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Munira A. Basrai, Ph.D.
Molecular Determinants of Faithful Chromosome Transmission and Cell Cycle Checkpoint Regulation.
A fundamental requirement of the cell division cycle is the maintenance, replication, and segregation of chromosomal DNA. Failure of complex mechanisms involved in maintaining genome integrity has been implicated in cancer, aging, and congenital birth defects. Research in our laboratory focuses on the molecular mechanisms of high fidelity chromosome transmission, the organization of chromatin structure, and the checkpoint regulatory mechanisms that ensure the proper execution of the cell cycle in Saccharomyces cerevisiae and the study of its human homologs. We are also interested in the application of genome technologies such as serial analysis of gene expression (SAGE) and DNA microarrays for analysis of transcription profiles and the identification of small non-annotated open reading frames (NORFs). We use a combination of genetic, biochemical, cell biology and whole genome approaches for analysis of gene function. The latter is made feasible through the application of a colony picking robot and whole genome arrays that we have in our laboratory.
Molecular Determinants of Faithful Chromosome Transmission. CEN DNA sequences and the trans-acting kinetochore components (centromere-specific DNA binding proteins) are required for high fidelity chromosome transmission. Additionally, a higher order chromatin structure provides a framework for interactions of histones, CEN DNA, and the kinetochore. We have characterized genes that are important for the structure/function of the kinetochore and studied the corresponding human homolog. We initiated our studies using a large reference set of chromosome transmission fidelity mutants, the ctf mutants of S. cerevisiae previously isolated by Spencer et al., using a colony color assay for chromosome loss. Using genetic screens we determined that mutations or deletions in S. cerevisiae SPT4 and NUP170 lead to defects in chromosome transmission fidelity and kinetochore integrity. In the first studies we established that Spt4p is a novel component of centromeric and heterochromatic chromatin, which is required for kinetochore function and gene silencing in S. cerevisiae. In cross-species approach we have also shown that the spt4, ctf, and silencing phenotypes of yeast are functionally complemented by a human homolog of SPT4. We established that the evolutionarily conserved centromeric histone H3 (CENH3)variant, Cse4p is mis-localized in spt4 mutants. Mis-localization and overexpression of mammalian CENH3 has been observed in colorectal cancer cell lines. We used budding yeast as a model to establish phenotypic consequences of mislocalized CENH3. Our results established that altered dosage and mis-localization of CENH3 and Histone H3 lead to genome instability. We are currently investigating molecular mechanisms that define the specific localization of CENH3 to the centromeres. In the second study we determined that the evolutionarily conserved Nup170p is a specialized component of the nucleopore complex (NPC) with roles in chromosome segregation and kinetochore function. These studies represent one of the first reports describing a functional relationship between the NPC and chromosome segregation and they have since been followed by similar observations in several other systems, including humans. In an extension of these results, we established an important functional and physical association between members of the Nup170p complex and the spindle checkpoint proteins Mad1p and Mad2p in S. cerevisiae. Prior to our analysis using live cell imaging no reports had been published on the localization of mitotic spindle checkpoint proteins in S. cerevisiae. We recently defined a minimal domain of Mad1p that is required for chromosome transmission and checkpoint functions. Our novel findings that S. cerevisiae Mad1p and Mad2p are localized to the NPC prompted us to investigate the localization of another spindle checkpoint protein Bub3p. We designed a novel genetically engineered reporter strain and showed preferential enrichment of Bub3p at defective kinetochores. This finding was an important observation because enrichment of a spindle checkpoint protein at kinetochores upon checkpoint activation had not previously reported in S. cerevisiae. Our results highlight the evolutionary conservation of pathways required for genome stability in yeast and humans and demonstrate that the yeast model system can be used to study the fundamental process of chromosome segregation.
Cell cycle checkpoint responses to DNA damage and replication arrest. In the first study we have studied a novel regulator of checkpoint response Hug1p that we identified using SAGE (Serial Analysis of Gene Expression). SAGE was used to identify, quantitate, and compare global gene expression patterns from hydroxyurea-arrested (S-phase), nocodozole-arrested (G2/M phase), and logarithmically growing cells of S. cerevisiae. SAGE analysis was done in collaborative efforts with Dr. Hieter, Dr. Kinzler, Dr. Velculescu and Dr. Vogelstein. SAGE permitted the identification of at least 302 previously unidentified transcripts from NORFs corresponding to proteins with <100 amino acids, some of which are expressed in a cell cycle-regulated manner. Further studies have shown that transcription of one of these, NORF5/HUG1 (hydroxyurea, ultraviolet, gamma induced), is induced by DNA damage and this induction requires MEC1, a homolog of the ataxia telangiectasia-mutated (ATM) gene and genes in the MEC1 pathway. Overexpression of HUG1 is lethal in the presence of DNA damage or replication arrest. A deletion of HUG1 rescues the lethality due to a mec1 null allele. Identification of a human homolog of HUG1 may further our understanding of similar pathways in humans. In a second project we have established an important relationship between oxidative stress genes SOD1 and LYS7 and the MEC1 mediated checkpoint pathway for DNA damage and replication arrest. We have shown that yeast strains lacking SOD1 or LYS7 are extremely sensitive to DNA damage and replication arrest agents when grown in the presence of air and show defective signaling through the MEC1 pathway. These studies have established a novel link between oxidative stress, redox state and checkpoint pathways in S. cerevisiae that may be applicable to other systems.
Functional genomics of genes with small open reading frames (sORFs) in S. cerevisiae. Genes with sORFs (<100 amino acids) represent an untapped source of important biology. sORFs largely escaped analysis since they were difficult to predict computationally and less likely to be targeted by genetic screens. Thus, the substantial number of sORFs and their potential importance has only recently become clear. To investigate sORF function, we undertook the first functional studies of sORFs in any system, using the model eukaryote Saccharomyces cerevisiae. These studies were done in collaboration with the groups of Dr. Jef Boeke, Dr. Mike Snyder and Dr. Ron Davis. Based on independent experimental approaches and computational analyses, evidence now exists for 299 sORFs in the S. cerevisiae genome representing about 5% of annotated S. cerevisiae ORFs. We determined that a similar percentage sORFs are annotated in other eukaryotes, including humans, and 184 of the S. cerevisiae sORFs exhibit significant similarity with ORFs in other organisms. To investigate sORF function, we constructed a collection of gene-deletion mutants of 141 newly identified sORFs, each of which contains a strain-specific 'molecular bar code', bringing the total number of sORF deletion strains to 248. Phenotypic analyses identified 20 sORFs required for haploid growth, growth at high temperature, growth in the presence of a non-fermentable carbon source or growth in the presence of DNA damage and replication arrest agents. We provide a collection of sORF deletion strains that can be integrated into the existing deletion collection as a resource for the yeast community for elucidating gene function. Our analyses of the S. cerevisiae sORFs establish that sORFs are conserved across eukaryotes and have important biological functions.
We have collaborations with Forrest Spencer, Johns Hopkins School of Medicine, Grant Hartzog, University of California, Santa Cruz; Oliver Rando, University of Massachusetts Med School; Richard Baker, University of Massachusetts, Amherst; Kerry Bloom, University of North Carolina, Chapel Hill; Maitreya Dunham, University of Washington, Seattle; Valeria Culotta, Johns Hopkins School of Medicine; Ken Belanger, Colgate University; Paul Meltzer, Genetics Branch, CCR/NIH; Natasha Caplen, Genetics Branch, CCR/NIH
This page was last updated on 3/12/2013.