The Human Stress-Activated Protein kin17 Belongs to the Multiprotein DNA Replication Complex and Associates In Vivo with Mammalian Replication Origins

ABSTRACT The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G1/S border and throughout the S phase and was negligible in both G0 and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase α. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an ∼600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.

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A Coordinated Temporal Interplay of Nucleosome Reorganization Factor, Sister Chromatin Cohesion Factor, and DNA Polymerase α Facilitates DNA Replication

Molecular and Cellular Biology ◽

10.1128/mcb.24.21.9568-9579.2004 ◽

2004 ◽

Vol 24 (21) ◽

pp. 9568-9579 ◽

Cited By ~ 43

Author(s):

Yanjiao Zhou ◽

Teresa S.-F. Wang

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

S Phase ◽

Terminal Region ◽

Replication Complex ◽

Dna Polymerase Α ◽

Chromatid Cohesion ◽

Phase Progression

ABSTRACT DNA replication depends critically upon chromatin structure. Little is known about how the replication complex overcomes the nucleosome packages in chromatin during DNA replication. To address this question, we investigate factors that interact in vivo with the principal initiation DNA polymerase, DNA polymerase α (Polα). The catalytic subunit of budding yeast Polα (Pol1p) has been shown to associate in vitro with the Spt16p-Pob3p complex, a component of the nucleosome reorganization system required for both replication and transcription, and with a sister chromatid cohesion factor, Ctf4p. Here, we show that an N-terminal region of Polα (Pol1p) that is evolutionarily conserved among different species interacts with Spt16p-Pob3p and Ctf4p in vivo. A mutation in a glycine residue in this N-terminal region of POL1 compromises the ability of Pol1p to associate with Spt16p and alters the temporal ordered association of Ctf4p with Pol1p. The compromised association between the chromatin-reorganizing factor Spt16p and the initiating DNA polymerase Pol1p delays the Pol1p assembling onto and disassembling from the late-replicating origins and causes a slowdown of S-phase progression. Our results thus suggest that a coordinated temporal and spatial interplay between the conserved N-terminal region of the Polα protein and factors that are involved in reorganization of nucleosomes and promoting establishment of sister chromatin cohesion is required to facilitate S-phase progression.

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Protein kinase D1 phosphorylation of KAT7 enhances its protein stability and promotes replication licensing and cell proliferation

Cell Death Discovery ◽

10.1038/s41420-020-00323-w ◽

2020 ◽

Vol 6 (1) ◽

Author(s):

Yao Liang ◽

Yuanyuan Su ◽

Chenzhong Xu ◽

Na Zhang ◽

Doudou Liu ◽

...

Keyword(s):

Cell Proliferation ◽

Dna Replication ◽

Protein Kinase ◽

Histone H4 ◽

Replication Complex ◽

Protein Kinase D1 ◽

Defective Mutant ◽

Histone H4 Acetylation

Abstract The histone acetyltransferase (HAT) KAT7/HBO1/MYST2 plays a crucial role in the pre-replication complex (pre-RC) formation, DNA replication and cell proliferation via acetylation of histone H4 and H3. In a search for protein kinase D1 (PKD1)-interacting proteins, we have identified KAT7 as a potential PKD1 substrate. We show that PKD1 directly interacts and phosphorylates KAT7 at Thr97 and Thr331 in vitro and in vivo. PKD1-mediated phosphorylation of KAT7 enhances its expression levels and stability by reducing its ubiquitination-mediated degradation. Significantly, the phospho-defective mutant KAT7-Thr97/331A attenuates histone H4 acetylation levels, MCM2/6 loading on the chromatin, DNA replication and cell proliferation. Similarly, PKD1 knockdown decreases, whereas the constitutive active mutant PKD1-CA increases histone H4 acetylation levels and MCM2/6 loading on the chromatin. Overall, these results suggest that PKD1-mediated phosphorylation of KAT7 may be required for pre-RC formation and DNA replication.

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The Schizosaccharomyces pombe cdt2+ Gene, a Target of G1-S Phase-Specific Transcription Factor Complex DSC1, Is Required for Mitotic and Premeiotic DNA Replication

Genetics ◽

10.1093/genetics/164.3.881 ◽

2003 ◽

Vol 164 (3) ◽

pp. 881-893 ◽

Cited By ~ 1

Author(s):

Shu-hei Yoshida ◽

Hiba Al-Amodi ◽

Taro Nakamura ◽

Christopher J McInerny ◽

Chikashi Shimoda

Keyword(s):

Dna Replication ◽

Schizosaccharomyces Pombe ◽

Mitotic Cell ◽

Life Cycles ◽

S Phase ◽

Growth Defect ◽

Replication Checkpoint ◽

Synthetically Lethal

Abstract We have defined five sev genes by genetic analysis of Schizosaccharomyces pombe mutants, which are defective in both proliferation and sporulation. sev1+/cdt2+ was transcribed during the G1-S phase of the mitotic cell cycle, as well as during the premeiotic S phase. The mitotic expression of cdt2+ was regulated by the MCB-DSC1 system. A mutant of a component of DSC1 affected cdt2+ expression in vivo, and a cdt2+ promoter fragment containing MCB motifs bound DSC1 in vitro. Cdt2 protein also accumulated in S phase and localized to the nucleus. cdt2 null mutants grew slowly at 30° and were unable to grow at 19°. These cdt2 mutants were also medially sensitive to hydroxyurea, camptothecin, and 4-nitroquinoline-1-oxide and were synthetically lethal in combination with DNA replication checkpoint mutations. Flow cytometry analysis and pulsed-field gel electrophoresis revealed that S-phase progression was severely retarded in cdt2 mutants, especially at low temperatures. Under sporulation conditions, premeiotic DNA replication was impaired with meiosis I blocked. Furthermore, overexpression of suc22+, a ribonucleotide reductase gene, fully complemented the sporulation defect of cdt2 mutants and alleviated their growth defect at 19°. These observations suggest that cdt2+ plays an important role in DNA replication in both the mitotic and the meiotic life cycles of fission yeast.

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Negative Regulation of Cdc18 DNA Replication Protein by Cdc2

Molecular Biology of the Cell ◽

10.1091/mbc.9.1.63 ◽

1998 ◽

Vol 9 (1) ◽

pp. 63-73 ◽

Cited By ~ 55

Author(s):

Antonia Lopez-Girona ◽

Odile Mondesert ◽

Janet Leatherwood ◽

Paul Russell

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Fission Yeast ◽

Phosphorylation Site ◽

Constitutive Expression ◽

S Phase ◽

Negative Regulation ◽

Chromosomal Dna

Fission yeast Cdc18, a homologue of Cdc6 in budding yeast and metazoans, is periodically expressed during the S phase and required for activation of replication origins. Cdc18 overexpression induces DNA rereplication without mitosis, as does elimination of Cdc2-Cdc13 kinase during G2 phase. These findings suggest that illegitimate activation of origins may be prevented through inhibition of Cdc18 by Cdc2. Consistent with this hypothesis, we report that Cdc18 interacts with Cdc2 in association with Cdc13 and Cig2 B-type cyclins in vivo. Cdc18 is phosphorylated by the associated Cdc2 in vitro. Mutation of a single phosphorylation site, T104A, activates Cdc18 in the rereplication assay. The cdc18-K9 mutation is suppressed by a cig2 mutation, providing genetic evidence that Cdc2-Cig2 kinase inhibits Cdc18. Moreover, constitutive expression of Cig2 prevents rereplication in cells lacking Cdc13. These findings identify Cdc18 as a key target of Cdc2-Cdc13 and Cdc2-Cig2 kinases in the mechanism that limits chromosomal DNA replication to once per cell cycle.

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Functionally homologous DNA replication genes in fission and budding yeast

Journal of Cell Science ◽

10.1242/jcs.112.14.2381 ◽

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.

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Cloned mouse DNA fragments can replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.5.3.563 ◽

1985 ◽

Vol 5 (3) ◽

pp. 563-568 ◽

Cited By ~ 6

Author(s):

H Ariga ◽

Z Tsuchihashi ◽

M Naruto ◽

M Yamada

Keyword(s):

Dna Replication ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Yeast Cells ◽

Dna Fragments ◽

Replication System ◽

Sv40 Dna

Mouse liver DNA was cut out with BamHI and cloned into YIp5, which contained the URA3 gene of Saccharomyces cerevisiae in pBR322. Of the several plasmids isolated, two plasmids, pMU65 and pMU111, could transform S. cerevisiae from the URA- to the URA+ phenotype and could replicate autonomously within the transformant, indicating that mouse DNA fragments present in pMU65 or pMU111 contain autonomously replicating sequences (ARS) for replication in S. cerevisiae. Furthermore, to determine the correlation between ARS function in yeast cells and that in much higher organisms, we tried to challenge these plasmids with the simian virus 40 (SV40) DNA replication system. Of the two plasmids tested, the EcoRI-BglII region of pMU65 could be hybridized with a chemically synthesized 13-nucleotide fragment corresponding to the origin region of SV40 DNA. Both pMU65 (the EcoRI-BglII region cloned in pBR322) and its subclone pMU65EB could replicate semiconservatively, and initiation of DNA replication started from the EcoRI-BglII region when the replicating activity of these plasmids was tested in the in vitro SV40 DNA replication system we have established before. Furthermore, pMU65 and pMU65EB could replicate autonomously within monkey Cos cells which produce SV40 T antigen constitutively. These results show that a 2.5-kilobase fragment of the EcoRI-BglII region in pMU65 contains the ARS needed for replication in the SV40 DNA replication system.

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Chromosomal replication origins in Candida albicans are genetically defined irrespective of their chromosomal context

10.1101/2020.10.15.341529 ◽

2020 ◽

Author(s):

Sreyoshi Mitra

Keyword(s):

Candida Albicans ◽

Dna Replication ◽

The Novel ◽

Replication Origins ◽

Chromosomal Replication ◽

Autonomously Replicating Sequence ◽

Replication Complex ◽

Intergenic Regions ◽

Novel Strategy

AbstractInitiation of DNA replication occurs at specialized regions along chromosomes called origins. The knowledge of replication origins is imperative to understand regulation of DNA replication. The properties of replication origins in the pathogenic budding yeast Candida albicans remain enigmatic in the absence of the knowledge of authentic chromosomal origins. Earlier, we identified centromere proximal (pCEN) chromosomal origins on chromosomes 5 and 7 of C. albicans. Here, we identify another centromere-distal (dCEN) chromosomal origin by two-dimensional agarose gel electrophoresis corresponding to a previously reported autonomously replicating sequence (ARS), CARS2. We show that all the identified chromosomal origins are bound by C. albicans homologs of conserved pre-replication complex proteins Orc2 and Mcm2. Previous reports from our lab and others have shown that there is a strong inter-relationship between centromere function and pericentric origin activity, suggesting that these origins are epigenetically regulated. However, we find that short intergenic regions corresponding to each of these origins functions as an ARS element in circular plasmids. Further, we use the novel strategy of in vivo gap repair to demonstrate that circular ARS plasmids can exist independently in vivo in C. albicans. Taken together, these results show that DNA sequence underlies the function of both centromere proximal and centromere distal chromosomal origins in C. albicans.

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

Biochemical Journal ◽

10.1042/bj20060363 ◽

2006 ◽

Vol 399 (1) ◽

pp. 151-160 ◽

Cited By ~ 27

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.22.13.4477-4490.2002 ◽

2002 ◽

Vol 22 (13) ◽

pp. 4477-4490 ◽

Cited By ~ 36

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.

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Cyclin B-Cdk1 Kinase Stimulates ORC- and Cdc6-Independent Steps of Semiconservative Plasmid Replication in Yeast Nuclear Extracts

Molecular and Cellular Biology ◽

10.1128/mcb.19.2.1226 ◽

1999 ◽

Vol 19 (2) ◽

pp. 1226-1241 ◽

Cited By ~ 18

Author(s):

Bernard P. Duncker ◽

Philippe Pasero ◽

Diego Braguglia ◽

Patrick Heun ◽

Michael Weinreich ◽

...

Keyword(s):

Dna Replication ◽

Origin Recognition Complex ◽

S Phase ◽

Autonomously Replicating Sequence ◽

Nuclear Extracts ◽

M Phase ◽

Efficient Replication ◽

Prereplicative Complex

ABSTRACT Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phase support the semiconservative replication of supercoiled plasmids in vitro. We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initiation in vivo. We found that replication in nuclear extracts initiates independently of the origin recognition complex (ORC), Cdc6p, and an autonomously replicating sequence (ARS) consensus. Nonetheless, quantitative density gradient analysis showed that S- and M-phase nuclear extracts consistently promote semiconservative DNA replication more efficiently than G1-phase extracts. The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extracts deficient for the Cdc7p kinase. In a cdc4-1 G1-phase extract, which accumulates high levels of the specific Clb-Cdk1 inhibitor p40 SIC1 , very low levels of semiconservative DNA replication were detected. Recombinant Clb5-Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G1-phase extract. In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40 SIC1 , restores efficient replication to both extracts. Our results suggest that in addition to its well-characterized role in regulating the origin-specific prereplication complex, the Clb-Cdk1 complex modulates the efficiency of the replication machinery itself.

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