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Antony M Carr

Publications and source records attributed to Antony M Carr.

15 recordsLinked to original sources
PubMed2003

Delineating the position of rad4+/cut5+ within the DNA-structure checkpoint pathways in Schizosaccharomyces pombe.

The fission yeast BRCT domain protein Rad4/Cut5 is required for genome integrity checkpoint responses and DNA replication. Here we address the position at which Rad4/Cut5 acts within the checkpoint response pathways. Rad4 is shown to act upstream of the effector kinases Chk1 and Cds1, as both Chk1 phosphorylation and Cds1 kinase activity require functional Rad4. Phosphorylation of Rad9, Rad26 and Hus1 in response to either DNA damage or inhibition of DNA replication are independent of Rad4/Cut5 checkpoint function. Further we show that a novel, epitope-tagged allele of rad4+/cut5+ acts as a dominant suppressor of the checkpoint deficiencies of rad3-, rad26- and rad17- mutants. Suppression results in the restoration of mitotic arrest and is dependent upon the remaining checkpoint Rad proteins and the two effector kinases. High-level expression of the rad4+/cut5+ allele in rad17 mutant cells restores the nuclear localization of Rad9, but this does not fully account for the observed suppression. We conclude from these data that Rad4/Cut5 acts with Rad3, Rad26 and Rad17 to effect the checkpoint response, and a model for its function is discussed.

Cell Cycle Proteins↗
PubMed2003

A subset of ATM- and ATR-dependent phosphorylation events requires the BRCA1 protein.

BRCA1 is a central component of the DNA damage response mechanism and defects in BRCA1 confer sensitivity to a broad range of DNA damaging agents. BRCA1 is required for homologous recombination and DNA damage-induced S and G(2)/M phase arrest. We show here that BRCA1 is required for ATM- and ATR-dependent phosphorylation of p53, c-Jun, Nbs1 and Chk2 following exposure to ionizing or ultraviolet radiation, respectively, and is also required for ATM phosphorylation of CtIP. In contrast, DNA damage-induced phosphorylation of the histone variant H2AX is independent of BRCA1. We also show that the presence of BRCA1 is dispensable for DNA damage-induced phosphorylation of Rad9, Hus1 and Rad17, and for the relocalization of Rad9 and Hus1. We propose that BRCA1 facilitates the ability of ATM and ATR to phosphorylate downstream substrates that directly influence cell cycle checkpoint arrest and apoptosis, but that BRCA1 is dispensable for the phosphorylation of DNA-associated ATM and ATR substrates.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2003

Cop9/signalosome subunits and Pcu4 regulate ribonucleotide reductase by both checkpoint-dependent and -independent mechanisms.

The signalosome is implicated in regulating cullin-dependent ubiquitin ligases. We find that two signalosome subunits, Csn1 and Csn2, are required to regulate ribonucleotide reductase (RNR) through the degradation of a small protein, Spd1, that acts to anchor the small RNR subunit in the nucleus. Spd1 destruction correlates with the nuclear export of the small RNR subunit, which, in turn, correlates with a requirement for RNR in replication and repair. Spd1 degradation is promoted by two separate CSN-dependent mechanisms. During unperturbed S phase, Spd1 degradation is independent of checkpoint proteins. In irradiated G2 cells, Spd1 degradation requires the DNA damage checkpoint. The signalosome copurifies with Pcu4 (cullin 4). Pcu4, Csn1, and Csn2 promote the degradation of Spd1, identifying a new function for the signalosome as a regulator of Pcu4-containing E3 ubiquitin ligase.

COP9 Signalosome Complex↗
PubMed2003

A simple Cre-loxP method for chromosomal N-terminal tagging of essential and non-essential Schizosaccharomyces pombe genes.

To facilitate the N-terminal tagging of essential genes at their genomic locus and under control of their own promoters we have developed a series of novel polymerase chain reaction templates. Initially, a 1.8 kb DNA fragment is integrated upstream of the ATG of the gene of interest. This fragment encodes the tag, a loxP site, a selectable marker, an exogenous nmt1 promoter and a second loxP site. In a single homologous integration event, the gene of interest is placed under control of the thiamine regulated nmt1 promoter, allowing identification of potential integrants on the basis of phenotype. Subsequently, this integrant strain is transformed with a plasmid expressing the Cre recombinase. This results in excision of the marker and nmt1 promoter and leaves sequences encoding an in-frame tag at the N-terminus of the gene of interest under the control of its native promoter. We have created TAP-cdc22, TAP-suc22 and TAP-rad50 strains using this N-tagging system, and developed a range of vectors for introducing TAP-, (His)10HA-, (His)6Myc- and EGFP.

Amino Acid Sequence↗
PubMed2003

Competition between the Rad50 complex and the Ku heterodimer reveals a role for Exo1 in processing double-strand breaks but not telomeres.

The Mre11-Rad50-Nbs1(Xrs2) complex and the Ku70-Ku80 heterodimer are thought to compete with each other for binding to DNA ends. To investigate the mechanism underlying this competition, we analyzed both DNA damage sensitivity and telomere overhangs in Schizosaccharomyces pombe rad50-d, rad50-d pku70-d, rad50-d exo1-d, and pku70-d rad50-d exo1-d cells. We found that rad50 exo1 double mutants are more methyl methanesulfonate (MMS) sensitive than the respective single mutants. The MMS sensitivity of rad50-d cells was suppressed by concomitant deletion of pku70+. However, the MMS sensitivity of the rad50 exo1 double mutant was not suppressed by the deletion of pku70+. The G-rich overhang at telomere ends in taz1-d cells disappeared upon deletion of rad50+, but the overhang reappeared following concomitant deletion of pku70+. Our data suggest that the Rad50 complex can process DSB ends and telomere ends in the presence of the Ku heterodimer. However, the Ku heterodimer inhibits processing of DSB ends and telomere ends by alternative nucleases in the absence of the Rad50-Rad32 protein complex. While we have identified Exo1 as the alternative nuclease targeting DNA break sites, the identity of the nuclease acting on the telomere ends remains elusive.

Antigens, Nuclear↗
PubMed2002

DNA structure dependent checkpoints as regulators of DNA repair.

Checkpoint proteins were initially identified because their loss of function resulted in defects in cell cycle arrest in response to genotoxic treatments. Initially, the analysis of checkpoint pathways concentrated on their function as signal transducers and how the checkpoint signals were communicated to the core cell cycle machinery and transcriptional apparatus. Although some of the early genetic analysis indicated a complex relationship between DNA replication, DNA repair and the checkpoint pathways, it is only now becoming apparent that checkpoint proteins regulate multiple DNA repair and replication functions. Furthermore, recent data suggest that some checkpoint proteins may participate directly in DNA repair events. In this review I summarise the current models for DNA structure-dependent checkpoint activation and review the evidence linking checkpoint proteins both directly and indirectly to DNA repair.

Cell Cycle Proteins↗
PubMed2002

Cytoplasmic poly(A) polymerases mediate cellular responses to S phase arrest.

The S-M checkpoint delays mitosis until DNA replication is complete; cells defective in this checkpoint lose viability when DNA replication is inhibited. This inviability can be suppressed in fission yeast by overexpression of Cid1 or the related protein Cid13. Fission yeast contain six cid1/cid13-like genes, whereas budding yeast has just two, TRF4 and TRF5. Trf4 and Trf5 were recently reported to comprise an essential DNA polymerase activity required for the establishment of sister chromatid cohesion. In contrast, we find that Cid1 is not a DNA polymerase but instead uses RNA substrates and has poly(A) polymerase activity. Unlike the previously characterized yeast poly(A) polymerase, which is a nuclear enzyme, Cid1 and Cid13 are constitutively cytoplasmic. Cid1 has a degree of substrate specificity in vitro, consistent with the notion that it targets a subset of cytoplasmic mRNAs for polyadenylation in vivo, hence increasing their stability and/or efficiency of translation. Preferred Cid1 targets presumably include mRNAs encoding components of the S-M checkpoint, whereas Cid13 targets are likely to be involved in dNTP metabolism. Cytoplasmic polyadenylation is known to be an important regulatory mechanism during early development in animals. Our findings in yeast suggest that this level of gene regulation is of more general significance in eukaryotic cells.

Amino Acid Sequence↗
PubMed2002

A single unbranched S-phase DNA damage and replication fork blockage checkpoint pathway.

The eukaryotic intra-S-phase checkpoint, which slows DNA synthesis in response to DNA damage, is poorly understood. Is DNA damage recognized directly, or indirectly through its effects on replication forks? Is the slowing of S phase in part because of competition between DNA synthesis and recombination/repair processes? The results of our genetic analyses of the intra-S-phase checkpoint in the fission yeast, Schizosaccharomyces pombe, suggest that the slowing of S phase depends weakly on the helicases Rqh1 and Srs2 but not on other recombination/repair pathways. The slowing of S phase depends strongly on the six checkpoint-Rad proteins, on Cds1, and on Rad4/Cut5 (similar to budding yeast Dpb11, which interacts with DNA polymerase epsilon) but not on Rhp9 (similar to budding yeast Rad9, necessary for direct damage recognition). These results suggest that, in fission yeast, the signal activating the intra-S-phase checkpoint is generated only when replication forks encounter DNA damage.

Cell Cycle Proteins↗
PubMed2002

Cdc2-cyclin B kinase activity links Crb2 and Rqh1-topoisomerase III.

The availability of a sister chromatid, and thus the cell cycle phase in which DNA double-strand breaks (DSBs) occur, influences the choice between homologous recombination (HR) or nonhomologous end joining (NHEJ). The sequential activation and destruction of CDK-cyclin activities controls progression through the cell cycle. Here we provide evidence that the major Schizosaccharomyces pombe CDK, Cdc2-cyclin B, influences recombinational repair of radiation-induced DSBs during the G(2) phase at two distinct stages. At an early stage in HR, a defect in Cdc2 kinase activity, which is caused by a single amino acid change in cyclin B, affects the formation of Rhp51 (Rad51(sp)) foci in response to ionizing radiation in a process that is redundant with the function of Rad50. At a late stage in HR, low Cdc2-cyclin B activity prevents the proper regulation of topoisomerase III (Top3) function, disrupting a recombination step that occurs after the assembly of Rhp51 foci. This effect of Cdc2-cyclin B kinase on Top3 function is mediated by the BRCT-domain-containing checkpoint protein Crb2, thus linking checkpoint proteins directly with recombinational repair in G(2). Our data suggest a model in which CDK activity links processing of recombination intermediates to cell cycle progression via checkpoint proteins.

CDC2 Protein Kinase↗
PubMed2002

Checkpoints: how to flag up double-strand breaks.

How checkpoint pathways recognise double-strand breaks has long been a mystery. Recent studies have found that two distinct checkpoint protein complexes associate independently with chromatin at the sites of DNA damage. Why do two distinct mechanisms recognise strand lesions, and what does this tell us about the checkpoint pathways?

Cell Cycle Proteins↗
PubMed2002

Deletion mutants in COP9/signalosome subunits in fission yeast Schizosaccharomyces pombe display distinct phenotypes.

The COP9/signalosome complex is highly conserved in evolution and possesses significant structural similarity to the 19S regulatory lid complex of the proteasome. It also shares limited similarity to the translation initiation factor eIF3. The signalosome interacts with multiple cullins in mammalian cells. In the fission yeast Schizosaccharomyces pombe, the Csn1 subunit is required for the removal of covalently attached Nedd8 from Pcu1, one of three S. pombe cullins. It remains unclear whether this activity is required for all the functions ascribed to the signalosome. We previously identified Csn1 and Csn2 as signalosome subunits in S. pombe. csn1 and csn2 null mutants are DNA damage sensitive and exhibit slow DNA replication. Two further putative subunits, Csn4 and Csn5, were identified from the S. pombe genome database. Herein, we characterize null mutations of csn4 and csn5 and demonstrate that both genes are required for removal of Nedd8 from the S. pombe cullin Pcu1 and that their protein products associate with Csn1 and Csn2. However, neither csn4 nor csn5 null mutants share the csn1 and csn2 mutant phenotypes. Our data suggest that the subunits of the signalosome cannot be considered as a distinct functional unit and imply that different subunits of the signalosome mediate distinct functions.

COP9 Signalosome Complex↗