scholarly journals Involvement of RAD9-Dependent Damage Checkpoint Control in Arrest of Cell Cycle, Induction of Cell Death, and Chromosome Instability Caused by Defects in Origin Recognition Complex in Saccharomyces cerevisiae

2002 ◽  
Vol 1 (2) ◽  
pp. 200-212 ◽  
Author(s):  
Keiichi Watanabe ◽  
Jun Morishita ◽  
Keiko Umezu ◽  
Katsuhiko Shirahige ◽  
Hisaji Maki
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Origin Recognition Complex ◽  
Chromosome Instability ◽  
Dna Lesions ◽  
Checkpoint Control ◽  
Origin Firing ◽  
Prolonged Incubation ◽  
Diploid Cells ◽  
Origin Recognition

ABSTRACT Perturbation of origin firing in chromosome replication is a possible cause of spontaneous chromosome instability in multireplicon organisms. Here, we show that chromosomal abnormalities, including aneuploidy and chromosome rearrangement, were significantly increased in yeast diploid cells with defects in the origin recognition complex. The cell cycle of orc1-4/orc1-4 temperature-sensitive mutant was arrested at the G2/M boundary, after several rounds of cell division at the restrictive temperature. However, prolonged incubation of the mutant cells at 37°C led to abrogation of G2 arrest, and simultaneously the cells started to lose viability. A sharp increase in chromosome instability followed the abrogation of G2 arrest. In orc1-4/orc1-4 rad9Δ/rad9Δ diploid cells grown at 37°C, G2 arrest and induction of cell death were suppressed, while chromosome instability was synergistically augmented. These findings indicated that DNA lesions caused by a defect in Orc1p function trigger the RAD9-dependent checkpoint control, which ensures genomic integrity either by stopping the cell cycle progress until lesion repair, or by inducing cell death when the lesion is not properly repaired. At semirestrictive temperatures, orc2-1/orc2-1 diploid cells demonstrated G2 arrest and loss of cell viability, both of which require RAD9-dependent checkpoint control. However, chromosome instability was not induced in orc2-1/orc2-1 cells, even in the absence of the checkpoint control. These data suggest that once cells lose the damage checkpoint control, perturbation of origin firing can be tolerated by the cells. Furthermore, although a reduction in origin-firing capacity does not necessarily initiate chromosome instability, the Orc1p possesses a unique function, the loss of which induces instability in the chromosome.

scholarly journals Control of DNA Rereplication via Cdc2 Phosphorylation Sites in the Origin Recognition Complex

2001 ◽  
Vol 21 (17) ◽  
pp. 5767-5777 ◽  
Author(s):  
Amit Vas ◽  
Winnie Mok ◽  
Janet Leatherwood
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Origin Recognition Complex ◽  
Safety Net ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Phosphorylation Sites ◽  
Cdc2 Kinase ◽  
Origin Recognition ◽  
Dna Rereplication

ABSTRACT Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G2 and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

scholarly journals DrosophilaHeterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing

2001 ◽  
Vol 12 (6) ◽  
pp. 1671-1685 ◽  
Author(s):  
Mohammed Momin Shareef ◽  
Chadwick King ◽  
Mona Damaj ◽  
RamaKrishna Badagu ◽  
Da Wei Huang ◽  
...  
Keyword(s):  
Dna Binding ◽  
Origin Recognition Complex ◽  
Polytene Chromosomes ◽  
Binding Activity ◽  
Hmg Proteins ◽  
Mitotic Chromosomes ◽  
Origin Firing ◽  
Dna Binding Activity ◽  
Origin Recognition

Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes inSaccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophilaembryo is associated with a multiprotein complex containingDrosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.

scholarly journals Phosphorylation of ORC2 Protein Dissociates Origin Recognition Complex from Chromatin and Replication Origins

2012 ◽  
Vol 287 (15) ◽  
pp. 11891-11898 ◽  
Author(s):  
Kyung Yong Lee ◽  
Sung Woong Bang ◽  
Sang Wook Yoon ◽  
Seung-Hoon Lee ◽  
Jong-Bok Yoon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Origin Recognition Complex ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Replication Origins ◽  
Eukaryotic Chromosome ◽  
M Phase ◽  
Cycle Dependent ◽  
Prereplicative Complex ◽  
Origin Recognition

During the late M to the G1 phase of the cell cycle, the origin recognition complex (ORC) binds to the replication origin, leading to the assembly of the prereplicative complex for subsequent initiation of eukaryotic chromosome replication. We found that the cell cycle-dependent phosphorylation of human ORC2, one of the six subunits of ORC, dissociates ORC2, -3, -4, and -5 (ORC2–5) subunits from chromatin and replication origins. Phosphorylation at Thr-116 and Thr-226 of ORC2 occurs by cyclin-dependent kinase during the S phase and is maintained until the M phase. Phosphorylation of ORC2 at Thr-116 and Thr-226 dissociated the ORC2–5 from chromatin. Consistent with this, the phosphomimetic ORC2 protein exhibited defective binding to replication origins as well as to chromatin, whereas the phosphodefective protein persisted in binding throughout the cell cycle. These results suggest that the phosphorylation of ORC2 dissociates ORC from chromatin and replication origins and inhibits binding of ORC to newly replicated DNA.

scholarly journals Characterization of a Novel Origin Recognition Complex-Like Complex: Implications for DNA Recognition, Cell Cycle Control, and Locus-Specific Gene Amplification

2003 ◽  
Vol 23 (14) ◽  
pp. 5005-5017 ◽  
Author(s):  
Mohammad Mohammad ◽  
Randall D. York ◽  
Jonathan Hommel ◽  
Geoffrey M. Kapler
Keyword(s):  
Cell Cycle ◽  
Gene Amplification ◽  
Origin Recognition Complex ◽  
Germ Line ◽  
Dna Recognition ◽  
Binding Activity ◽  
Specific Gene ◽  
Type I ◽  
Dependent Manner ◽  
Origin Recognition

ABSTRACT The origin recognition complex (ORC) plays a central role in eukaryotic DNA replication. Here we describe a unique ORC-like complex in Tetrahymena thermophila, TIF4, which bound in an ATP-dependent manner to sequences required for cell cycle-controlled replication and gene amplification (ribosomal DNA [rDNA] type I elements). TIF4's mode of DNA recognition was distinct from that of other characterized ORCs, as it bound exclusively to single-stranded DNA. In contrast to yeast ORCs, TIF4 DNA binding activity was cell cycle regulated and peaked during S phase, coincident with the redistribution of the Orc2-related subunit, p69, from the cytoplasm to the macronucleus. Origin-binding activity and nuclear p69 immunoreactivity were further regulated during development, where they distinguished replicating from nonreplicating nuclei. Both activities were lost from germ line micronuclei following the programmed arrest of micronuclear replication. Replicating macronuclei stained with Orc2 antibodies throughout development in wild-type cells but failed to do so in the amplification-defective rmm11 mutant. Collectively, these findings indicate that the regulation of TIF4 is intimately tied to the cell cycle and developmentally programmed replication cycles. They further implicate TIF4 in rDNA gene amplification. As type I elements interact with other sequence-specific single-strand breaks (in vitro and in vivo), the dynamic interplay of Orc-like (TIF4) and non-ORC-like proteins with this replication determinant may provide a novel mechanism for regulation.

scholarly journals RPA shields inherited DNA lesions for post-mitotic DNA synthesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Aleksandra Lezaja ◽  
Andreas Panagopoulos ◽  
Yanlin Wen ◽  
Edison Carvalho ◽  
Ralph Imhof ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cancer Cells ◽  
Cell Cycle Progression ◽  
Protein A ◽  
Low Frequency ◽  
Replication Stress ◽  
Dna Lesions ◽  
Checkpoint Control ◽  
Cycle Progression

AbstractThe paradigm that checkpoints halt cell cycle progression for genome repair has been challenged by the recent discovery of heritable DNA lesions escaping checkpoint control. How such inherited lesions affect genome function and integrity is not well understood. Here, we identify a new class of heritable DNA lesions, which is marked by replication protein A (RPA), a protein primarily known for shielding single-stranded DNA in S/G2. We demonstrate that post-mitotic RPA foci occur at low frequency during unperturbed cell cycle progression, originate from the previous cell cycle, and are exacerbated upon replication stress. RPA-marked inherited ssDNA lesions are found at telomeres, particularly of ALT-positive cancer cells. We reveal that RPA protects these replication remnants in G1 to allow for post-mitotic DNA synthesis (post-MiDAS). Given that ALT-positive cancer cells exhibit high levels of replication stress and telomere fragility, targeting post-MiDAS might be a new therapeutic opportunity.

scholarly journals Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phase

2020 ◽  
Author(s):  
Mark C. Johnson ◽  
Geylani Can ◽  
Miguel Santos ◽  
Diana Alexander ◽  
Philip Zegerman
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
S Phase ◽  
Dna Lesions ◽  
Replication Initiation ◽  
Origin Firing ◽  
Initiation Factors ◽  
Dependent Inhibition ◽  
Rad53 Kinase ◽  
S Phase Checkpoint

AbstractAcross eukaryotes, checkpoints maintain the order of cell cycle events in the face of DNA damage or incomplete replication. Although a wide array of DNA lesions activates the checkpoint kinases, whether and how this response differs in different phases of the cell cycle remains poorly understood. The S-phase checkpoint for example results in the slowing of replication, which in the budding yeast Saccharomyces cerevisiae is caused by Rad53 kinase-dependent inhibition of the initiation factors Sld3 and Dbf4. Despite this, we show here that Rad53 phosphorylates both of these substrates throughout the cell cycle at the same sites as in S-phase, suggesting roles for this pathway beyond S-phase. Indeed we show that Rad53-dependent inhibition of Sld3 and Dbf4 limits re-replication in G2/M phase, preventing inappropriate gene amplification events. In addition we show that inhibition of Sld3 and Dbf4 after DNA damage in G1 phase prevents premature replication initiation at all origins at the G1/S transition. This study redefines the scope and specificity of the ‘S-phase checkpoint’ with implications for understanding the roles of this checkpoint in the majority of cancers that lack proper cell cycle controls.

scholarly journals A novel role for Cdc5p in DNA replication.

1996 ◽  
Vol 16 (12) ◽  
pp. 6775-6782 ◽  
Author(s):  
C F Hardy ◽  
A Pautz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Replication ◽  
Origin Recognition Complex ◽  
Budding Yeast ◽  
Yeast Saccharomyces Cerevisiae ◽  
Plasmid Maintenance ◽  
Multiple Origins ◽  
Synthetically Lethal ◽  
Origin Recognition

DNA replication initiates from specific chromosomal sites called origins, and in the budding yeast Saccharomyces cerevisiae these sites are occupied by the origin recognition complex (ORC). Dbf4p is proposed to play a role in targeting the G1/S kinase Cdc7p to initiation complexes late in G1. We report that Dbf4p may also recruit Cdc5p to origin complexes. Cdc5p is a member of the Polo family of kinases that is required for the completion of mitosis. Cdc5p and Cdc7p each interact with a distinct domain of Dbf4p. cdc5-1 mutants have a plasmid maintenance defect that can be suppressed by the addition of multiple origins. cdc5-1 orc2-1 double mutants are synthetically lethal. Levels of Cdc5p were found to be cell cycle regulated and peaked in G2/M. These results suggest a role for Cdc5p and possibly Polo-like kinases at origin complexes.

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