia Montagnoli ◽  
Barbara Valsasina ◽  
Valter Croci ◽  
Maria Menichincheri ◽  
Sonia Rainoldi ◽  
...  
Keyword(s):  
Dna Replication ◽  
Antitumor Activity ◽  
Kinase Inhibitor ◽  
Cdc7 Kinase ◽  
Initiation Of Dna Replication
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