scholarly journals Girdin: an essential component of pre-replicative complex in human cells

2019 ◽  
Author(s):  
Lihong Wu ◽  
Yu Hua ◽  
Feiran Chang ◽  
Daochun Kong
Keyword(s):  
Dna Replication ◽  
Essential Role ◽  
Human Cells ◽  
Replication Initiation ◽  
The Other ◽  
Initiation Factor ◽  
Replication Origins ◽  
Initiation Factors ◽  
Initiation Of Dna Replication ◽  
Dna Replication Origins

AbstractA central event in the initiation of DNA replication in eukaryotes is the assembly of pre-replicative complex (pre-RC) on specific chromatin sites known as DNA replication origins. The pre-RC assembly process differs between budding and fission yeasts. In fission yeast, Sap1 directly participates in pre-RC assembly, together with the four initiation factors: ORC, Cdc18/Cdc6, Cdt1, and MCM. In metazoans, the nature of DNA replication origins is not defined and the mechanism of pre-RC assembly remains incompletely known. In this study, Girdin was identified as an essential replication initiation factor in human cells. Similar to the activity of Sap1, human Girdin binds to DNA origins, interacts with ORC, and is required for pre-RC assembly due to its essential role in recruitment of Cdc6 to DNA origins. Thus, DNA origins in human or metazoans are defined as including two elements, one bound by ORC and the other bound by Girdin.

scholarly journals A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks through chromosomal replication origins in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (6) ◽  
pp. 3261-3271 ◽  
Author(s):  
A M Merchant ◽  
Y Kawasaki ◽  
Y Chen ◽  
M Lei ◽  
B K Tye
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Replication Origins ◽  
Minichromosome Maintenance ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Dna Replication Initiation ◽  
Initiation Factors

We describe a new minichromosome maintenance factor, Mcm10, and show that this essential protein is involved in the initiation of DNA replication in Saccharomyces cerevisiae. The mcm10 mutant has an autonomously replicating sequence-specific minichromosome maintenance defect and arrests at the nonpermissive temperature with dumbbell morphology and 2C DNA content. Mcm10 is a nuclear protein that physically interacts with several members of the MCM2-7 family of DNA replication initiation factors. Cloning and sequencing of the MCM10 gene show that it is identical to DNA43, a gene identified independently for its putative role in replicating DNA. Two-dimensional DNA gel analysis reveals that the mcm10-1 lesion causes a dramatic reduction in DNA replication initiation at chromosomal origins, including ORI1 and ORI121. Interestingly, the mcm10-1 lesion also causes replication forks to pause during elongation through these same loci. This novel phenotype suggests a unique role for the Mcm10 protein in the initiation of DNA synthesis at replication origins.

scholarly journals Autophosphorylation of Archaeal Cdc6 Homologues Is Regulated by DNA

2001 ◽  
Vol 183 (18) ◽  
pp. 5459-5464 ◽  
Author(s):  
Beatrice Grabowski ◽  
Zvi Kelman
Keyword(s):  
Dna Replication ◽  
Essential Role ◽  
Biochemical Properties ◽  
Replication Initiation ◽  
Winged Helix ◽  
Initiator Protein ◽  
C Terminus ◽  
Eukaryotic Dna ◽  
Initiation Of Dna Replication ◽  
Winged Helix Domain

ABSTRACT The initiator protein Cdc6 (Cdc18 in fission yeast) plays an essential role in the initiation of eukaryotic DNA replication. In yeast the protein is expressed before initiation of DNA replication and is thought to be essential for loading of the helicase onto origin DNA. The biochemical properties of the protein, however, are largely unknown. Using three archaeal homologues of Cdc6, it was found that the proteins are autophosphorylated on Ser residues. The winged-helix domain at the C terminus of Cdc6 interacts with DNA, which apparently regulates the autophosphorylation reaction. Yeast Cdc18 was also found to autophosphorylate, suggesting that this function of Cdc6 may play a widely conserved and essential role in replication initiation.

scholarly journals DNA Replication Origins Fire Stochastically in Fission Yeast

2006 ◽  
Vol 17 (1) ◽  
pp. 308-316 ◽  
Author(s):  
Prasanta K. Patel ◽  
Benoit Arcangioli ◽  
Stephen P. Baker ◽  
Aaron Bensimon ◽  
Nicholas Rhind
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Replication Initiation ◽  
Epigenetic Mechanism ◽  
Replication Origins ◽  
Origin Firing ◽  
Cell Cycles ◽  
Initiation Of Replication ◽  
Dna Combing ◽  
Dna Replication Origins

DNA replication initiates at discrete origins along eukaryotic chromosomes. However, in most organisms, origin firing is not efficient; a specific origin will fire in some but not all cell cycles. This observation raises the question of how individual origins are selected to fire and whether origin firing is globally coordinated to ensure an even distribution of replication initiation across the genome. We have addressed these questions by determining the location of firing origins on individual fission yeast DNA molecules using DNA combing. We show that the firing of replication origins is stochastic, leading to a random distribution of replication initiation. Furthermore, origin firing is independent between cell cycles; there is no epigenetic mechanism causing an origin that fires in one cell cycle to preferentially fire in the next. Thus, the fission yeast strategy for the initiation of replication is different from models of eukaryotic replication that propose coordinated origin firing.

scholarly journals Transcription drives DNA replication initiation and termination in human cells

2018 ◽  
Author(s):  
Yu-Hung Chen ◽  
Sarah Keegan ◽  
Malik Kahli ◽  
Peter Tonzi ◽  
David Fenyö ◽  
...  
Keyword(s):  
Dna Replication ◽  
Rna Polymerase Ii ◽  
Replication Fork ◽  
Human Cells ◽  
Replication Initiation ◽  
Origin Firing ◽  
Dna Replication Initiation ◽  
Origin Activation ◽  
Dna Replication Origins ◽  
The Impact

ABSTRACTThe locations of active DNA replication origins in the human genome, and the determinants of origin activation, remain controversial. Additionally, neither the predominant sites of replication termination nor the impact of transcription on replication-fork mobility have been defined. We demonstrate that replication initiation occurs preferentially in the immediate vicinity of the transcription start site of genes occupied by high levels of RNA polymerase II, ensuring co-directional replication of the most highly transcribed genes. Further, we demonstrate that dormant replication origin firing represents the global activation of pre-existing origins. We also show that DNA replication naturally terminates at the polyadenylation site of transcribed genes. During replication stress, termination is redistributed to gene bodies, generating a global reorientation of replication relative to transcription. Our analysis provides a unified model for the coupling of transcription with replication initiation and termination in human cells.

scholarly journals Multiple pathways can bypass the essential role of fission yeast Hsk1 kinase in DNA replication initiation

2011 ◽  
Vol 195 (3) ◽  
pp. 387-401 ◽  
Author(s):  
Seiji Matsumoto ◽  
Motoshi Hayano ◽  
Yutaka Kanoh ◽  
Hisao Masai
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Replication Fork ◽  
Essential Role ◽  
Replication Initiation ◽  
Origin Firing ◽  
Physiological Conditions ◽  
Dna Replication Initiation ◽  
Initiation Of Dna Replication

Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication that potentially regulates timing and locations of replication origin firing. Here, we show that viability of fission yeast hsk1Δ cells can be restored by loss of mrc1, which is required for maintenance of replication fork integrity, by cds1Δ, or by a checkpoint-deficient mutant of mrc1. In these mutants, normally inactive origins are activated in the presence of hydroxyurea and binding of Cdc45 to MCM is stimulated. mrc1Δ bypasses hsk1Δ more efficiently because of its checkpoint-independent inhibitory functions. Unexpectedly, hsk1Δ is viable at 37°C. More DNA is synthesized, and some dormant origins fire in the presence of hydroxyurea at 37°C. Furthermore, hsk1Δ bypass strains grow poorly at 25°C compared with higher temperatures. Our results show that Hsk1 functions for DNA replication can be bypassed by different genetic backgrounds as well as under varied physiological conditions, providing additional evidence for plasticity of the replication program in eukaryotes.

scholarly journals A Positive Amplification Mechanism Involving a Kinase and Replication Initiation Factor Helps Assemble the Replication Fork Helicase

2017 ◽  
Vol 292 (8) ◽  
pp. 3062-3073 ◽  
Author(s):  
Irina Bruck ◽  
Nalini Dhingra ◽  
Daniel L. Kaplan
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Budding Yeast ◽  
S Phase ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Minichromosome Maintenance ◽  
Initiation Factors ◽  
Complex Protein ◽  
Amplification Mechanism

The assembly of the replication fork helicase during S phase is key to the initiation of DNA replication in eukaryotic cells. One step in this assembly in budding yeast is the association of Cdc45 with the Mcm2–7 heterohexameric ATPase, and a second step is the assembly of the tetrameric GINS (GG-Ichi-Nii-San) complex with Mcm2–7. Dbf4-dependent kinase (DDK) and S-phase cyclin-dependent kinase (S-CDK) are two S phase-specific kinases that phosphorylate replication proteins during S phase, and Dpb11, Sld2, Sld3, Pol ϵ, and Mcm10 are factors that are also required for replication initiation. However, the exact roles of these initiation factors in assembly of the replication fork helicase remain unclear. We show here that Dpb11 stimulates DDK phosphorylation of the minichromosome maintenance complex protein Mcm4 alone and also of the Mcm2–7 complex and the dsDNA-loaded Mcm2–7 complex. We further demonstrate that Dpb11 can directly recruit DDK to Mcm4. A DDK phosphomimetic mutant of Mcm4 bound Dpb11 with substantially higher affinity than wild-type Mcm4, suggesting a mechanism to recruit Dpb11 to DDK-phosphorylated Mcm2–7. Furthermore, dsDNA-loaded Mcm2–7 harboring the DDK phosphomimetic Mcm4 mutant bound GINS in the presence of Dpb11, suggesting a mechanism for how GINS is recruited to Mcm2–7. We isolated a mutant of Dpb11 that is specifically defective for binding to Mcm4. This mutant, when expressed in budding yeast, diminished cell growth and DNA replication, substantially decreased Mcm4 phosphorylation, and decreased association of GINS with replication origins. We conclude that Dpb11 functions with DDK and Mcm4 in a positive amplification mechanism to trigger the assembly of the replication fork helicase.

scholarly journals A predictable conserved DNA base composition signature defines human core DNA replication origins

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ildem Akerman ◽  
Bahar Kasaai ◽  
Alina Bazarova ◽  
Pau Biak Sang ◽  
Isabelle Peiffer ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Sequence ◽  
Cell Types ◽  
Replication Initiation ◽  
Replication Origins ◽  
Dna Base Composition ◽  
Dna Replication Initiation ◽  
Sequence Elements ◽  
Dna Replication Origins ◽  
Genomic Regions

Abstract DNA replication initiates from multiple genomic locations called replication origins. In metazoa, DNA sequence elements involved in origin specification remain elusive. Here, we examine pluripotent, primary, differentiating, and immortalized human cells, and demonstrate that a class of origins, termed core origins, is shared by different cell types and host ~80% of all DNA replication initiation events in any cell population. We detect a shared G-rich DNA sequence signature that coincides with most core origins in both human and mouse genomes. Transcription and G-rich elements can independently associate with replication origin activity. Computational algorithms show that core origins can be predicted, based solely on DNA sequence patterns but not on consensus motifs. Our results demonstrate that, despite an attributed stochasticity, core origins are chosen from a limited pool of genomic regions. Immortalization through oncogenic gene expression, but not normal cellular differentiation, results in increased stochastic firing from heterochromatin and decreased origin density at TAD borders.

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 A DNA Replication-Independent Function of the pre-Replication Complex during Cell Invasion in C. elegans

2021 ◽  
Author(s):  
Evelyn Lattmann ◽  
Ting Deng ◽  
Michael Walser ◽  
Patrizia Widmer ◽  
Charlotte Rexha-Lambert ◽  
...  
Keyword(s):  
Dna Replication ◽  
Cell Invasion ◽  
Pi3k Pathway ◽  
Replication Initiation ◽  
Proliferating Cells ◽  
Replication Complex ◽  
Dna Replication Initiation ◽  
C Elegans ◽  
Initiation Factors ◽  
Dna Replication Origins

Cell invasion is an initiating event during tumor cell metastasis and an essential process during development. A screen of  C. elegans  orthologs of genes over-expressed in invasive human melanoma cells has identified several components of the conserved DNA pre-replication complex (pre-RC) as positive regulators of anchor cell (AC) invasion. The pre-RC functions cell-autonomously in the G1-arrested AC to promote invasion, independently of its role in licensing DNA replication origins in proliferating cells. While the helicase activity of the pre-RC is necessary for AC invasion, the downstream acting DNA replication initiation factors are not required. The pre-RC promotes the invasive fate by regulating the expression of extracellular matrix genes and components of the PI3K signaling pathway. Increasing PI3K pathway activity partially suppressed the AC invasion defects caused by pre-RC depletion, suggesting that the PI3K pathway is one critical pre-RC target. We propose that the pre-RC acts in the non-proliferating AC as a transcriptional regulator that facilitates the switch to an invasive phenotype.

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