Stephen E. Kearsey

Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas

Many individuals with multiple or large colorectal adenomas or early-onset colorectal cancer (CRC) have no detectable germline mutations in the known cancer predisposition genes. Using whole-genome sequencing, supplemented by linkage and association analysis, we identified specific heterozygous POLE or POLD1 germline variants in several multiple-adenoma and/or CRC cases but in no controls. The variants associated with susceptibility, POLE p.Leu424Val and POLD1 p.Ser478Asn, have high penetrance, and POLD1 mutation was also associated with endometrial cancer predisposition. The mutations map to equivalent sites in the proofreading (exonuclease) domain of DNA polymerases ɛ and δ and are predicted to cause a defect in the correction of mispaired bases inserted during DNA replication. In agreement with this prediction, the tumors from mutation carriers were microsatellite stable but tended to acquire base substitution mutations, as confirmed by yeast functional assays. Further analysis of published data showed that the recently described group of hypermutant, microsatellite-stable CRCs is likely to be caused by somatic POLE mutations affecting the exonuclease domain.

MCM proteins: evolution, properties, and role in DNA replication

5. Regulation of MCM proteins during the cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 5.1. MCM protein levels in proliferating and non-proliferating cells . . . . . . . . . . . . . . . . . 125 5.2. Involvement of ORC and Cdc6rcdc18 in binding of MCM proteins to chromatin . . . . . . 126 5.3. Role of phosphorylation in regulating MCM function . . . . . . . . . . . . . . . . . . . . . . . 127 5.4. Relevance of the nuclear membrane to MCM function . . . . . . . . . . . . . . . . . . . . . . 129

G1-phase and B-type cyclins exclude the DNA-replication factor Mcm4 from the nucleus

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.

Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18

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.

Global Role for Polyadenylation-Assisted Nuclear RNA Degradation in Posttranscriptional Gene Silencing

ABSTRACT Fission yeast Cid14, a component of the TRAMP (Cid14/Trf4-Air1-Mtr4 polyadenylation) complex, polyadenylates nuclear RNA and stimulates degradation by the exosome for RNA quality control. Here, we analyze patterns of global gene expression in cells lacking the Cid14 or the Dis3/Rpr44 subunit of the nuclear exosome. We found that transcripts from many genes induced during meiosis, including key regulators, accumulated in the absence of Cid14 or Dis3. Moreover, our data suggest that additional substrates include transcripts involved in heterochromatin assembly. Mutant cells lacking Cid14 and/or Dis3 accumulate transcripts corresponding to naturally silenced repeat elements within heterochromatic domains, reflecting defects in centromeric gene silencing and derepression of subtelomeric gene expression. We also uncover roles for Cid14 and Dis3 in maintaining the genomic integrity of ribosomal DNA. Our data indicate that polyadenylation-assisted nuclear RNA turnover functions in eliminating a variety of RNA targets to control diverse processes, such as heterochromatic gene silencing, meiotic differentiation, and maintenance of genomic integrity.

Enigmatic variations: divergent modes of regulating eukaryotic DNA replication.

Proteins involved in DNA replication are conserved from yeast to mammals, suggesting that the mechanism was established at an early stage of eukaryotic evolution. In spite of this common origin, recent findings have revealed surprising variations in how replication initiation is controlled, implying that a conserved mechanism has not necessarily resulted in regulatory conservation.

DNA damage induces Cdt1 proteolysis in fission yeast through a pathway dependent on Cdt2 and Ddb1

Cdt1 is an essential protein required for licensing of replication origins. Here, we show that in Schizosaccharomyces pombe, Cdt1 is proteolysed in M and G1 phases in response to DNA damage and that this mechanism seems to be conserved from yeast to Metazoa. This degradation does not require Rad3 and Cds1, indicating that it is independent of classic DNA damage and replication checkpoint pathways. Damage‐induced degradation of Cdt1 is dependent on Cdt2 and Ddb1, which are components of a Cul4 ubiquitin ligase. We also show that Cdt2 and Ddb1 are needed for cell‐cycle changes in Cdt1 levels in the absence of DNA damage. Cdt2 and Ddb1 have been shown to be involved in the degradation of the Spd1 inhibitor of ribonucleotide reductase after DNA damage, and we speculate that Cdt1 downregulation might contribute to genome stability by reducing demand on dNTP pools during DNA repair.

The Kinetochore Proteins Pcs1 and Mde4 and Heterochromatin Are Required to Prevent Merotelic Orientation

Summary Background Accurate chromosome segregation depends on the establishment of correct—amphitelic—kinetochore orientation. Merotelic kinetochore orientation is an error that occurs when a single kinetochore attaches to microtubules emanating from opposite spindle poles, a condition that hinders segregation of the kinetochore to a spindle pole in anaphase. To avoid chromosome missegregation resulting from merotelic kinetochore orientation, cells have developed mechanisms to prevent or correct merotelic attachment. A protein called Pcs1 has been implicated in preventing merotelic attachment in mitosis and meiosis II in the fission yeast S. pombe. Results We report that Pcs1 forms a complex with a protein called Mde4. Both Pcs1 and Mde4 localize to the central core of centromeres. Deletion of mde4+, like that of pcs1+, causes the appearance of lagging chromosomes during the anaphases of mitotic and meiosis II cells. We provide evidence that the kinetochores of lagging chromosomes in both pcs1 and mde4 mutant cells are merotelically attached. In addition, we find that lagging chromosomes in cells with defective centromeric heterochromatin also display features consistent with merotelic attachment. Conclusions We suggest that the Pcs1/Mde4 complex is the fission yeast counterpart of the budding yeast monopolin subcomplex Csm1/Lrs4, which promotes the segregation of sister kinetochores to the same pole during meiosis I. We propose that the Pcs1/Mde4 complex acts in the central kinetochore domain to clamp microtubule binding sites together, the centromeric heterochromatin coating the flanking domains provides rigidity, and both systems contribute to the prevention of merotelic attachment.

Nuclear distribution and chromatin association of DNA polymerase α-primase is affected by TEV protease cleavage of Cdc23 (Mcm10) in fission yeast

BackgroundCdc23/Mcm10 is required for the initiation and elongation steps of DNA replication but its biochemical function is unclear. Here, we probe its function using a novel approach in fission yeast, involving Cdc23 cleavage by the TEV protease.ResultsInsertion of a TEV protease cleavage site into Cdc23 allows in vivo removal of the C-terminal 170 aa of the protein by TEV protease induction, resulting in an S phase arrest. This C-terminal fragment of Cdc23 is not retained in the nucleus after cleavage, showing that it lacks a nuclear localization signal and ability to bind to chromatin. Using an in situ chromatin binding procedure we have determined how the S phase chromatin association of DNA polymerase α-primase and the GINS (Sld5-Psf1-Psf2-Psf3) complex is affected by Cdc23 inactivation. The chromatin binding and sub-nuclear distribution of DNA primase catalytic subunit (Spp1) is affected by Cdc23 cleavage and also by inactivation of Cdc23 using a degron allele, implying that DNA polymerase α-primase function is dependent on Cdc23. In contrast to the effect on Spp1, the chromatin association of the Psf2 subunit of the GINS complex is not affected by Cdc23 inactivation.ConclusionAn important function of Cdc23 in the elongation step of DNA replication may be to assist in the docking of DNA polymerase α-primase to chromatin.

Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA.

To investigate how a defective replicon might acquire replication competence, we have studied the reversion of autonomously replicating sequence (ARS) mutations. By mutagenesis of a Saccharomyces cerevisiae plasmid lacking a functional origin of replication, we have obtained a series of cis-acting mutations which confer ARS activity on the plasmid. The original plasmid contained an ARS element inactivated by point mutation, but surprisingly only 1 of the 10 independent Ars+ revertants obtained shows a back mutation in this element. In the remainder of the revertants, sequence changes in the M13 vector DNA generate new ARSs. In two cases, a single nucleotide change results in an improved match to the ARS consensus, while six other cases show small duplications of vector sequence creating additional matches to the ARS consensus. These results suggest that changes in replication origin distribution may arise de novo by point mutation rather than by transposition of preexisting origin sequences.