Biomedical subjects
Publications and source records attributed to S A MacNeill.
The correct processing of Okazaki fragments during lagging-strand DNA replication has a vital role in maintaining genome integrity. Recent findings suggest that, in eukaryotes, the processing of Okazaki fragments occurs by a two-step mechanism governed by the single-stranded DNA binding factor RPA.
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The analysis of completed archaeal genome sequences led to the identification of a set of approximately 10-20 genes whose protein products were inferred to be involved in chromosomal DNA replication. Until recently, however, little was known of the biochemical properties of these proteins. Here, I review recent progress in this area brought about by biochemical and structural analysis. Aside from shedding considerable new light on the molecular machinery of DNA replication in the archaea, the results of these studies also present new opportunities for understanding the molecular events of chromosomal DNA replication in eukaryotic cells.
Direct interaction between DNA polymerase delta and its processivity factor proliferating cell nuclear antigen (PCNA) is essential for effective replication of the eukaryotic genome, yet the precise manner by which this occurs is unclear. We show that the 54 kDa subunit of DNA polymerase delta from Schizosaccharomyces pombe interacts directly with Pcn1 (PCNA) both in vivo and in vitro. Binding is effected via a short sequence at the C-terminus of Cdc27 with significant similarity to the canonical PCNA binding motif first identified in the mammalian p21(Cip1) protein. This motif is both necessary and sufficient for binding of Pcn1 by Cdc27 in vitro and is essential for Cdc27 function in vivo. We also show that the Pcn1 binding motif in Cdc27 is distinct from its binding site for Cdc1, the 55 kDa B-subunit of polymerase delta, and present evidence that Cdc27 can bind to Pcn1 and Cdc1 simultaneously. Finally, we show that Cdc27 performs at least two distinct essential functions, one of which is independent of Pcn1 binding.
In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe replication factor C (RF-C) plays key roles both in chromosomal DNA replication and in DNA replication checkpoint function. At the replication fork, the five-subunit RF-C complex functions to load the trimeric polymerase accessory factor PCNA onto DNA. PCNA then acts as a sliding clamp, tethering Pol delta to the DNA to maximise its processivity. Here we describe the cloning of the S. pombe rfc3+ gene, encoding a homologue of the S. cerevisiae Rfc3 and human hRFC36 proteins. The 1026 bp rfc3+ ORF is interrupted by five introns, ranging in size from 49 to 165 bp. The spliced ORF is predicted to encode a 342 amino-acid protein that is approximately 50% identical at the amino acid sequence level to the S. cerevisiae Rfc3 and human hRFC36 proteins. As expected, S. pombe rfc3+ is an essential gene, with rfc3delta cells being defective for DNA replication. Loss of rfc3+ function can be rescued by heterologous expression of either the S. cerevisiae Rfc3 or human hRFC36 proteins in S. pombe.
In this report, we investigated the phenotypes caused by temperature-sensitive (ts) mutant alleles of dna2(+) of Schizosaccharomyces pombe, a homologue of DNA2 of budding yeast, in an attempt to further define its function in vivo with respect to lagging-strand synthesis during the S-phase of the cell cycle. At the restrictive temperature, dna2 (ts) cells arrested at late S-phase but were unaffected in bulk DNA synthesis. Moreover, they exhibited aberrant mitosis when combined with checkpoint mutations, in keeping with a role for Dna2 in Okazaki fragment maturation. Similarly, spores in which dna2(+) was disrupted duplicated their DNA content during germination and also arrested at late S-phase. Inactivation of dna2(+) led to chromosome fragmentation strikingly similar to that seen when cdc17(+), the DNA ligase I gene, is inactivated. The temperature-dependent lethality of dna2 (ts) mutants was suppressed by overexpression of genes encoding subunits of polymerase delta (cdc1(+) and cdc27(+)), DNA ligase I (cdc17(+)), and Fen-1 (rad2(+)). Each of these gene products plays a role in the elongation or maturation of Okazaki fragments. Moreover, they all interacted with S. pombe Dna2 in a yeast two-hybrid assay, albeit to different extents. On the basis of these results, we conclude that dna2(+) plays a direct role in the Okazaki fragment elongation and maturation. We propose that dna2(+) acts as a central protein to form a complex with other proteins required to coordinate the multienzyme process for Okazaki fragment elongation and maturation.
DNA polymerase delta (Pol delta), which plays keys roles in DNA replication, repair and recombination in eukaryotic cells, comprises at least two essential subunits - a large catalytic subunit (PolD1) possessing both DNA polymerase and 3'-5' exonuclease activities, and a smaller subunit (PolD2) whose function is not yet clear. Here we describe the cloning and sequencing of a Xenopus cDNA encoding a homologue of the PolD2 subunit. This protein (designated XlCdc1) is 69% identical to the human PolD2 protein and 34% identical to fission yeast Cdc1. Alignment of PolD2 protein sequences across ten eukaryotic species identifies 36 invariant amino-acid positions. These 36 residues are located within ten conserved regions (designated I-X) likely to have key functional roles. Consistent with this, the mutations in six previously identified yeast mutant PolD2 proteins map within conserved regions III, VI, VII and VIII. Several of the invariant amino acids are also conserved across the archaeal DNA polymerase II DP1 protein family.
Replication factor C (RF-C) is a five subunit DNA polymerase (Pol) delta/straightepsilon accessory factor required at the replication fork for loading the essential processivity factor PCNA onto the 3'-ends of nascent DNA strands. Here we describe the genetic analysis of the rfc2 +gene of the fission yeast Schizosaccharomyces pombe encoding a structural homologue of the budding yeast Rfc2p and human hRFC37 proteins. Deletion of the rfc2 + gene from the chromosome is lethal but does not result in the checkpoint-dependent cell cycle arrest seen in cells deleted for the gene encoding PCNA or for those genes encoding subunits of either Pol delta or Pol straightepsilon. Instead, rfc2 Delta cells proceed into mitosis with incompletely replicated DNA, indicating that the DNA replication checkpoint is inactive under these conditions. Taken together with recent results, these observations suggest a simple model in which assembly of the RF-C complex onto the 3'-end of the nascent RNA-DNA primer is the last step required for the establishment of a checkpoint-competent state.
Highly purified DNA polymerase delta from the fission yeast Schizosaccharomyces pombe is a complex of at least four distinct subunits. Genes encoding three of these (pol3+/cdc6+, cdc1+ and cdc27+) have been characterised previously. Here we describe the isolation and characterisation of cdm1+, the gene encoding the smallest (22kDa) subunit of the Pol delta complex. Over-expression of cdm1+, which encodes a 160 amino-acid protein with no significant sequence similarity to proteins in current databases, is able to rescue cells carrying temperature-sensitive mutations in either pol3+/cdc6+, cdc1+ or cdc27+. Cells deleted for cdm1+ are viable, indicating that cdm1+ is non-essential for mitotic growth, and are no more sensitive to a variety of DNA replication inhibitors and DNA damaging agents than are wild-type cells. In addition, over-expression of cdm1+ suppresses the temperature-sensitive cdc24-M38 mutant suggesting that cdc24+ may also have a role in DNA polymerase delta function.
DNA polymerase delta (pol delta) plays an essential role in DNA replication, repair, and recombination. We have purified pol delta from Schizosaccharomyces pombe more than 10(3)-fold and demonstrated that the polymerase activity of purified S. pombe pol delta is completely dependent on proliferating cell nuclear antigen and replication factor C. SDS/PAGE analysis of the purified fraction indicated that the pol delta complex consists of five subunits that migrate with apparent molecular masses of 125, 55, 54, 42, and 22 kDa. Western blot analysis indicated that the 125, 55, and 54 kDa proteins are the large catalytic subunit (Pol3), Cdc1, and Cdc27, respectively. The identity of the other two subunits, p42 and p22, was determined following proteolytic digestion and sequence analysis of the resulting peptides. The peptide sequences derived from the p22 subunit indicated that this subunit is identical to Cdm1, previously identified as a multicopy suppressor of the temperature-sensitive cdc1-P13 mutant, whereas peptide sequences derived from the p42 subunit were identical to a previously uncharacterized ORF located on S. pombe chromosome 1.
Studies on DNA replication in S. pombe have provided powerful insights into the way in which the genome of this model eukaryote is replicated and how the replication process is controlled. These studies have been facilitated by the simplicity and range of methods available in this organism for physiological and genetic analysis of DNA replication mutants. In the future, continued focus on the analysis of such mutants, coupled with increasingly sophisticated biochemical investigation of the processes of DNA replication in both wild-type and mutant cells, will ensure continued rapid progress in this area.
cdc1+ is required for cell cycle progression in Schizosaccharomyces pombe. Cells carrying temperature-sensitive cdc1 mutants undergo cell cycle arrest when shifted to the restrictive temperature, becoming highly elongated. Here we describe the cloning and sequencing of cdc1+, which is shown to encode a 462 residue protein that displays significant sequence similarity to the small subunit of mammalian DNA polymerase delta. cdc1+ interacts genetically with pol3+, which encodes the large subunit of DNA polymerase delta in fission yeast, and the Cdc1 protein binds to Pol3 in vitro, strongly suggesting that Cdc1 is likely to be the small subunit of Pol delta. In addition, we show that cdc1+ overexpression is sufficient to rescue cells carrying temperature-sensitive cdc27 alleles and that the Cdc1 and Cdc27 proteins interact in vivo and in vitro. Deletion of either cdc1+ or cdc27+ results in cell cycle arrest with the arrested cells having a single nucleus with 2C DNA content. No evidence was obtained for a cut phenotype, indicating that neither cdc1+ nor cdc27+ is required for checkpoint function. cdc1 mutant cells are supersensitive to the DNA synthesis inhibitor hydroxyurea and to the DNA damaging agent MMS, display increased frequency of mini-chromosome loss and have an extended S phase.
The p34cdc2 protein serine-threonine kinase plays an essential role in the life cycle of fission yeast, being required for both the G1-S and G2-M transitions during mitotic growth, and also for the second meiotic nuclear division. Functional homologues of p34cdc2 (each ca. 60% identical to the fission yeast prototype) have been isolated from organisms as diverse as humans, insects and plants, and there is now considerable evidence supporting the view that fundamental aspects of the cell cycle controls uncovered in fission yeast will prove to be conserved in all eukaryotes. By comparing the amino acid sequences of fission yeast p34cdc2 with its higher eukaryotic counterparts it is possible to identify conserved residues that are likely to be centrally important for p34cdc2 function. Here the effects are described of mutating a number of these conserved residues. Twenty-three new mutant alleles have been constructed and tested. We show that replacing cysteine 67 with tryptophan renders the resulting mutant protein p80cdc25-independent (while neither leucine, isoleucine nor valine has this effect) and that several of the amino acids within the highly conserved PSTAIRE region are not absolutely required for p34cdc2 function. Five acidic amino acids have also been mutated within p34cdc2, which are invariant across the eukaryotic protein kinase family. Acid-to-base mutations at three of these residues resulted in a dominant-negative, cell cycle arrest phenotype while similar mutations at the other two simply abolished p34cdc2 protein function. The results are discussed with reference to the predicted tertiary structure of the p34cdc2 enzyme.
The p34cdc2 protein kinase plays a key role in the control of the mitotic cell cycle of fission yeast, being required for both entry into S-phase and for entry into mitosis in the mitotic cell cycle, as well as for the initiation of the second meiotic nuclear division. In recent years, structural and functional homologues of p34cdc2, as well as several of the proteins that interact with and regulate p34cdc2 function in fission yeast, have been identified in a wide range of higher eukaryotic cell types, suggesting that the control mechanisms uncovered in this simple eukaryote are likely to be well conserved across evolution. Here we describe the construction and characterisation of a fission yeast strain in which the endogenous p34cdc2 protein is entirely absent and is replaced by its human functional homologue p34CDC2. We have used this strain to analyse aspects of the function of the human p34CDC2 protein genetically. We show that the function of the human p34CDC2 protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80cdc25, that it responds to altered levels of both the mitotic inhibitor p107wee1 and the p34cdc2-binding protein p13suc1, and is lethal in combination with the mutant B-type cyclin p56cdc13-117. In addition, we demonstrate that the human p34CDC2 protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34CDC2 proteins in fission yeast.
The p34cdc2 protein kinase plays a central role in the regulation of the eukaryotic cell cycle, being required both in late G1 for the commitment to S-phase and in late G2 for the initiation of mitosis. p34cdc2 also determines the precise timing of entry into mitosis in fission yeast, where a number of gene products that regulate p34cdc2 activity have been identified and characterised. To investigate further the mitotic role of p34cdc2 in this organism we have isolated new cold-sensitive p34cdc2 mutants. These are defective only in their G2 function and are extragenic suppressors of the lethal premature entry into mitosis brought about by mutating the mitotic inhibitor p107wee1 and overproducing the mitotic activator p80cdc25. One of the mutant proteins p34cdc2-E8 is only functional in the absence of p107wee1, and all the mutant strains have reduced histone H1 kinase activity in vitro. Each mutant allele has been cloned and sequenced, and the lesions responsible for the cold-sensitive phenotypes identified. All the mutations were found to map to regions that are conserved between the fission yeast p34cdc2 and functional homologues from higher eukaryotes.
The cell division cycle gene cdc27+ of the fission yeast Schizosaccharomyces pombe is required for the transition from G2 into mitosis. Genetic and physiological experiments suggest a close relationship between cdc27+ and the cdc2+ gene, a key regulator of mitosis in yeast and also in higher eukaryotic cells. We isolated the cdc27+ gene by complementation of a temperature-sensitive cdc27 mutant. The DNA sequence of this gene predicts a 1116 nucleotide open reading frame split by five short introns, ranging in size from 49 to 74 nucleotides. Analysis of cDNA clones confirmed the structure of the gene. The deduced cdc27+ gene product consists of 372 amino acids with a predicted Mr of 43 kDa. No homology of the predicted protein with known proteins could be found, thus the cdc27+ gene encodes a novel function required for the G2-M transition. Northern analysis revealed two mRNAs of 1.4 and 2.2 kb transcribed from this gene, the smaller transcript being approximately tenfold more abundant than the larger. The level of cdc27+ mRNAs remained constant through the cell cycle indicating that the time of action of the cdc27+ gene, which is known to be regulated by elements of the mitotic control, is not determined by periodic accumulation of its transcripts.
The protein serine-threonine kinase p34cdc2+ plays a central role in the control of the mitotic cell cycle of the fission yeast Schizosaccharomyces pombe. p34cdc2+ function is required both for the initiation of DNA replication and for entry into mitosis, and is also required for the initiation of the second meiotic nuclear division. Recent extensive analysis of p34cdc2+ homologue proteins in higher eukaryotes has demonstrated that p34cdc2+ function is likely to be conserved in all eukaryotic cells. Here we report the isolation and characterisation of five new temperature-sensitive alleles of the cdc2+ gene. All five have been cloned and sequenced, together with the meiotically defective cdc2-N22 allele, bringing the total of p34cdc2+ mutants cloned in this and previous reports to seventeen. The five temperature-sensitive alleles define four separate mutations within the p34cdc2+ protein sequence, two of which give rise to cell cycle arrest in G2 only, when shifted to the restrictive temperature. The nature of the mutation in each protein is described and possible implications for the structure and function of p34cdc2+ discussed.
The suitability of fission yeast as a model for understanding the eukaryotic cell cycle has been validated in five years of exciting developments. We review recent advances in understanding the nature of the controls that regulate progression through the cell cycle and the coordination of DNA replication and mitosis.