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||Ph.D.: 1977, Indian Institute of Science;
M.Sc.: 1971, Indian Agricultural Research Institute;
B.Sc.: 1969, Kerala
||California Institute of Technology; State University of New York, Stony Brook
||Site-specific DNA recombination; Molecular symbiosis in yeast
The University of Texas at Austin
Molecular Genetics & Microbiology
1 University Station A5000
Austin TX 78712-0162
|Laboratory home page: http://www.sbs.utexas.edu/jayaram/jayaramlab.htm
The object of our study is a selfish DNA molecule, the 2 micron plasmid, present nearly ubiquitously in Saccharomyces yeast strains. The plasmid persists stably in yeast cell populations at high copy number by combining a stability system and an amplification system. The former partitions replicated plasmid molecules roughly equally between daughter cells during division; the latter restores copy number when it drops from the steady state value due to a rare missegregation event.
The amplification pathway involves site-specific DNA recombination mediated by the Flp recombinase enzyme. The mechanism of this reaction has held our attention for several years, and is now well elucidated. Our current research employs methods of directed evolution to obtain Flp variants that recognize and recombine novel target sites that are quite diverged from the native recombination site. We utilize the evolved variants in biochemical and structural studies to understand the details of macromolecular recognition and subunit cooperatiivty during recombination. These studies have implications in engineering genomes at pre-selected genetic loci to effect directed DNA rearrangements.
We also devote our attention to understanding how the partitioning system confers nearly chromosome-like stability to the 2 micron plasmid. Recent results indicate that the plasmid purloins components of the host segregation machinery for its equal partitioning during cell division. We are investigating further mechanistic details of this molecular poaching.
We wish to understand how the partitioning and amplification systems communicate and interact with each other to maintain the plasmid as a benign parasite element in yeast. To know more about the Jayaram laboratory, its members and activities, go to http://www.sbs.utexas.edu/jayaram/jayaramlab.htm
A novel role for the mitotic spindle during DNA segregation in yeast: Promoting 2 micron plasmid-cohesin association
Mehta, S., Yang, X., Jayaram, M., and Velmurugan, S.
Mol Cell Biol 25: 4283-4298.
ABSTRACT: The 2 micron circle plasmid in Saccharomyces cerevisiae is a model for a stable, high-copy-number, extrachromosomal "selfish" DNA element. By combining a partitioning system and an amplification system, the plasmid ensures its stable propagation and copy number maintenance, even though it does not provide any selective advantage to its host. Recent evidence suggests that the partitioning system couples plasmid segregation to chromosome segregation. We now demonstrate an unexpected and unconventional role for the mitotic spindle in the plasmid-partitioning pathway. The spindle specifies the nuclear address of the 2 micron circle and promotes recruitment of the cohesin complex to the plasmid-partitioning locus STB. Only the nuclear microtubules, and not the cytoplasmic ones, are required for loading cohesin at STB. In cells recovering from nocodazole-induced spindle depolymerization and G(2)/M arrest, cohesin-STB association can be established coincident with spindle restoration. This postreplication recruitment of cohesin is not functional in equipartitioning. However, normally acquired cohesin can be inactivated after replication without causing plasmid missegregation. In the mtw1-1 mutant yeast strain, the plasmid cosegregates with the spindle and the spindle-associated chromosomes; by contrast, a substantial number of the chromosomes are not associated with the spindle. These results are consistent with a model in which the spindle promotes plasmid segregation in a chromosome-linked fashion.
The 2 micron plasmid causes cell death in Saccharomyces cerevisiae with a mutation in the Ulp1 protease
Dobson, M. J., Pickett, A. J., Velmurugan, S., Pinder, J. B., Barrett, L. A., Jayaram, M. , and Chew, J. S.
Mol Cell Biol 25: 4299-4310.
ABSTRACT: The 2 micron circle plasmid confers no phenotype in wild-type Saccharomyces cerevisiae but in a nib1 mutant, an elevated plasmid copy number is associated with cell death. Complementation was used to identify nib1 as a mutant allele of the ULP1 gene that encodes a protease required for removal of a ubiquitin-like protein, Smt3/SUMO, from protein substrates. The nib1 mutation replaces conserved tryptophan 490 with leucine in the protease domain of Ulp1. Complete deletion of ULP1 is lethal, even in a strain that lacks the 2micron circle. Partial deletion of ULP1, like the nib1 mutation, results in clonal variations in plasmid copy number. In addition, a subset of these mutant cells produces lineages in which all cells have reduced proliferative capacity, and this phenotype is dependent upon the presence of the 2 micron circle. Segregation of the 2 micron circle requires two plasmid-encoded proteins, Rep1 and Rep2, which are found to colocalize with Ulp1 protein in the nucleus and interact with Smt3 in a two-hybrid assay. These associations and the observation of missegregation of a fluorescently tagged 2mum circle reporter plasmid in a subset of ulp1 mutant cells suggest that Smt3 modification plays a role in both plasmid copy number control and segregation.
The Mu transposase interwraps distant DNA sites within a functional transpososome in the absence of DNA supercoiling
Yin, Z., Jayaram, M., Pathania, S., and Harshey, R. M.
J Biol Chem 280: 6149-6156.
ABSTRACT: A Mu transpososome assembled on negatively supercoiled DNA traps five supercoils by intertwining the left (L) and right (R) ends of Mu with an enhancer element (E). To investigate the contribution of DNA supercoiling to this elaborate synapse in which E and L cross once, E and R twice, and L and R twice, we have analyzed DNA crossings in a transpososome assembled on nicked substrates under conditions that bypass the supercoiling requirement for transposition. We find that the transposase MuA can recreate an essentially similar topology on nicked substrates, interwrapping both E-R and L-R twice but being unable to generate the single E-L crossing. In addition, we deduce that the functional MuA tetramer must contribute to three of the four observed crossings and, thus, to restraining the enhancer within the complex. We discuss the contribution of both MuA and DNA supercoiling to the 5-noded Mu synapse built at the 3-way junction.
Mutations in a partitioning protein and altered chromatin structure at the partitioning locus prevent cohesin recruitment by the Saccharomyces cerevisiae plasmid and cause plasmid missegregation
Yang, X-M, Mehta, S., Uzri, D., Jayaram, M., and Velmurugan, S.
Mol Cell Biol 24: 5290-5303.
ABSTRACT: The 2 micron circle is a highly persistent "selfish" DNA element resident in the Saccharomyces cerevisiae nucleus whose stability approaches that of the chromosomes. The plasmid partitioning system, consisting of two plasmid-encoded proteins, Rep1p and Rep2p, and a cis-acting locus, STB, apparently feeds into the chromosome segregation pathway. The Rep proteins assist the recruitment of the yeast cohesin complex to STB during the S phase, presumably to apportion the replicated plasmid molecules equally to daughter cells. The DNA-protein and protein-protein interactions of the partitioning system, as well as the chromatin organization at STB, are important for cohesin recruitment. Rep1p variants that are incompetent in binding to Rep2p, STB, or both fail to assist the assembly of the cohesin complex at STB and are nonfunctional in plasmid maintenance. Preventing the cohesin-STB association without impeding Rep1p-Rep2p-STB interactions also causes plasmid missegregation. During the yeast cell cycle, the Rep1p and Rep2p proteins are expelled from STB during a short interval between the late G(1) and early S phases. This dissociation and reassociation event ensures that cohesin loading at STB is replication dependent and is coordinated with chromosomal cohesin recruitment. In an rsc2 deletion yeast strain, lacking a specific chromatin remodeling complex and exhibiting a high degree of plasmid loss, neither Rep1p nor the cohesin complex can be recruited to STB. The phenotypes of the Rep1p mutations and of the rsc2 deletion mutant are consistent with the role of cohesin in plasmid partitioning being analogous to that in chromosome partitioning.
Recombination of hybrid target sites by binary combinations of Flp variants: mutations that foster interprotomer collaboration and enlarge substrate tolerance.
Konieczka, J. H, Paek A., Jayaram M., and Voziyanov Y.
J Mol Biol 339: 365-378.
ABSTRACT: Strategies of directed evolution and combinatorial mutagenesis applied to the Flp site-specific recombinase have yielded recombination systems that utilize bi-specific hybrid target sites. A hybrid site is assembled from two half-sites, each harboring a distinct binding specificity. Satisfying the two specificities by a binary combination of Flp variants, while necessary, may not be sufficient to elicit recombination. We have identified amino acid substitutions that foster interprotomer collaboration between partner Flp variants to potentiate strand exchange in hybrid sites. One such substitution, A35T, acts specifically in cis with one of the two partners of a variant pair, Flp(K82M) and Flp(A35T, R281V). The same A35T mutation is also present within a group of mutations that rescue a Flp variant, Flp(Y60S), that is defective in establishing monomer-monomer interactions on the native Flp target site. Strikingly, these mutations are localized to peptide regions involved in interdomain and interprotomer interactions within the recombination complex. The same group of mutations, when transferred to the context of wild-type Flp, can relax its specificity to include non-native target sites. The hybrid Flp systems described here mimic the naturally occurring XerC/XerD recombination system that utilizes two recombinases with distinct DNA binding specificities. The ability to overcome the constraints of binding site symmetry in Flp recombination has important implications in the targeted manipulations of genomes.
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