The 2 micron plasmid purloins the yeast cohesin complex: a mechanism for coupling plasmid partitioning and chromosome segregation?

The yeast 2 micron plasmid achieves high fidelity segregation by coupling its partitioning pathway to that of the chromosomes. Mutations affecting distinct steps of chromosome segregation cause the plasmid to missegregate in tandem with the chromosomes. In the absence of the plasmid stability system, consisting of the Rep1 and Rep2 proteins and the STB DNA, plasmid and chromosome segregations are uncoupled. The Rep proteins, acting in concert, recruit the yeast cohesin complex to the STB locus. The periodicity of cohesin association and dissociation is nearly identical for the plasmid and the chromosomes. The timely disassembly of cohesin is a prerequisite for plasmid segregation. Cohesin-mediated pairing and unpairing likely provides a counting mechanism for evenly partitioning plasmids either in association with or independently of the chromosomes

Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites

As a tool in directed genome manipulations, site-specific recombination is a double-edged sword. Exquisite specificity, while highly desirable, makes it imperative that the target site be first inserted at the desired genomic locale before it can be manipulated. We describe a combination of computational and experimental strategies, based on the tyrosine recombinase Flp and its target site FRT, to overcome this impediment. We document the systematic evolution of Flp variants that can utilize, in a bacterial assay, two sites from the human interleukin 10 gene, IL10, as recombination substrates. Recombination competence on an end target site is acquired via chimeric sites containing mixed sequences from FRT and the genomic locus. This is the first time that a tyrosine site-specific recombinase has been coaxed successfully to perform DNA exchange within naturally occurring sequences derived from a foreign genomic context. We demonstrate the ability of an Flp variant to mediate integration of a reporter cassette in Escherichia coli via recombination at one of the IL10-derived sites

The selfish yeast plasmid uses the nuclear motor Kip1p but not Cin8p for its localization and equal segregation

The 2 micron plasmid of Saccharomyces cerevisiae uses the Kip1 motor, but not the functionally redundant Cin8 motor, for its precise nuclear localization and equal segregation. The timing and lifetime of Kip1p association with the plasmid partitioning locus STB are consistent with Kip1p being an authentic component of the plasmid partitioning complex. Kip1–STB association is not blocked by disassembling the mitotic spindle. Lack of Kip1p disrupts recruitment of the cohesin complex at STB and cohesion of replicated plasmid molecules. Colocalization of a 2 micron reporter plasmid with Kip1p in close proximity to the spindle pole body is reminiscent of that of a CEN reporter plasmid. Absence of Kip1p displaces the plasmid from this nuclear address, where it has the potential to tether to a chromosome or poach chromosome segregation factors. Exploiting Kip1p, which is subsidiary to Cin8p for chromosome segregation, to direct itself to a “partitioning center” represents yet another facet of the benign parasitism of the yeast plasmid

Reactions of Cre with Methylphosphonate DNA: Similarities and Contrasts with Flp and Vaccinia Topoisomerase

Chien-Hui Ma is with UT Austin, Aashiq H. Kachroo is with UT Austin, Anna Macieszak is with Polish Academy of Sciences, Tzu-Yang Chen is with UT Austin, Piotr Guga is with Polish Academy of Sciences, Makkuni Jayaram is with UT Austin.Background -- Reactions of vaccinia topoisomerase and the tyrosine site-specific recombinase Flp with methylphosphonate (MeP) substituted DNA substrates, have provided important insights into the electrostatic features of the strand cleavage and strand joining steps catalyzed by them. A conserved arginine residue in the catalytic pentad, Arg-223 in topoisomerase and Arg-308 in Flp, is not essential for stabilizing the MeP transition state. Topoisomerase or its R223A variant promotes cleavage of the MeP bond by the active site nucleophile Tyr-274, followed by the rapid hydrolysis of the MeP-tyrosyl intermediate. Flp(R308A), but not wild type Flp, mediates direct hydrolysis of the activated MeP bond. These findings are consistent with a potential role for phosphate electrostatics and active site electrostatics in protecting DNA relaxation and site-specific recombination, respectively, against abortive hydrolysis. Methodology/Principal Findings -- We have examined the effects of DNA containing MeP substitution in the Flp related Cre recombination system. Neutralizing the negative charge at the scissile position does not render the tyrosyl intermediate formed by Cre susceptible to rapid hydrolysis. Furthermore, combining the active site R292A mutation in Cre (equivalent to the R223A and R308A mutations in topoisomerase and Flp, respectively) with MeP substitution does not lead to direct hydrolysis of the scissile MeP bond in DNA. Whereas Cre follows the topoisomerase paradigm during the strand cleavage step, it follows the Flp paradigm during the strand joining step. Conclusions/Significance -- Collectively, the Cre, Flp and topoisomerase results highlight the contribution of conserved electrostatic complementarity between substrate and active site towards transition state stabilization during site-specific recombination and DNA relaxation. They have potential implications for how transesterification reactions in nucleic acids are protected against undesirable abortive side reactions. Such protective mechanisms are significant, given the very real threat of hydrolytic genome damage or disruption of RNA processing due to the cellular abundance and nucleophilicity of water.This work was supported by the NIH award GM035654 to M. J. Partial support was provided by the Robert F. Welch Foundation (F-1274) and a Faculty Research Award from the University of Texas at Austin. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Microbiolog

Role of DNA Topology in Biological Machines and Evolutionarily Related Biological Functions: Chemical Chirality in Site-Specific DNA Recombination

<b style='mso-bidi-font-weight: normal'>Difference topology<span lang=EN-US style='font-family:Arial'>: The DNA path within a high-order DNA-protein assembly can be revealed from the topology of DNA knots and links formed by the action of Flp or Cre site-specific recombinase on target sites placed close to (and flanking) the assembly. This â analysis is experimentally simple, and has broad applicability. Choice between alternative reaction mechanisms: Recombination product topologies resulting from a fixed synaptic topology can help distinguish between alternative modes of site arrangement and strand exchange by a given recombinase enzyme. <b style='mso-bidi-font-weight:normal'>Conserved biological functions revealed by conserved topology of DNA loci in vivo:<span style="mso-spacerun:yes">&nbsp; DNA topology can reveal evolutionary relationships between two diverged DNA loci that perform analogous but distinct biological functions. The unusual positive chromatin writhe at the centromeres of yeast chromosomes is shared by the partitioning locus of an extrachromosomal circular DNA element present in the yeast nucleus. This finding lends credence to the possible origin of these loci from a common ancestor that once directed the segregation of both the chromosomes and the plasmid during cell division. <b style='mso-bidi-font-weight:normal'>Chemical chirality in DNA recombination: The active site of the Flp/Cre recombinase contains two conserved positively charged side-chains (<span class=SpellE>Arg-I and Arg-II) responsible for compensating the negative charge on the non-brridging oxygen atoms of the scissile phosphate group. When one of the oxygens is replaced by a neutral group, one of the arginine residues becomes dispensable. The reactivity of DNA substrates containing substitutions at either of the two oxygens with Flp/Cre lacking either of the two arginines reveals the chirality of Arg-oxygen interactions, and defines the stereochemical course of the recombination reaction. Non UBCUnreviewedAuthor affiliation: University of Texas at AustinFacult

Selfishness in Moderation: Evolutionary Success of the Yeast Plasmid

The yeast plasmid 2-μm circle is an extrachromosomal selfish DNA element whose genetic endowments are devoted to its stable, high copy propagation. The mean steady state plasmid copy number of approximately 60 per cell appears to be evolutionarily optimized at its permissible maximum value. A plasmid-encoded negative regulatory mechanism prevents a rise in copy number that might imperil normal host metabolism and thus indirectly reduce plasmid fitness. The plasmid utilizes the host replication machinery for its own duplication. A plasmid-encoded partitioning system mediates even distribution of the replicated molecules to daughter cells, apparently by feeding into the chromosome segregation pathway. The plasmid also harbors an amplification system as a potential safeguard against a fall in copy number due to an occasional missegregation event. The 2-μm circle provides a model for how moderation of selfishness can ensure the successful persistence of an extrachromosomal element without compromising the fitness of its host. © 2003 Elsevier Inc. All rights reserved