Biomedical subjects
Publications and source records attributed to K Labib.
The MCM2-7 complex is essential for both the initiation and elongation phases of eukaryotic chromosome replication. There is some evidence that MCM2-7 proteins may act as a DNA helicase; at the same time, a variety of other DNA helicases have also been implicated in the replication of eukaryotic chromosomes.
A prereplicative complex (pre-RC) of proteins is assembled at budding yeast origins of DNA replication during the G1-phase of the cell cycle, as shown by genomic footprinting. The proteins responsible for this prereplicative footprint have yet to be identified but are likely to be involved in the earliest stages of the initiation step of chromosome replication. Here we show that MCM2-7 proteins are essential for both the formation and maintenance of the pre-RC footprint at the origin ARS305. It is likely that pre-RCs contain heteromeric complexes of MCM2-7 proteins, since degradation of Mcm2, 3, 6, or 7 during G1-phase, after pre-RC formation, causes loss of Mcm4 from the nucleus. It has been suggested that pre-RCs on unreplicated chromatin may generate a checkpoint signal that inhibits premature mitosis during S-phase. We show that, although mitosis does indeed occur in the absence of replication if MCM proteins are degraded during G1-phase, anaphase is prevented if MCMs are degraded during S-phase. Our data indicate that pre-RCs do not play a direct role in checkpoint control during chromosome replication.
Little is known about the DNA helicases required for the elongation phase of eukaryotic chromosome replication. Minichromosome maintenance (MCM) protein complexes have DNA helicase activity but have only been functionally implicated in initiating DNA replication. Using an improved method for constructing conditional degron mutants, we show that depletion of MCMs after initiation irreversibly blocks the progression of replication forks in Saccharomyces cerevisiae. Like the Escherichia coli dnaB and SV40 T antigen helicases, therefore, the MCM complex is loaded at origins before initiation and is essential for elongation. Restricting MCM loading to the G(1) phase ensures that initiation and elongation occur just once per cell cycle.
Cdc45p assembles at replication origins before initia tion and is required for origin firing in Saccharomyces cerevisiae. A heat-inducible cdc45 degron mutant was constructed that promotes rapid degradation of Cdc45p at the restrictive temperature. Consistent with a role in initiation, loss of Cdc45p in G(1) prevents all detectable DNA replication without preventing subsequent entry into mitosis. Loss of Cdc45p activity during S-phase blocks S-phase completion but not activation of replication checkpoints. Using density substitution, we show that after allowing replication fork establishment, Cdc45p inactivation prevents the subsequent progression of individual replication forks. This provides the first direct functional evidence that Cdc45p plays an essential role during elongation. Thus, like the large T antigen in SV40 replication, Cdc45p plays a central role in both initiation and elongation phases of chromosomal DNA replication.
We describe an in situ technique for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe. After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent. Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence. Cell cycle changes in the chromatin association of proteins can therefore be determined from individual cells in asynchronous cultures. We have applied this method to the DNA replication factor mcm4/cdc21, and find that chromatin association occurs during anaphase B, significantly earlier than is the case in budding yeast. Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). Release of mcm4 from chromatin occurs during S phase and requires DNA replication. Upon overexpressing cdc18, we show that mcm4 is required for re-replication of the genome in the absence of mitosis and is associated with chromatin in cells undergoing re-replication.
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In two independent screens we isolated fission yeast mutations with phenotypes suggesting defects in B-cyclin function or expression. These mutations define a single gene which we call ded1. We show that ded1 encodes a general translation factor that is related in sequence and function to RNA helicases required for translation in other species. Levels of the B-cyclins Cig2 and Cdc13 are dramatically reduced upon inactivation of Ded1, and this reduction is independent of degradation by the anaphase promoting complex. When a ded1 mutant is grown under semi-restrictive conditions, the translation of Cig2 (and to a lesser extent Cdc13), is impaired relative to other proteins. We show that B-cyclin translation is specifically inhibited upon nitrogen starvation of wild-type cells, when B-cyclin/Cdc2 inactivation is a prerequisite for G(1) arrest and subsequent mating. Our data suggest that translational inhibition of B-cyclin expression represents a third mechanism, in addition to cyclin degradation and Rum1 inhibition, that contributes to Cdc2 inactivation as cells exit from the mitotic cell cycle and prepare for meiosis.
Cyclin-dependent kinases (CDKs) activate the firing of replication origins during the S phase of the cell cycle. They also block re-initiation of DNA replication within a single cell cycle, by preventing the assembly of prereplicative complexes at origins. We show here that, in budding yeast, CDKs exclude the essential prereplicative-complex component Mcm4 from the nucleus. Although origin firing can be triggered by the B-type cyclins only, both G1-phase and B-type cyclins cause exit of Mcm4 from the nucleus. These results suggest that G1 cyclins may diminish the cell's capacity to assemble prereplicative complexes before B-type cyclins trigger origin firing during S phase.
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The onset of S phase in fission yeast is regulated at Start, the point of commitment to the mitotic cell cycle. The p34cdc2 kinase is essential for G1 progression past Start, but until now its regulation has been poorly understood. Here we show that the cig2/cyc17 B-type cyclin has an important role in G1 progression, and demonstrate that p34cdc2 kinase activity is periodically associated with cig2 in G1. Cells lacking cig2 are defective in G1 progression, and this is particularly clear in small cells that must regulate Start with respect to cell size. We also find that the cig1 B-type cyclin can promote G1 progression. Whilst p25rum1 can inhibit cig2/cdc2 activity in vitro, and may transiently inhibit this complex in vivo, cig1 is regulated independently of p25rum1. Since cig1/cdc2 kinase activity peaks in mitotic cells, and decreases after mitosis with similar kinetics to cdc13-associated kinase activity, we suggest that cig2 is likely to be the principal fission yeast G1 cyclin. cig2 protein levels accumulate in G1 cells, and we propose that p25rum1 may transiently inhibit cig2-associated p34cdc2 activity until the critical cell size required for Start is reached.
A clearer picture of replication control is emerging through the characterization of proteins, such as cdc18/Cdc6 and members of the mini-chromosome maintenance (MCM) protein family, that are involved in the initiation step. Cyclin B dependent kinases have conserved roles in both Saccharomyces cerevisiae and Schizosaccharomyces pombe, switching on DNA replication in G1 and preventing re-replication in G2. A model is suggested where MCMs and CDKs play complementary roles to ensure 'once-per-cell-cycle' replication, with CDKs maintaining a G1 or G2 state, whereas MCMs provide a cis-acting control on chromatin to prevent reinitiation during a single S phase.
A large number of dominant mutants have been generated in the fission yeast cdc2 gene, causing lethality when expressed in wild-type cells. The mutants interfere with distinct aspects of p34cdc2 function, producing one of four different phenotypes: mitotic arrest, multiple rounds of S phase in the absence of mitosis, premature mitosis or G2 arrest. The mitotic mutants DL41, DL45 and DL50 are characterized in this paper. Over-expression of DL41 or DL45 causes mitotic arrest, specifically interfering with sister chromatid separation, without preventing spindle elongation. This suggests a role for p34cdc2 in triggering sister chromatid separation at anaphase. DL41 and DL45 also cause abnormal septum formation, suggesting that p34cdc2 may also be involved in regulating this process in fission yeast. These mitotic aspects of p34cdc2 function may involve interaction with p13suc1, since increased expression of suc1 partially suppresses DL41 and DL45. Over-expression of DL50 causes premature mitotic entry in cells that have not completed S phase, resulting in lethality. DL41, DL45 and DL50 correspond to mutation of p34cdc2 residues predicted to be on the surface of the protein, identifying potential sites of interaction with mitotic regulators of p3cdc2, and these residues are conserved amongst cdc2 proteins found in other eukaryotes.
The p34cdc2 kinase is essential for progression past Start in the G1 phase of the fission yeast cell cycle, and also acts in G2 to promote mitotic entry. Whilst very little is known about the G1 function of cdc2, the rum1 gene has recently been shown to encode an important regulator of Start in fission yeast, and a model for rum1 function suggests that it inhibits p34cdc2 activity. Here we present genetic data suggesting that rum1 maintains p34cdc2 in a pre-Start G1 form, inhibiting its activity until the cell achieves the critical mass required for Start, and find that in the absence of rum1 p34cdc2 has increased Start activity in vivo. It is also known that mutation of cdc2, or overexpression of rum1, can disrupt the dependency of S-phase upon mitosis, resulting in an extra round of S-phase in the absence of mitosis. We show that cdc2 and rum1 interact in this process, and describe dominant cdc2 mutants causing multiple rounds of S-phase in the absence of mitosis. We suggest that interaction of rum1 and cdc2 regulates Start, and this interaction is important for the regulation of S-phase within the cell cycle.
We have identified the rum1+ gene as a new regulator of the G1-phase of the fission yeast cell cycle. rum1+ determines the cell cycle timing of Start, by maintaining cells in a pre-Start state until they have attained a minimal critical mass. Cells lacking rum1+ are unable to arrest in pre-Start G1 in response to nitrogen starvation and are subsequently sterile. In addition, rum1+ prevents entry into mitosis from pre-Start G1, as shown by the fact that cdc10 mutants in the absence of rum1+ undergo lethal mitosis without entering S-phase.
Five monoclonal antibodies raised against rat PCNA cross-reacted with a similar protein in the fission yeast Schizosaccharomyces pombe. One of these was used to screen an S.pombe cDNA expression library. An incomplete cDNA was isolated and used to screen a genomic library, identifying a single gene, designated pcn1+ (proliferating cell nuclear antigen). The gene encodes a protein of 260 amino acids, with a deduced sequence 52% identical to human and rat PCNAs, which are 98.5% identical to each other. The budding yeast PCNA homologue POL30 is only 35% identical to the human and rat proteins. Pcn1 has a region near the C-terminus of particularly high homology to higher eukaryotic PCNA proteins. pcn1+ is essential for viability and delta pcn1 cells undergo aberrant DNA replication before cell cycle arrest. Overproduction of the protein leads to cell cycle delay in G2. Disruption of pcn1+ is complemented by the human PCNA gene, demonstrating that these genes are functional homologues.
A quadriplegic patient with a C6 lesion had a stroke with aphasia and right hemiplegia 20 years after his cervical cord injury. The combination of hemiplegia and quadriplegia created unusual rehabilitation problems, the most disabling of which was a painful flexion contracture of the right elbow that prevented any useful right arm function. A sequence of phenol nerve blocks produced almost complete resolution of this contracture and was a key factor in his successful return to independent living. Although phenol block is much less commonly performed in the upper than lower extremities, its judicious use in carefully selected cases may be beneficial to the patient's functional outcome.