Chien Hui Ma

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 2 micron plasmid of Saccharomyces cerevisiae: A miniaturized selfish genome with optimized functional competence

The 2 micron plasmid of Saccharomyces cerevisiae is a relatively small multi-copy selfish DNA element that resides in the yeast nucleus at a copy number of 40-60 per haploid cell. The plasmid is able to persist in host populations with almost chromosome-like stability with the help of a partitioning system and a copy number control system. The first part of this article describes the properties of the partitioning system comprising two plasmid coded proteins, Rep1 and Rep2, and a partitioning locus STB. Current evidence supports a model in which the Rep-STB system couples plasmid segregation to chromosome segregation by promoting the physical association of plasmid molecules with chromosomes. In the second part, the focus is on the Flp site-specific recombination system housed by the plasmid, which plays a critical role in maintaining steady state plasmid copy number. The Flp system corrects any decrease in plasmid population by promoting plasmid amplification via a recombination induced rolling circle replication mechanism. Appropriate plasmid amplification, without runaway increase in copy number, is ensured by positive and negative regulation of FLP gene expression by plasmid coded proteins and by the control of Flp level/activity through post-translational modification of Flp by the cellular sumoylation system. The Flp system has been successfully utilized to understand mechanisms of site-specific recombination and to bring about directed genetic alterations for addressing fundamental problems in biology and for accomplishing bio-engineering objectives. A particularly interesting, and perhaps less well known and underappreciated, application of Flp in revealing unique DNA topologies required to confer functional competence to DNA-protein machines is discussed.

Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site‐specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′‐phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg‐308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti‐hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site‐specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns.

Real-time single-molecule tethered particle motion analysis reveals mechanistic similarities and contrasts of Flp site-specific recombinase with Cre and λ Int

Flp, a tyrosine site-specific recombinase coded for by the selfish two micron plasmid of Saccharomyces cerevisiae, plays a central role in the maintenance of plasmid copy number. The Flp recombination system can be manipulated to bring about a variety of targeted DNA rearrangements in its native host and under non-native biological contexts. We have performed an exhaustive analysis of the Flp recombination pathway from start to finish by using single-molecule tethered particle motion (TPM). The recombination reaction is characterized by its early commitment and high efficiency, with only minor detraction from ‘non-productive’ and ‘wayward’ complexes. The recombination synapse is stabilized by strand cleavage, presumably by promoting the establishment of functional interfaces between adjacent Flp monomers. Formation of the Holliday junction intermediate poses a rate-limiting barrier to the overall reaction. Isomerization of the junction to the conformation favoring its resolution in the recombinant mode is not a slow step. Consistent with the completion of nearly every initiated reaction, the chemical steps of strand cleavage and exchange are not reversible during a recombination event. Our findings demonstrate similarities and differences between Flp and the mechanistically related recombinases λ Int and Cre. The commitment and directionality of Flp recombination revealed by TPM is consistent with the physiological role of Flp in amplifying plasmid DNA.

Reactions of Cre with methylphosphonate DNA: similarities and contrasts with Flp and vaccinia topoisomerase.

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.

Temporal sequence and cell cycle cues in the assembly of host factors at the yeast 2 micron plasmid partitioning locus

The Saccharomyces cerevisiae 2 micron plasmid exemplifies a benign but selfish genome, whose stability approaches that of the chromosomes of its host. The plasmid partitioning locus STB (stability locus) displays certain functional analogies with centromeres along with critical distinctions, a significant one being the absence of the kinetochore complex at STB. The remodels the structure of chromatin (RSC) chromatin remodeling complex, the nuclear motor Kip1, the histone H3 variant Cse4 and the cohesin complex associate with both loci. These factors appear to contribute to plasmid segregation either directly or indirectly through their roles in chromosome segregation. Assembly and disassembly of the plasmid-coded partitioning proteins Rep1 and Rep2 and host factors at STB follow a temporal hierarchy during the cell cycle. Assembly is initiated by STB association of [Rsc8-Rsc58], followed by [Rep1-Rep2-Kip1] and [Cse4-Rsc2-Sth1] recruitment, and culminates in cohesin assembly. Disassembly starts with dissociation of RSC components, is followed by cohesin disassembly and Cse4 exit during anaphase and late telophase, respectively. [Rep1-Rep2-Kip1] persists through G1 of the ensuing cell cycle. The de novo assembly of the ‘partitioning complex’ is cued by the innate cell cycle clock and is dependent on DNA replication. Shared functional attributes of STB and centromere (CEN) are consistent with a potential evolutionary link between them.

Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase

Freeze-frame synthetic proteins trap DNA reaction intermediates in single live cells, revealing origins of genome instability. DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. However, the main kinds and sources of DNA damage repaired by HR in somatic cells and the roles of important HR proteins remain elusive. We present engineered proteins that trap, map, and quantify Holliday junctions (HJs), a central DNA intermediate in HR, based on catalytically deficient mutant RuvC protein of Escherichia coli. We use RuvCDefGFP (RDG) to map genomic footprints of HR at defined DNA breaks in E. coli and demonstrate genome-scale directionality of double-strand break (DSB) repair along the chromosome. Unexpectedly, most spontaneous HR-HJ foci are instigated, not by DSBs, but rather by single-stranded DNA damage generated by replication. We show that RecQ, the E. coli ortholog of five human cancer proteins, nonredundantly promotes HR-HJ formation in single cells and, in a novel junction-guardian role, also prevents apparent non-HR–HJs promoted by RecA overproduction. We propose that one or more human RecQ orthologs may act similarly in human cancers overexpressing the RecA ortholog RAD51 and find that cancer genome expression data implicate the orthologs BLM and RECQL4 in conjunction with EME1 and GEN1 as probable HJ reducers in such cancers. Our results support RecA-overproducing E. coli as a model of the many human tumors with up-regulated RAD51 and provide the first glimpses of important, previously elusive reaction intermediates in DNA replication and repair in single living cells.

Electrostatic Suppression Allows Tyrosine Site-specific Recombination in the Absence of a Conserved Catalytic Arginine

The active site of the tyrosine family site-specific recombinase Flp contains a conserved catalytic pentad that includes two arginine residues, Arg-191 and Arg-308. Both arginines are essential for the transesterification steps of strand cleavage and strand joining in DNA substrates containing a phosphate group at the scissile position. During strand cleavage, the active site tyrosine supplies the nucleophile to form a covalent 3′-phosphotyrosyl intermediate. The 5′-hydroxyl group produced by cleavage provides the nucleophile to re-form a 3′-5′ phosphodiester bond in a recombinant DNA strand. In previous work we showed that substitution of the scissile phosphate (P) by the charge neutral methylphosphonate (MeP) makes Arg-308 dispensable during the catalytic activation of the MeP diester bond. However, in the Flp(R308A) reaction, water out-competes the tyrosine nucleophile (Tyr-343) to cause direct hydrolysis of the MeP diester bond. We now report that for MeP activation Arg-191 is also not required. In contrast to Flp(R308A), Flp(R191A) primarily mediates normal cleavage by Tyr-343 but also exhibits a weaker direct hydrolytic activity. The cleaved MeP-tyrosyl intermediate formed by Flp(R191A) can be targeted for nucleophilic attack by a 5′-hydroxyl or water and channeled toward strand joining or hydrolysis, respectively. In collaboration with wild type Flp, Flp(R191A) promotes strand exchange between MeP- and P-DNA partners. Loss of a catalytically crucial positively charged side chain can thus be suppressed by a compensatory modification in the DNA substrate that neutralizes the negative charge on the scissile phosphate.

Restoration of catalytic functions in Cre recombinase mutants by electrostatic compensation between active site and DNA substrate

Two conserved catalytic arginines, Arg-173 and Arg-292, of the tyrosine site-specific recombinase Cre are essential for the transesterification steps of strand cleavage and joining in native DNA substrates containing scissile phosphate groups. The active site tyrosine (Tyr-324) provides the nucleophile for the cleavage reaction, and forms a covalent 3′-phosphotyrosyl intermediate. The 5′-hydroxyl group formed during cleavage provides the nucleophile for the joining reaction between DNA partners, yielding strand exchange. Previous work showed that substitution of the scissile phosphate (P) by methylphosphonate (MeP) permits strand cleavage by a Cre variant lacking Arg-292. We now demonstrate that MeP activation and cleavage are not blocked by substitution of Arg-173 or even simultaneous substitutions of Arg-173 and Arg-292 by alanine. Furthermore, Cre(R173A) and Cre(R292A) are competent in strand joining, Cre(R173A) being less efficient. No joining activity is detected with Cre(R173A, R292A). Consistent with their ability to cleave and join strands, Cre(R173A) and Cre(R292A) can promote recombination between two MeP-full-site DNA partners. These findings shed light on the overall contribution of active site electrostatics, and tease apart distinctive contributions of the individual arginines, to the chemical steps of recombination. They have general implications in active site mechanisms that promote important phosphoryl transfer reactions in nucleic acids.

Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.