Shakil Ahmed

Wat1/pop3, a Conserved WD Repeat Containing Protein Acts Synergistically with Checkpoint Kinase Chk1 to Maintain Genome Ploidy in Fission Yeast S. pombe

Aberrant chromosome segregation defects can lead to aneuploidy, a common characteristic of human solid tumors. Aneuploidy is generated due to defects in the mitotic spindle or due to inefficient mitotic checkpoint response. We have isolated a novel mutant allele of wat1, a WD repeat containing protein that exhibits conditional synthetic lethality with chk1 knock out. We observed only a marginal decrease in the level of α tubulin protein level in wat1-17 mutants after prolong exposure at semi permissive temperature. Interestingly the protein level of α-tubulin was reduced in the chk1Δ wat1-17 double mutant at 18°C with defective microtubule structure. Consistent with loss of microtubule structure in the chk1 deletion background, the double mutant of wat1-17 chk1Δ was hypersensitive to the microtubule destabilizing agent TBZ suggesting severe defects in microtubule integrity in wat1-17 mutant in the absence of Chk1. Combination of wat1-17 with the chk1 deletion also aggravates the defects in the maintenance of genome ploidy. The mutation in wat1-17 was mapped to Cys 233 that was changed to tyrosine. Based on the molecular modeling studies, we hypothesize that the substitution of the bulky Tyr residue at Cys233 position in wat1-17 mutant results in conformational changes. This in turn can affect its intercations with other interacting partners and perturb the overall functions of the Wat1 protein.

Fission yeast Drp1 is an essential protein required for recovery from DNA damage and chromosome segregation.

DNA double strand breaks (DSBs) are the most critical types of DNA damage that can leads to chromosomal aberrations, genomic instability and cancer. Several genetic disorders such as Xeroderma pigmentosum are linked with defects in DNA repair. Human Rint1, a TIP1 domain containing protein is involved in membrane trafficking but its role in DNA damage response is elusive. In this study we characterized the role of Drp1 (damage responsive protein 1), a Rint1 family protein during DNA damage response in fission yeast. We identified that Drp1 is an essential protein and indispensable for survival and growth. Using in vitro random mutagenesis approach we isolated a temperature sensitive mutant allele of drp1 gene (drp1-654) that exhibits sensitivity to DNA damaging agents, in particular to alkylation damage and UV associated DNA damage. The drp1-654 mutant cells are also sensitive to double strand break inducing agent bleomycin. Genetic interaction studies identified that Rad50 and Drp1 act in the same pathway during DNA damage response and the physical interaction of Drp1 with Rad50 was unaffected in drp1-654 mutant at permissive as well as non permissive temperature. Furthermore Drp1 was found to be required for the recovery from MMS induced DNA damage. We also demonstrated that the Drp1 protein localized to nucleus and was required to maintain the chromosome stability.

DNA topoisomerase 2 mutant allele mildly delays the mitotic progression and activates the checkpoint protein kinase Chk1 in fission yeast Schizosaccharomyces pombe

DNA topoisomerases are specialized nuclear enzymes that perform topological modifications on double-stranded DNA (dsDNA) and hence are essential for DNA metabolism such as replication, transcription, recombination, condensation and segregation. In a genetic screen, we identified a temperature-sensitive mutant allele of topoisomerase 2 that exhibits conditional synthetic lethality with a chk1 knockout strain. The mutant allele of topoisomerase 2 is defective in chromosome segregation at a non-permissive temperature and there was increase in chromosome segregation defects in the double mutant of top2-10 and chk1 delete at a non-permissive temperature. More importantly, topoisomearse 2 mutant cells mildly delay the mitotic progression at non-permissive temperature that is mediated by checkpoint protein kinase Chk1. Additionally, top2-10 mutant cells also activate the Chk1 at a non-permissive temperature and this activation of Chk1 takes place at the time of mitosis. Interestingly, top2-10 mutant cells retain their viability at a non-permissive temperature if the cells are not allowed to enter into mitosis. Taking together our results, we speculate that in the top2-10 mutant, the segregation of entangled chromatids during mitosis could result in delaying the mitotic progression through the activation of Chk1 kinase.

Skp2 inhibits osteogenesis by promoting ubiquitin-proteasome degradation of Runx2.

Osteogenic transcription factor Runx2 is essential for osteoblast differentiation. The activity of Runx2 is tightly regulated at transcriptional as well as post-translational level. However, regulation of Runx2 stability by ubiquitin mediated proteasomal degradation by E3 ubiquitin ligases is little-known. Here, for the first time we demonstrate that Skp2, an SCF family E3 ubiquitin ligase negatively targets Runx2 by promoting its polyubiquitination and proteasome dependent degradation. Co-immunoprecipitation studies revealed that Skp2 physically interacts with Runx2 both in a heterologous as well as physiologically relevant system. Functional consequences of Runx2-Skp2 physical interaction were then assessed by promoter reporter assay. We show that Skp2-mediated downregulation of Runx2 led to reduced Runx2 transactivation and osteoblast differentiation. On the contrary, inhibition of Skp2 restored Runx2 levels and promoted osteoblast differentiation. We further show that Skp2 and Runx2 proteins are co-expressed and show inverse relation in vivo such as in lactating, ovariectomized and estrogen-treated ovariectomized animals. Together, these data demonstrate that Skp2 targets Runx2 for ubiquitin mediated degradation and hence negatively regulate osteogenesis. Therefore, the present study provides a plausible therapeutic target for osteoporosis or cleidocranial dysplasia caused by the heterozygous mutation of Runx2 gene.

Role of swi7H4 mutant allele of DNA polymerase α in the DNA damage checkpoint response.

Besides being a mediator of initiation of DNA replication, DNA polymerase α plays a key role in chromosome maintenance. Swi7H4, a novel temperature sensitive mutant of DNA polymerase α was shown to be defective in transcriptional silencing at the mating type centromere and telomere loci. It is also required for the establishment of chromatin state that can recruit the components of the heterochromatin machinery at these regions. Recently the role of DNA polymerase α in the S-phase alkylation damage response in S. pombe has also been studied. Here we investigate whether defects generated by swi7H4, a mutant allele of DNA polymerase α can activate a checkpoint response. We show that swi7H4 exhibit conditional synthetic lethality with chk1 null mutant and the double mutant of swi7H4 with chk1 deletion aggravate the chromosome segregation defects. More importantly swi7H4 mutant cells delay the mitotic progression at non permissive temperature that is mediated by checkpoint protein kinase Chk1. In addition we show that, in the swi7H4 mutant background, cells accumulate DNA damage at non permissive temperature activating the checkpoint kinase protein Chk1. Further, we observed synthetic lethality between swi7H4 and a number of genes involved in DNA repair pathway at semi permissive temperature. We summarize that defects in swi7H4 mutant results in DNA damage that delay mitosis in a Chk1 dependent manner that also require the damage repair pathway for proper recovery.

C-terminal region of Mad2 plays an important role during mitotic spindle checkpoint in fission yeast Schizosaccharomyces pombe

The mitotic arrest deficiency 2 (Mad2) protein is an essential component of the spindle assembly checkpoint that interacts with Cdc20/Slp1 and inhibit its ability to activate anaphase promoting complex/cyclosome (APC/C). In bladder cancer cell line the C-terminal residue of the mad2 gene has been found to be deleted. In this study we tried to understand the role of the C-terminal region of mad2 on the spindle checkpoint function. To envisage the role of C-terminal region of Mad2, we truncated 25 residues of Mad2 C-terminal region in fission yeast S.pombe and characterized its effect on spindle assembly checkpoint function. The cells containing C-terminal truncation of Mad2 exhibit sensitivity towards microtubule destabilizing agent suggesting perturbation of spindle assembly checkpoint. Further, the C-terminal truncation of Mad2 exhibit reduced viability in the nda3-KM311 mutant background at non-permissive temperature. Truncation in mad2 gene also affects its foci forming ability at unattached kinetochore suggesting that the mad2-∆CT mutant is unable to maintain spindle checkpoint activation. However, in response to the defective microtubule, only brief delay of mitotic progression was observed in Mad2 C-terminal truncation mutant. In addition we have shown that the deletion of two β strands of Mad2 protein abolishes its ability to interact with APC activator protein Slp1/Cdc20. We purpose that the truncation of two β strands (β7 and β8) of Mad2 destabilize the safety belt and affect the Cdc20-Mad2 interaction leading to defects in the spindle checkpoint activation.

Mutant allele of rna14 in fission yeast affects pre-mRNA splicing.

Spliceosome and 3′

Fission yeast cells overproducing HSET/KIFC1 provides a useful tool for identification and evaluation of human kinesin-14 inhibitors

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Phosphorylation of Wat1, human Lst8 homolog is critical for the regulation of TORC2 –Gad8 dependent pathway in fission yeast Schizosacchromyces pombe

-end processing complexes are necessary for the precursor mRNA (pre-mRNA) maturation. Spliceosome complex removes noncoding introns, while 3′

Fission yeast Ctf1, a cleavage and polyadenylation factor subunit is required for the maintenance of genomic integrity

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