Separation-of-Function Mutations in Saccharomyces cerevisiae MSH2 That Confer Mismatch Repair Defects but Do Not Affect Nonhomologous-Tail Removal during Recombination

Yeast Msh2p forms complexes with Msh3p and Msh6p to repair DNA mispairs that arise during DNA replication. In addition to their role in mismatch repair (MMR), the MSH2 and MSH3 gene products are required to remove 3' nonhomologous DNA tails during genetic recombination. The mismatch repair genes MSH6, MLH1, and PMS1, whose products interact with Msh2p, are not required in this process. We have identified mutations in MSH2 that do not disrupt genetic recombination but confer a strong defect in mismatch repair. Twenty-four msh2 mutations that conferred a dominant negative phenotype for mismatch repair were isolated. A subset of these mutations mapped to residues in Msh2p that were analogous to mutations identified in human nonpolyposis colorectal cancer msh2 kindreds. Approximately half of the these MMR-defective mutations retained wild-type or nearly wild-type activity for the removal of nonhomologous DNA tails during genetic recombination. The identification of mutations in MSH2 that disrupt mismatch repair without affecting recombination provides a first step in dissecting the Msh-effector protein complexes that are thought to play different roles during DNA repair and genetic recombination.     

Gross chromosomal rearrangements and elevated recombination at an inducible site-specific replication fork barrier

Genomic rearrangements linked to aberrant recombination are associated with cancer and human genetic diseases. Such recombination has indirectly been linked to replication fork stalling. Using fission yeast, we have developed a genetic system to block replication forks at nonhistone/DNA complexes located at a specific euchromatic site. We demonstrate that stalled replication forks lead to elevated intrachromosomal and ectopic recombination promoting site-specific gross chromosomal rearrangements. We show that recombination is required to promote cell viability when forks are stalled, that recombination proteins associate with sites of fork stalling, and that recombination participates in deleterious site-specific chromosomal rearrangements. Thus, recombination is a "double-edged sword," preventing cell death when the replisome disassembles at the expense of genetic stability.     

Intrasubunit and intersubunit interactions controlling assembly of active synaptic complexes during Hin-catalyzed DNA recombination

Serine recombinases, which generate double-strand breaks in DNA, must be carefully regulated to ensure that chemically active DNA complexes are assembled correctly. In the Hin-catalyzed site-specific DNA inversion reaction, two inversely oriented recombination sites on the same DNA molecule assemble into a synaptic complex that uniquely generates inversion products. The Fis-bound recombinational enhancer, together with topological constraints directed by DNA supercoiling, functions to regulate Hin synaptic complex formation and activity. We have isolated a collection of gain-of-function mutants in 22 positions within the catalytic and oligomerization domains of Hin using two genetic screens and by site-directed mutagenesis. One genetic screen measured recombination in the absence of Fis and the other assessed SOS induction as a readout of increased DNA cleavage. These mutations, together with molecular modeling, identify important sites of dynamic intrasubunit and intersubunit interactions that regulate assembly of the active tetrameric recombination complex. Of particular interest are interactions between the oligomerization helix (helix E) and the catalytic domain of the same subunit that function to hold the dimer in an inactive state in the absence of the Fis/enhancer system. Among these is a relay involving a triad of phenylalanines that are proposed to switch positions during the transition from dimers to the catalytically active tetramer. Novel Hin mutants that generate synaptic complexes that are blocked at steps prior to DNA cleavage are also described.     

A biotin interference assay highlights two different asymmetric interaction profiles for lambda integrase arm-type binding sites in integrative versus excisive recombination

The site-specific recombinase integrase encoded by bacteriophage lambda promotes integration and excision of the viral chromosome into and out of its Escherichia coli host chromosome through a Holliday junction recombination intermediate. This intermediate contains an integrase tetramer bound via its catalytic carboxyl-terminal domains to the four "core-type" sites of the Holliday junction DNA and via its amino-terminal domains to distal "arm-type" sites. The two classes of integrase binding sites are brought into close proximity by an ensemble of accessory proteins that bind and bend the intervening DNA. We have used a biotin interference assay that probes the requirement for major groove protein binding at specified DNA loci in conjunction with DNA protection, gel mobility shift, and genetic experiments to test several predictions of the models derived from the x-ray crystal structures of minimized and symmetrized surrogates of recombination intermediates lacking the accessory proteins and their cognate DNA targets. Our data do not support the predictions of "non-canonical" DNA targets for the N-domain of integrase, and they indicate that the complexes used for x-ray crystallography are more appropriate for modeling excisive rather than integrative recombination intermediates. We suggest that the difference in the asymmetric interaction profiles of the N-domains and arm-type sites in integrative versus excisive recombinogenic complexes reflects the regulation of recombination, whereas the asymmetry of these patterns within each reaction contributes to directionality.     

Recombinational repair and restart of damaged replication forks

Genome duplication necessarily involves the replication of imperfect DNA templates and, if left to their own devices, replication complexes regularly run into problems. The details of how cells overcome these replicative 'hiccups' are beginning to emerge, revealing a complex interplay between DNA replication, recombination and repair that ensures faithful passage of the genetic material from one generation to the next.     

Processing of recombination intermediates in vitro

Genetic recombination involves the exchange of genetic material between chromosomes to produce new assortments of alleles. As such, it affects one of the most fundamental and important components of heredity--the genome itself. To understand the molecular basis of recombination, efforts have been directed to try to determine how simple organisms recombine their DNA. One approach involves the development of in vitro systems in which recombination reactions can be studied using purified enzymes. Detailed studies of these systems, using enzymes isolated from bacteria and bacterial viruses, indicate the formation of unique protein-DNA complexes. The structure of the DNA within these complexes has important consequences for the subsequent formation of recombinant products.     

Sae2 is an endonuclease that processes hairpin DNA cooperatively with the Mre11/Rad50/Xrs2 complex

Mre11/Rad50 complexes in all organisms function in the repair of DNA double-strand breaks. In budding yeast, genetic evidence suggests that the Sae2 protein is essential for the processing of hairpin DNA intermediates and meiotic double-strand breaks by Mre11/Rad50 complexes, but the biochemical basis of this functional relationship is not known. Here we demonstrate that recombinant Sae2 binds DNA and exhibits endonuclease activity on single-stranded DNA independently of Mre11/Rad50 complexes, but hairpin DNA structures are cleaved cooperatively in the presence of Mre11/Rad50 or Mre11/Rad50/Xrs2. Hairpin structures are not processed at the tip by Sae2 but rather at single-stranded DNA regions adjacent to the hairpin. Truncation and missense mutants of Sae2 inactivate this endonuclease activity in vitro and fail to complement Deltasae2 strains in vivo for meiosis and recombination involving hairpin intermediates, suggesting that the catalytic activities of Sae2 are important for its biological functions.     

Effects of XRCC2 and RAD51B mutations on somatic and meiotic recombination in Arabidopsis thaliana

Homologous recombination is key to the maintenance of genome integrity and the creation of genetic diversity. At the mechanistic level, recombination involves the invasion of a homologous DNA template by broken DNA ends, repair of the break and exchange of genetic information between the two DNA molecules. Invasion of the template in eukaryotic cells is catalysed by the RAD51 and DMC1 recombinases, assisted by a number of accessory proteins, including the RAD51 paralogues. Eukaryotic genomes encode a variable number of RAD51 paralogues, ranging from two in yeast to five in animals and plants. The RAD51 paralogues form at least two distinct protein complexes, believed to play roles in the assembly and stabilization of the RAD51‐DNA nucleofilament. Somatic recombination assays and immunocytology confirm that the three ‘non‐meiotic’ paralogues of Arabidopsis, RAD51B, RAD51D and XRCC2, are involved in somatic homologous recombination, and that they are not required for the formation of radioinduced RAD51 foci. Given the presence of all five proteins in meiotic cells, the apparent absence of a meiotic role for RAD51B, RAD51D and XRCC2 is surprising, and perhaps simply the result of a more subtle meiotic phenotype in the mutants. Analysis of meiotic recombination confirms this, showing that the absence of XRCC2, and to a lesser extent RAD51B, but not RAD51D, increases rates of meiotic crossing over. The roles of RAD51B and XRCC2 in recombination are thus not limited to mitotic cells.     

Initiation of recombination during transformation of Bacillus subtilis requires no extensive homologous sequences

Summary Lysates obtained shortly after entry of transforming DNA to Bacillus subtilis contain donor-recipient DNA complexes, in which the donor moiety is associated with the recipient DNA in an unstable way. The complexes could be artificially stabilized by crosslinking with 4,5,8-trimethylpsoralen. The unstable complexes dissociated upon helix-destabilizing treatments, such as heating at 70°C, and CsCl gradient centrifugation at pH 11.2, but remained stable during CsCl gradient centrifugation at pH 10. Donor-recipient DNA complexes were not formed after entry of heterologous pUB110 DNA. These observations suggest that base-pairing is involved in the unstable association. The donor moiety of the unstable complexes was completely, or almost completely, digestible by nuclease S₁, indicating that the donor and recipient base-sequences are only paired over very short distances.The unstable donor-recipient DNA complexes are true recombination intermediates because (i) strain 7G224 (recE4) was impaired in the formation of the unstable complexes, and (ii) the unstable complexes were rapidly converted to stable complexes in recombination proficient strains, whereas their conversion was delayed in the recombination deficient strain 7G84.Unstable complexes were also formed with Escherichia coli donor DNA, but to a lesser extent. Apparently a limited degree of base-sequence homology is sufficient to initiate recombination.     

Intermediates in Hin-mediated DNA inversion: a role for Fis and the recombinational enhancer in the strand exchange reaction.

The site-specific inversion reaction controlling flagellin synthesis in Salmonella involves the function of three proteins: Hin, Fis and HU. The DNA substrate must be supercoiled and contain a recombinational enhancer sequence in addition to the two recombination sites. Using mutant substrates or modified reaction conditions, large amounts of complexes can be generated which are recognized by double-stranded breaks within both recombination sites upon quenching. The cleaved molecules contain 2-bp staggered cuts within the central dinucleotide of the recombination site. Hin is covalently associated with the 5' end while the protruding 3' end contains a free hydoxyl. We demonstrate that complexes generated in the presence of an active enhancer are intermediates that have advanced past the major rate limiting step(s) of the reaction. In the absence of a functional enhancer, Hin is also able to assemble and catalyze site-specific cleavages within the two recombination sites. However, these complexes are kinetically distinct from the complexes assembled with a functional enhancer and cannot generate inversion without an active enhancer. The results suggest that strand exchange leading to inversion is mediated by double-stranded cleavage of DNA at both recombination sites followed by the rotation of strands to position the DNA into the recombinant configuration. The role of the enhancer and DNA supercoiling in these reactions is discussed.     

Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair

Genetic data have revealed that the absence of Bacillus subtilis RecO and one of the end-processing avenues (AddAB or RecJ) renders cells as sensitive to DNA damaging agents as the null recA, suggesting that both end-resection pathways require RecO for recombination. RecA, in the rATP·Mg²⁺ bound form (RecA·ATP), is inactive to catalyze DNA recombination between linear double-stranded (ds) DNA and naked complementary circular single-stranded (ss) DNA. We showed that RecA·ATP could not nucleate and/or polymerize on SsbA·ssDNA or SsbB·ssDNA complexes. RecA·ATP nucleates and polymerizes on RecO·ssDNA·SsbA complexes more efficiently than on RecO·ssDNA·SsbB complexes. Limiting SsbA concentrations were sufficient to stimulate RecA·ATP assembly on the RecO·ssDNA·SsbB complexes. RecO and SsbA are necessary and sufficient to ‘activate’ RecA·ATP to catalyze DNA strand exchange, whereas the AddAB complex, RecO alone or in concert with SsbB was not sufficient. In presence of AddAB, RecO and SsbA are still necessary for efficient RecA·ATP-mediated three-strand exchange recombination. Based on genetic and biochemical data, we proposed that SsbA and RecO (or SsbA, RecO and RecR in vivo) are crucial for RecA activation for both, AddAB and RecJ–RecQ (RecS) recombinational repair pathways.     

RecO-mediated DNA homology search and annealing is facilitated by SsbA

Bacillus subtilis RecO plays a central role in recombinational repair and genetic recombination by (i) stimulating RecA filamentation onto SsbA-coated single-stranded (ss) DNA, (ii) modulating the extent of RecA-mediated DNA strand exchange and (iii) promoting annealing of complementary DNA strands. Here, we report that RecO-mediated strand annealing is facilitated by cognate SsbA, but not by a heterologous one. Analysis of non-productive intermediates reveals that RecO interacts with SsbA-coated ssDNA, resulting in transient ternary complexes. The self-interaction of ternary complexes via RecO led to the formation of large nucleoprotein complexes. In the presence of homology, SsbA, at the nucleoprotein, removes DNA secondary structures, inhibits spontaneous strand annealing and facilitates RecO loading onto SsbA–ssDNA complex. RecO relieves SsbA inhibition of strand annealing and facilitates transient and random interactions between homologous naked ssDNA molecules. Finally, both proteins lose affinity for duplex DNA. Our results provide a mechanistic framework for rationalizing protein release and dsDNA zippering as coordinated events that are crucial for RecA-independent plasmid transformation.     

Unveiling novel RecO distant orthologues involved in homologous recombination

The generation of a RecA filament on single-stranded DNA is a critical step in homologous recombination. Two main pathways leading to the formation of the nucleofilament have been identified in bacteria, based on the protein complexes mediating RecA loading: RecBCD (AddAB) and RecFOR. Many bacterial species seem to lack some of the components involved in these complexes. The current annotation of the Helicobacter pylori genome suggests that this highly diverse bacterial pathogen has a reduced set of recombination mediator proteins. While it is now clear that homologous recombination plays a critical role in generating H. pylori diversity by allowing genomic DNA rearrangements and integration through transformation of exogenous DNA into the chromosome, no complete mediator complex is deduced from the sequence of its genome. Here we show by bioinformatics analysis the presence of a RecO remote orthologue that allowed the identification of a new set of RecO proteins present in all bacterial species where a RecR but not RecO was previously identified. HpRecO shares less than 15% identity with previously characterized homologues. Genetic dissection of recombination pathways shows that this novel RecO and the remote RecB homologue present in H. pylori are functional in repair and in RecA-dependent intrachromosomal recombination, defining two initiation pathways with little overlap. We found, however, that neither RecOR nor RecB contributes to transformation, suggesting the presence of a third, specialized, RecA-dependent pathway responsible for the integration of transforming DNA into the chromosome of this naturally competent bacteria. These results provide insight into the mechanisms that this successful pathogen uses to generate genetic diversity and adapt to changing environments and new hosts.     

Smc5/6 is required for repair at collapsed replication forks

In eukaryotes, three pairs of structural-maintenance-of-chromosome (SMC) proteins are found in conserved multisubunit protein complexes required for chromosomal organization. Cohesin, the Smc1/3 complex, mediates sister chromatid cohesion while two condensin complexes containing Smc2/4 facilitate chromosome condensation. Smc5/6 scaffolds an essential complex required for homologous recombination repair. We have examined the response of smc6 mutants to the inhibition of DNA replication. We define homologous recombination-dependent and -independent functions for Smc6 during replication inhibition and provide evidence for a Rad60-independent function within S phase, in addition to a Rad60-dependent function following S phase. Both genetic and physical data show that when forks collapse (i.e., are not stabilized by the Cds1Chk2 checkpoint), Smc6 is required for the effective repair of resulting lesions but not for the recruitment of recombination proteins. We further demonstrate that when the Rad60-dependent, post-S-phase Smc6 function is compromised, the resulting recombination-dependent DNA intermediates that accumulate following release from replication arrest are not recognized by the G2/M checkpoint.     

Exchange of DNA base pairs that coincides with recognition of homology promoted by E. coli RecA protein

The unresolved mechanism by which a single strand of DNA recognizes homology in duplex DNA is central to understanding genetic recombination and repair of double-strand breaks. Using stopped-flow fluorescence we monitored strand exchange catalyzed by E. coli RecA protein, measuring simultaneously the rate of exchange of A:T base pairs and the rates of formation and dissociation of the three-stranded intermediates called synaptic complexes. The rate of exchange of A:T base pairs was indistinguishable from the rate of formation of synaptic complexes, whereas the rate of displacement of a single strand from complexes was five to ten times slower. This physical evidence shows that a subset of bases exchanges at a rate that is fast enough to account for recognition of homology. Together, several studies suggest that a mechanism governed by the dynamic structure of DNA and catalyzed by diverse enzymes underlies both recognition of homology and initiation of strand exchange.     

Various properties of DNA from isolated fractions of the mouse synaptonemal complexes

A fraction of synaptonemal complexes (SC) isolated from mouse spermatocytes has been electrophoretically purified in agarose gel. The DNA from the SC fraction constitutes approximately 0.5% of total nuclear DNA, and its molecules have length heterogeneity from 1 k.b. to 20 k.b. The content of beta-globin gene is the same in DNA from the SC fraction and in total nuclear DNA. The specificity of DNA from the SC fraction is manifested by higher contents of the repeated alternative sequences GT/CA and B1-sequence that is probably due to the processes of genetic meiotic recombination.     

Homomeric interaction of the mouse Rad52 protein

The Rad52 protein plays a crucial role in repairing DNA damage and homologous recombination, possibly by virtue of its ability to catalyze annealing of single-stranded DNA. In agreement with recent genetic data, we here present results based on the two-hybrid system, demonstrating that mouse Rad52p is able to form homomeric complexes. A small domain necessary and sufficient for the self-interaction is located in the conserved N-terminus of the protein. These data contribute to the important insights into the architecture of the multi-protein complex involved in recombinational DNA repair.     

p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction.

The tumour suppressor p53 prevents tumour formation after DNA damage by halting cell cycle progression to allow DNA repair or by inducing apoptotic cell death. Loss of wild-type p53 function renders cells resistant to DNA damage-induced cell cycle arrest and ultimately leads to genomic instabilities including gene amplifications, translocations and aneuploidy. Some of these chromosomal lesions are based on mechanisms that involve recombinatorial events. Here we report that p53 physically interacts with key factors of homologous recombination: the human RAD51 protein and its prokaryotic homologue RecA. In vitro, wild-type p53 inhibits defined biochemical activities of RecA protein, such as three-way DNA strand exchange and single strand DNA-dependent ATPase activity. In vivo, temperature-sensitive p53 forms complexes with RAD51 only in wild-type but not in mutant conformation. These observations suggest that functional wild-type p53 may select directly the appropriate pathway for DNA repair and control the extent and timing of the production of genetic variation via homologous recombination. Gene amplification an other types of chromosome rearrangements involved in tumour progression might occur not only as result of inappropriate cell proliferation but as a direct consequence of a defect in p53-mediated control of homologous recombination processes due to mutations in the p53 gene.     

Mechanisms of distinctive mismatch tolerance between Rad51 and Dmc1 in homologous recombination

Homologous recombination (HR) is a primary DNA double-strand breaks (DSBs) repair mechanism. The recombinases Rad51 and Dmc1 are highly conserved in the RecA family; Rad51 is mainly responsible for DNA repair in somatic cells during mitosis while Dmc1 only works during meiosis in germ cells. This spatiotemporal difference is probably due to their distinctive mismatch tolerance during HR: Rad51 does not permit HR in the presence of mismatches, whereas Dmc1 can tolerate certain mismatches. Here, the cryo-EM structures of Rad51–DNA and Dmc1–DNA complexes revealed that the major conformational differences between these two proteins are located in their Loop2 regions, which contain invading single-stranded DNA (ssDNA) binding residues and double-stranded DNA (dsDNA) complementary strand binding residues, stabilizing ssDNA and dsDNA in presynaptic and postsynaptic complexes, respectively. By combining molecular dynamic simulation and single-molecule FRET assays, we identified that V273 and D274 in the Loop2 region of human RAD51 (hRAD51), corresponding to P274 and G275 of human DMC1 (hDMC1), are the key residues regulating mismatch tolerance during strand exchange in HR. This HR accuracy control mechanism provides mechanistic insights into the specific roles of Rad51 and Dmc1 in DNA double-strand break repair and may shed light on the regulatory mechanism of genetic recombination in mitosis and meiosis.