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.

scholarly journals Saccharomyces cerevisiae Ime2 phosphorylates Sic1 at multiple PXS/T sites but is insufficient to trigger Sic1 degradation

2006 ◽  
Vol 399 (1) ◽  
pp. 151-160 ◽  
Author(s):  
Chantelle Sedgwick ◽  
Matthew Rawluk ◽  
James Decesare ◽  
Sheetal Raithatha ◽  
James Wohlschlegel ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Phase Boundary ◽  
S Phase ◽  
Phosphorylation Sites ◽  
Cyclin Dependent Kinase ◽  
Proliferating Cells ◽  
Initiation Of Dna Replication ◽  
Cdk Phosphorylation

The initiation of DNA replication in Saccharomyces cerevisiae depends upon the destruction of the Clb–Cdc28 inhibitor Sic1. In proliferating cells Cln–Cdc28 complexes phosphorylate Sic1, which stimulates binding of Sic1 to SCFCdc4 and triggers its proteosome mediated destruction. During sporulation cyclins are not expressed, yet Sic1 is still destroyed at the G1-/S-phase boundary. The Cdk (cyclin dependent kinase) sites are also required for Sic1 destruction during sporulation. Sic1 that is devoid of Cdk phosphorylation sites displays increased stability and decreased phosphorylation in vivo. In addition, we found that Sic1 was modified by ubiquitin in sporulating cells and that SCFCdc4 was required for this modification. The meiosis-specific kinase Ime2 has been proposed to promote Sic1 destruction by phosphorylating Sic1 in sporulating cells. We found that Ime2 phosphorylates Sic1 at multiple sites in vitro. However, only a subset of these sites corresponds to Cdk sites. The identification of multiple sites phosphorylated by Ime2 has allowed us to propose a motif for phosphorylation by Ime2 (PXS/T) where serine or threonine acts as a phospho-acceptor. Although Ime2 phosphorylates Sic1 at multiple sites in vitro, the modified Sic1 fails to bind to SCFCdc4. In addition, the expression of Ime2 in G1 arrested haploid cells does not promote the destruction of Sic1. These data support a model where Ime2 is necessary but not sufficient to promote Sic1 destruction during sporulation.

scholarly journals Role of Papillomavirus E1 Initiator Dimerization in DNA Replication

2005 ◽  
Vol 79 (13) ◽  
pp. 8661-8664 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Severe Effect ◽  
Origin Of Dna Replication ◽  
Initiation Of Dna Replication

ABSTRACT Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.

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 Multiple Phosphorylation Sites of DNA Polymerase α-Primase Cooperate to Regulate the Initiation of DNA Replication in Vitro

2001 ◽  
Vol 276 (41) ◽  
pp. 38076-38083
Author(s):  
Oliver Schub ◽  
Gabor Rohaly ◽  
Richard W.P. Smith ◽  
Annerose Schneider ◽  
Silke Dehde ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Phosphorylation Sites ◽  
Dna Polymerase Α ◽  
Initiation Of Dna Replication

Functionally homologous DNA replication genes in fission and budding yeast

1999 ◽  
Vol 112 (14) ◽  
pp. 2381-2390
Author(s):  
M. Sanchez ◽  
A. Calzada ◽  
A. Bueno
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Kinase Activity ◽  
Budding Yeast ◽  
S Phase ◽  
Yeast Gene ◽  
Cdc2 Kinase ◽  
Initiation Of Dna Replication

The cdc18(+) gene of the fission yeast Schizosaccharomyces pombe is involved in the initiation of DNA replication as well as in coupling the S phase to mitosis. In this work, we show that the Saccharomyces cerevisiae CDC6 gene complements cdc18-K46 ts and cdc18 deletion mutant S. pombe strains. The budding yeast gene suppresses both the initiation and the checkpoint defects associated with the lack of cdc18(+). The Cdc6 protein interacts in vivo with Cdc2 kinase complexes. Interestingly, Cdc6 is an in vitro substrate for Cdc13/Cdc2 and Cig1/Cdc2, but not for Cig2/Cdc2-associated kinases. Overexpression of Cdc6 in fission yeast induces multiple rounds of S-phase in the absence of mitosis and cell division. This CDC6-dependent continuous DNA synthesis phenotype is independent of the presence of a functional cdc18(+) gene product and, significantly, requires only Cig2/Cdc2-associated kinase activity. Finally, these S. pombe over-replicating cells do not require any protein synthesis other than that of Cdc6. Our data strongly suggest that CDC6 and cdc18(+) are functional homologues and also support the idea that controls restricting genome duplication diverge in fission and budding yeast.

scholarly journals Phosphorylation of Sic1, a Cyclin-dependent Kinase (Cdk) Inhibitor, by Cdk Including Pho85 Kinase Is Required for Its Prompt Degradation

1998 ◽  
Vol 9 (9) ◽  
pp. 2393-2405 ◽  
Author(s):  
Masafumi Nishizawa ◽  
Masaoki Kawasumi ◽  
Marie Fujino ◽  
Akio Toh-e
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Cdk Inhibitor ◽  
Phosphorylation Sites ◽  
Cyclin Dependent Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cdk Phosphorylation ◽  
Cdc28 Kinase

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 andCLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G1cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable inpho85Δ cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G1Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G1.

scholarly journals Geminin Inhibits a Late Step in the Formation of Human Pre-replicative Complexes

2014 ◽  
Vol 289 (44) ◽  
pp. 30810-30821 ◽  
Author(s):  
Min Wu ◽  
Wenyan Lu ◽  
Ruth E. Santos ◽  
Mark G. Frattini ◽  
Thomas J. Kelly
Keyword(s):  
Dna Replication ◽  
Origin Recognition Complex ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Initial Step ◽  
High Salt ◽  
Late Step ◽  
Eukaryotic Dna ◽  
Initiation Of Dna Replication

The initial step in initiation of eukaryotic DNA replication involves the assembly of pre-replicative complexes (pre-RCs) at origins of replication during the G1 phase of the cell cycle. In metazoans initiation is inhibited by the regulatory factor Geminin. We have purified the human pre-RC proteins, studied their interactions in vitro with each other and with origin DNA, and analyzed the effects of HsGeminin on formation of DNA-protein complexes. The formation of an initial complex containing the human origin recognition complex (HsORC), HsCdt1, HsCdc6, and origin DNA is cooperative, involving all possible binary interactions among the components. Maximal association of HsMCM2–7, a component of the replicative helicase, requires HsORC, HsCdc6, HsCdt1, and ATP, and is driven by interactions of HsCdt1 and HsCdc6 with multiple HsMCM2–7 subunits. Formation of stable complexes, resistant to high salt, requires ATP hydrolysis. In the absence of HsMCM proteins, HsGeminin inhibits the association of HsCdt1 with DNA or with HsORC-HsCdc6-DNA complexes. However, HsGeminin does not inhibit recruitment of HsMCM2–7 to DNA to form complexes containing all of the pre-RC proteins. In fact, HsGeminin itself is a component of such complexes, and interacts directly with the HsMcm3 and HsMcm5 subunits of HsMCM2–7, as well as with HsCdt1. Although HsGeminin does not prevent the initial formation of DNA-protein complexes containing the pre-RC proteins, it strongly inhibits the formation of stable pre-RCs that are resistant to high salt. We suggest that bound HsGeminin prevents transition of the pre-RC to a state that is competent for initiation of DNA replication.

scholarly journals A Conserved Domain of Schizosaccharomyces pombe dfp1+ Is Uniquely Required for Chromosome Stability following Alkylation Damage during S Phase

2002 ◽  
Vol 22 (13) ◽  
pp. 4477-4490 ◽  
Author(s):  
Amy D. Fung ◽  
Jiongwen Ou ◽  
Stephanie Bueler ◽  
Grant W. Brown
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Critical Role ◽  
S Phase ◽  
Alkylation Damage ◽  
Conserved Regions ◽  
Initiation Of Dna Replication ◽  
S Phase Checkpoint

ABSTRACT The fission yeast Dbf4 homologue Dfp1 has a well-characterized role in regulating the initiation of DNA replication. Sequence analysis of Dfp1 homologues reveals three highly conserved regions, referred to as motifs N, M, and C. To determine the roles of these conserved regions in Dfp1 function, we have generated dfp1 alleles with mutations in these regions. Mutations in motif N render cells sensitive to a broad range of DNA-damaging agents and replication inhibitors, yet these mutant proteins are efficient activators of Hsk1 kinase in vitro. In contrast, mutations in motif C confer sensitivity to the alkylating agent methyl methanesulfonate (MMS) but, surprisingly, not to UV, ionizing radiation, or hydroxyurea. Motif C mutants are poor activators of Hsk1 in vitro but can fulfill the essential function(s) of Dfp1 in vivo. Strains carrying dfp1 motif C mutants have an intact mitotic and intra-S-phase checkpoint, and epistasis analysis indicates that dfp1 motif C mutants function outside of the known MMS damage repair pathways, suggesting that the observed MMS sensitivity is due to defects in recovery from DNA damage. The motif C mutants are most sensitive to MMS during S phase and are partially suppressed by deletion of the S-phase checkpoint kinase cds1. Following treatment with MMS, dfp1 motif C mutants exhibit nuclear fragmentation, chromosome instability, precocious recombination, and persistent checkpoint activation. We propose that Dfp1 plays at least two genetically separable roles in the DNA damage response in addition to its well-characterized role in the initiation of DNA replication and that motif C plays a critical role in the response to alkylation damage, perhaps by restarting or stabilizing stalled replication forks.

Cyclin-Dependent Kinases Phosphorylate AML1 (RUNX1) and Regulate AML1 Transactivation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2721-2721
Author(s):  
Joseph R. Biggs ◽  
Luke F. Peterson ◽  
Youhong Zhang ◽  
Andrew S. Kraft ◽  
Dong-Er Zhang
Keyword(s):  
Transcriptional Activation ◽  
Critical Role ◽  
Extramedullary Hematopoiesis ◽  
Conditional Knockout ◽  
Cyclin Dependent Kinases ◽  
Adult Mice ◽  
Blood Neutrophils ◽  
Cdk Phosphorylation

Abstract The transcription factor AML1 (RUNX1) plays a critical role in normal hematopoiesis and in the development of leukemia. The phenotype generated by knockout of the AML1 gene demonstrates that AML1 is necessary for normal development of fetal hematopoietic cells. Conditional knockout of AML1 expression in adult bone marrow leads to lineage-specific effects on B- and T-cell maturation and pronounced inhibition of common lymphocyte progenitor production. In addition, the AML1-deficient adult mice exhibited inefficient platelet production and a mild myeloproliferative phenotype characterized by an increase in peripheral blood neutrophils, an increase in myeloid progenitor populations, and extramedullary hematopoiesis composed of maturing myeloid and erythroid elements. Previous studies have shown that phosphorylation of AML1, particularly at serines 276 and 303, can affect transcriptional activation. We have now shown that phosphorylation of AML1 serines 276 and 303 can be blocked in vivo by inhibitors of the cyclin-dependent kinases (Cdks) Cdk1 and Cdk2. In addition, these two sites can be phosphorylated in vitro by purified, active Cdk1/cyclin B and Cdk2/cyclin A, but not by Cdk4/cyclin D. Mutation of AML1 serines 276 and 303 reduces AML1 activity during most phases of the cell cycle. In addition, mutation of serines 276 and 303 prevents phosphorylation from occurring at two nearby sites (serine 293 and threonine 300). Mutation of serine 293 and threonine 300 causes an increase of AML1 activity, especially in cells arrested in G2/M by treatment with nocodazole. Since Cdk phosphorylation can be stimulatory (serines 276 and 303) or inhibitory (serine 293, threonine 300), the interplay of Cdk phosphorylation at different sites might confer a very fine level of regulation of AML1 activity.

scholarly journals Role of the Escherichia coli Nucleotide Excision Repair Proteins in DNA Replication

2000 ◽  
Vol 182 (20) ◽  
pp. 5706-5714 ◽  
Author(s):  
Geri F. Moolenaar ◽  
Celine Moorman ◽  
Nora Goosen
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Inhibitory Effect ◽  
Binding Domain ◽  
Nucleotide Excision ◽  
Polymerase I

ABSTRACT DNA polymerase I (PolI) functions both in nucleotide excision repair (NER) and in the processing of Okazaki fragments that are generated on the lagging strand during DNA replication.Escherichia coli cells completely lacking the PolI enzyme are viable as long as they are grown on minimal medium. Here we show that viability is fully dependent on the presence of functional UvrA, UvrB, and UvrD (helicase II) proteins but does not require UvrC. In contrast, ΔpolA cells grow even better when theuvrC gene has been deleted. Apparently UvrA, UvrB, and UvrD are needed in a replication backup system that replaces the PolI function, and UvrC interferes with this alternative replication pathway. With specific mutants of UvrC we could show that the inhibitory effect of this protein is related to its catalytic activity that on damaged DNA is responsible for the 3′ incision reaction. Specific mutants of UvrA and UvrB were also studied for their capacity to support the PolI-independent replication. Deletion of the UvrC-binding domain of UvrB resulted in a phenotype similar to that caused by deletion of the uvrC gene, showing that the inhibitory incision activity of UvrC is mediated via binding to UvrB. A mutation in the N-terminal zinc finger domain of UvrA does not affect NER in vivo or in vitro. The same mutation, however, does give inviability in combination with the ΔpolA mutation. Apparently the N-terminal zinc-binding domain of UvrA has specifically evolved for a function outside DNA repair. A model for the function of the UvrA, UvrB, and UvrD proteins in the alternative replication pathway is discussed.

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