Rad52 Inverse Strand Exchange Drives RNA-Templated DNA Double-Strand Break Repair.

RNA can serve as a template for DNA double-strand break repair in yeast cells, and Rad52, a member of the homologous recombination pathway, emerged as an important player in this process. However, the exact mechanism of how Rad52 contributes to RNA-dependent DSB repair remained unknown. Here, we report an unanticipated activity of yeast and human Rad52: inverse strand exchange, in which Rad52 forms a complex with dsDNA and promotes strand exchange with homologous ssRNA or ssDNA. We show that in eukaryotes, inverse strand exchange between homologous dsDNA and RNA is a distinctive activity of Rad52; neither Rad51 recombinase nor the yeast Rad52 paralog Rad59 has this activity. In accord with our in vitro results, our experiments in budding yeast provide evidence that Rad52 inverse strand exchange plays an important role in RNA-templated DSB repair in vivo.

Linear dichroism reveals the perpendicular orientation of DNA bases in the RecA and Rad51 recombinase filaments: A possible mechanism for the strand exchange reaction.

Linear dichroism spectroscopy is used to investigate the structure of RecA family recombinase filaments (RecA and Rad51 proteins) with DNA for clarifying the molecular mechanism of DNA strand exchange promoted by these proteins and its activation. The measurements show that the recombinases promote the perpendicular base orientation of single-stranded DNA only in the presence of activators, indicating the importance of base orientation in the reaction. We summarize the results and discuss the role of DNA base orientation.

Human Rad51 Protein Requires Higher Concentrations of Calcium Ions for D-Loop Formation than for Oligonucleotide Strand Exchange.

Human Rad51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. Both the activator protein Swi5/Sfr1 and Ca2+ ions stimulate different reaction steps and induce perpendicular DNA base alignment in the presynaptic complex. To investigate the role of base orientation in the strand exchange reaction, we examined the Ca2+ concentration dependence of strand exchange activities and structural changes in the presynaptic complex. Our results show that optimal D-loop formation (strand exchange with closed circular DNA) required Ca2+ concentrations greater than 5 mM, whereas 1 mM Ca2+ was sufficient for strand exchange between two oligonucleotides. Structural changes indicated by increased fluorescence intensity of poly(dεA) (a poly(dA) analog) reached a plateau at 1 mM Ca2+. Ca2+ > 2 mM was required for saturation of linear dichroism signal intensity at 260 nm, associated with rigid perpendicular DNA base orientation, suggesting a correlation with the stimulation of D-loop formation. Therefore, Ca2+ exerts two different effects. Thermal stability measurements suggest that HsRad51 binds two Ca2+ ions with KD values of 0.2 and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. Our results indicate parallels between the Mg2+ activation of RecA and the Ca2+ activation of HsRad51.

Mismatch detection in homologous strand exchange amplified by hydrophobic effects.

In contrast to DNA replication and transcription where nucleotides are added and matched one by one, homologous recombination by DNA strand exchange tests whole sequences for complementarity, which requires elimination of mismatched yet thermodynamically stable intermediates. To understand the remarkable sequence specificity of homologous recombination, we have studied strand exchange between a 20-mer duplex containing one single mismatch (placed at varied positions) with the matching single strand in presence of poly(ethylene glycol) representing a semi-hydrophobic environment. A FRET-based assay shows that rates and yields of strand exchange from mismatched to matched strands rapidly increase with semi-hydrophobic co-solute concentration, contrasting previously observed general strand exchange accelerating effect of ethyl glycol ethers. We argue that this effect is not caused simply by DNA melting or solvent-induced changes of DNA conformation but is more complex involving several mechanisms. The catalytic effects, we propose, involve strand invasion facilitated by reduced duplex stability due to weakened base stacking ("longitudinal breathing"). Secondly, decreased water activity makes base-pair hydrogen bonds stronger, increasing the relative energy penalty per mismatch. Finally, unstacked mismatched bases (gaps) are stabilized through partly intercalated hydrophobic co-solvent molecules, assisting nucleation of strand invasion at the point of mismatch. We speculate that nature long ago discovered, and now exploits in various enzymes, that sequence recognition power of nucleic acids may be modulated in a hydrophobic environment.

RecA kinetically selects homologous DNA by testing a five- or six-nucleotide matching sequence and deforming the second DNA.

RecA family proteins pair two DNAs with the same sequence to promote strand exchange during homologous recombination. To understand how RecA proteins search for and recognize homology, we sought to determine the length of homologous sequence that permits RecA to start its reaction. Specifically, we analyzed the effect of sequence heterogeneity on the association rate of homologous DNA with RecA/single-stranded DNA complex. We assumed that the reaction can start with equal likelihood at any point in the DNA, and that sequence heterogeneity abolishes some possible initiation sites. This analysis revealed that the effective recognition size is five or six nucleotides, larger than the three nucleotides recognized by a RecA monomer. Because the first DNA is elongated 1.5-fold by intercalation of amino acid residues of RecA every three bases, the second bound DNA must be elongated to pair with the first. Because this length is similar to estimates based on the strand-exchange reaction or DNA pair formation, the homology test is likely to occur primarily at the association step. The energetic difference due to the absence of hydrogen bonding is too small to discriminate single-nucleotide heterogeneity over a five- or six-nucleotide sequence. The selection is very likely to be made kinetically, and probably involves some structural factor other than Watson-Crick hydrogen bonding. It would be valuable to determine whether this is also the case for other biological reactions involving DNA base complementarity, such as replication, transcription, and translation.

Main steps in DNA double-strand break repair: an introduction to homologous recombination and related processes.

DNA double-strand breaks arise accidentally upon exposure of DNA to radiation and chemicals or result from faulty DNA metabolic processes. DNA breaks can also be introduced in a programmed manner, such as during the maturation of the immune system, meiosis, or cancer chemo- or radiotherapy. Cells have developed a variety of repair pathways, which are fine-tuned to the specific needs of a cell. Accordingly, vegetative cells employ mechanisms that restore the integrity of broken DNA with the highest efficiency at the lowest cost of mutagenesis. In contrast, meiotic cells or developing lymphocytes exploit DNA breakage to generate diversity. Here, we review the main pathways of eukaryotic DNA double-strand break repair with the focus on homologous recombination and its various subpathways. We highlight the differences between homologous recombination and end-joining mechanisms including non-homologous end-joining and microhomology-mediated end-joining and offer insights into how these pathways are regulated. Finally, we introduce noncanonical functions of the recombination proteins, in particular during DNA replication stress.

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange.

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity.

Structure of a filament of stacked octamers of human DMC1 recombinase.

Eukaryal DMC1 proteins play a central role in homologous recombination in meiosis by assembling at the sites of programmed DNA double-strand breaks and carrying out a search for allelic DNA sequences located on homologous chromatids. They are close homologs of eukaryal Rad51 and archaeal RadA proteins and are remote homologs of bacterial RecA proteins. These recombinases (also called DNA strand-exchange proteins) promote a pivotal strand-exchange reaction between homologous single-stranded and double-stranded DNA substrates. An octameric form of a truncated human DMC1 devoid of its small N-terminal domain (residues 1-83) has been crystallized. The structure of the truncated DMC1 octamer is similar to that of the previously reported full-length DMC1 octamer, which has disordered N-terminal domains. In each protomer, only the ATP cap regions (Asp317-Glu323) show a noticeable conformational difference. The truncated DMC1 octamers further stack with alternate polarity into a filament. Similar filamentous assemblies of DMC1 have been observed to form on DNA by electron microscopy.

DNA Strand Exchange to Monitor Human RAD51-Mediated Strand Invasion and Pairing.

The homologous recombination (HR) pathway maintains genomic integrity by repairing DNA double-strand breaks (DSBs), single-strand DNA gaps, and collapsed replication forks. The process of HR involves strand invasion, homology search, and DNA strand exchange between paired DNA molecules. HR is critical for the high-fidelity repair of DNA DSBs in mitotic cells and for the exchange of genetic information during meiosis. Here we describe a DNA strand exchange reaction in vitro utilizing purified proteins and defined DNA substrates to measure the strand invasion and pairing activities of the human RAD51 protein. We further discuss how this reaction can be catalytically stimulated by the mediator protein BRCA2.

Recombinational repair in the absence of holliday junction resolvases in E. coli.

Cells possess two major DNA damage tolerance pathways that allow them to duplicate their genomes despite the presence of replication blocking lesions: translesion synthesis (TLS) and daughter strand gap repair (DSGR). The TLS pathway involves specialized DNA polymerases that are able to synthesize past DNA lesions while DSGR relies on Recombinational Repair (RR). At least two mechanisms are associated with RR: Homologous Recombination (HR) and RecA Mediated Excision Repair (RAMER). While HR and RAMER both depend on RecFOR and RecA, only the HR mechanism should involve Holliday Junctions (HJs) resolvase reactions. In this study we investigated the role of HJ resolvases, RuvC, TopIII and RusA on the balance between RAMER and HR in E. coli MG1655 derivatives. Using UV survival measurements, we first clearly establish that, in this genetic background, topB and ruvC define two distinct pathways of HJ resolution. We observed that a recA mutant is much more sensitive to UV than the ruvC topB double mutant which is deficient in HR because of its failure to resolve HJs. This difference is independent of RAMER, the SOS system, RusA, and the three TLS DNA polymerases, and may be accounted for by Double Strand Break repair mechanisms such as Synthesis Dependent Strand Annealing, Single Strand Annealing, or Break Induced Replication, which are independent of HJ resolvases. We then used a plasmid-based assay, in which RR is triggered by a single blocking lesion present on a plasmid molecule, to establish that while HR requires topB, ruvC or rusA, RAMER is independent of these genes and, as expected, requires a functional UvrABC excinuclease. Surprisingly, analysis of the RR events in a strain devoid of HJ resolvases reveals that the UvrABC dependent repair of the single lesion present on the plasmid molecule can generate an excision track potentially extending to dozens of nucleotides.

Understanding Rad51 function is a prerequisite for progress in cancer research.

The human protein Rad51 is double-edged in cancer contexts: on one hand, preventing tumourigenesis by eliminating potentially carcinogenic DNA damage and, on the other, promoting tumours by introducing new mutations. Understanding mechanistic details of Rad51 in homologous recombination (HR) and repair could facilitate design of novel methods, including CRISPR, for Rad51-targeted cancer treatment. Despite extensive research, however, we do not yet understand the mechanism of HR in sufficient detail, partly due to complexity, a large number of Rad51 protein units being involved in the exchange of long DNA segments. Another reason for lack of understanding could be that current recognition models of DNA interactions focus only on hydrogen bond-directed base pair formation. A more complete model may need to include, for example, the kinetic effects of DNA base stacking and unstacking ('longitudinal breathing'). These might explain how Rad51 can recognize sequence identity of DNA over several bases long stretches with high accuracy, despite the fact that a single base mismatch could be tolerated if we consider only the hydrogen bond energy. We here propose that certain specific hydrophobic effects, recently discovered destabilizing stacking of nucleobases, may play a central role in this context for the function of Rad51.

The extended N-terminus of Mycobacterium smegmatis RecX potentiates its ability to antagonize RecA functions.

The members of the RecX family of proteins have a unique capacity to regulate the catalytic activities of RecA/Rad51 proteins in both prokaryotic and eukaryotic organisms. However, our understanding of the functional roles of RecX in pathogenic and non-pathogenic mycobacteria has been limited by insufficient knowledge of the molecular mechanisms of its activity and regulation. Moreover, the significance of a unique 14 amino acid N-terminal extension in Mycobacterium smegmatis RecX (MsRecX) to its function remains unknown. Here, we advance our understanding of the antagonistic roles of mycobacterial RecX proteins and the functional significance of the extended N-terminus of MsRecX. The full-length MsRecX acts as an antagonist of RecA, negatively regulating RecA promoted functions, including DNA strand exchange, LexA cleavage and ATP hydrolysis, but not binding of ATP. The N-terminally truncated MsRecX variants retain the RecA inhibitory activity, albeit with lower efficiencies compared to the full-length protein. Perhaps most importantly, direct visualization of RecA nucleoprotein filaments, which had been incubated with RecX proteins, showed that they promote disassembly of nucleoprotein filaments primarily within the filaments. In addition, interaction of RecX proteins with the RecA nucleoprotein filaments results in the formation of stiff and irregularly shaped nucleoprotein filaments. Thus, these findings add an additional mechanism by which RecX disassembles RecA nucleoprotein filaments. Overall, this study provides strong evidence for the notion that the N-terminal 14 amino acid region of MsRecX plays an important role in the negative regulation of RecA functions and new insights into the molecular mechanism underlying RecX function.

In Vitro Assay for Plasmid Length DNA Strand Exchange by Human DMC1.

Meiosis is a specialized cell division that generates gametes. Meiotic recombination is essential not only to generate diversity in offspring, but also to hold homologous chromosomes together through chiasma allowing proper chromosome segregation. This process requires the meiosis-specific recombinase, DMC1. DMC1 facilitates the search for homology between the homologous chromosomes and is followed by DNA strand invasion and strand exchange to produce a linkage between the two homologous chromosomes. The development of biochemical in vitro assays and the purification of the requisite proteins factors has led to a better understanding of the molecular mechanisms of meiotic homologous recombination. In this chapter, a detailed in vitro assay to examine DNA strand exchange over 5000 bases of DNA catalyzed by human DMC1 is described. This method has proved to be valuable for examining the catalytic potential of hDMC1 and delineating the functional interaction with other HR factors.

Gel-Based Assays for Measuring DNA Unwinding, Annealing, and Strand Exchange.

Efficient replication and repair of the genome requires a multitude of protein-DNA transactions. These interactions can result in a variety of consequences for DNA such as the unwinding of double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA), the annealing of complementary ssDNAs, or the exchange of ssDNA with one strand of a dsDNA duplex. Some DNA helicases possess all three activities, but many DNA-interacting proteins can also catalyze one or more of these reactions. Assays that quantify these activities are an important first step in characterizing these protein-DNA interactions in vitro. Here, we describe methods for the formation of dsDNA substrates and the assays that can be used to biochemically characterize proteins that can unwind, anneal, and/or exchange DNA strands.

RecA Regulation by RecU and DprA During Bacillus subtilis Natural Plasmid Transformation.

Natural plasmid transformation plays an important role in the dissemination of antibiotic resistance genes in bacteria. During this process, Bacillus subtilis RecA physically interacts with RecU, RecX, and DprA. These three proteins are required for plasmid transformation, but RecA is not. In vitro, DprA recruits RecA onto SsbA-coated single-stranded (ss) DNA, whereas RecX inhibits RecA filament formation, leading to net filament disassembly. We show that a null recA (ΔrecA) mutation suppresses the plasmid transformation defect of competent ΔrecU cells, and that RecU is essential for both chromosomal and plasmid transformation in the ΔrecX context. RecU inhibits RecA filament growth and facilitates RecA disassembly from preformed filaments. Increasing SsbA concentrations additively contributes to RecU-mediated inhibition of RecA filament extension. DprA is necessary and sufficient to counteract the negative effect of both RecU and SsbA on RecA filament growth onto ssDNA. DprA-SsbA activates RecA to catalyze DNA strand exchange in the presence of RecU, but this effect was not observed if RecU was added prior to RecA. We propose that DprA contributes to RecA filament growth onto any internalized SsbA-coated ssDNA. When the ssDNA is homologous to the recipient, DprA antagonizes the inhibitory effect of RecU on RecA filament growth and helps RecA to catalyze chromosomal transformation. On the contrary, RecU promotes RecA filament disassembly from a heterologous (plasmid) ssDNA, overcoming an unsuccessful homology search and favoring plasmid transformation. The DprA-DprA interaction may promote strand annealing upon binding to the complementary plasmid strands and facilitating thereby plasmid transformation rather than through a mediation of RecA filament growth.

Expression, Purification, and Biochemical Evaluation of Human RAD51 Protein.

The RAD51 DNA strand exchange protein plays an important role in maintaining the integrity of the human genome. It promotes homology-directed DNA repair by exchanging strands between the damaged and the intact DNA molecules. It also plays an important role in stabilizing distressed DNA replication forks. When overexpressed or misregulated, however, RAD51 contributes to "rogue," genome destabilizing events that can lead to cancer, cell death, and to acquisition of chemotherapy resistance by cancerous cells. Human RAD51 is, therefore, an important and highly coveted anticancer drug target. Biochemical, biophysical, and structural studies of the human RAD51 and establishment of its structure-activity relationship require purification of large quantities of protein. In this chapter we describe a robust method for expression and purification of human RAD51 and the methods for assessing its activity based on the single-strand DNA-binding stoichiometry and its capacity to carry out the DNA strand exchange reaction.

Reconstituting the 4-Strand DNA Strand Exchange.

Proteins of the Rad51 family play a key role in homologous recombination by carrying out DNA strand exchange. Here, we present the methodology and the protocols for the 4-strand exchange between gapped circular DNA and homologous linear duplex DNA promoted by human Rad51 and Escherichia coli RecA orthologs. This reaction includes formation of joint molecules and their extension by branch migration in a polar manner. The presented methodology may be used for reconstitution of the medial-to-late stages of homologous recombination in vitro as well as for investigation of the mechanisms of branch migration by helicase-like proteins, e.g., Rad54, BLM, or RecQ1.

Observation and Analysis of RAD51 Nucleation Dynamics at Single-Monomer Resolution.

Human RAD51 promotes accurate DNA repair by homologous recombination and is involved in protection and repair of damaged DNA replication forks. The active species of RAD51 and related recombinases in all organisms is a nucleoprotein filament assembled on single-stranded DNA (ssDNA). The formation of a nucleoprotein filament competent for the recombination reaction, or for DNA replication support, is a delicate and strictly regulated process, which occurs through filament nucleation followed by filament extension. The rates of these two phases of filament formation define the capacity of RAD51 to compete with the ssDNA-binding protein RPA, as well as the lengths of the resulting filament segments. Single-molecule approaches can provide a wealth of quantitative information on the kinetics of RAD51 nucleoprotein filament assembly, internal dynamics, and disassembly. In this chapter, we describe how to set up a single-molecule total internal reflection fluorescence microscopy experiment to monitor the initial steps of RAD51 nucleoprotein filament formation in real-time and at single-monomer resolution. This approach is based on the unique, stretched-ssDNA conformation within the recombinase nucleoprotein filament and follows the efficiency of Förster resonance energy transfer (EFRET) between two DNA-conjugated fluorophores. We will discuss the practical aspects of the experimental setup, extraction of the FRET trajectories, and how to analyze and interpret the data to obtain information on RAD51 nucleation kinetics, the mechanism of nucleation, and the oligomeric species involved in filament formation.

Dissecting the Recombination Mediator Activity of BRCA2 Using Biochemical Methods.

Homologous recombination (HR) is an essential pathway to restart stalled replication forks, repair spontaneous DNA double-strand breaks, and generate genetic diversity. Together with genetic studies in model organisms, the development of purification protocols and biochemical assays has allowed investigators to begin to understand how the complex machinery of HR functions. At the core of the HR process is the recombination enzyme RecA in bacteria or RAD51 and DMC1 in eukaryotes. The main steps of HR can be reconstituted in vitro and involve: (1) The formation of a ssDNA-RAD51 complex into a helical structure termed the nucleoprotein filament after one DNA strand has been resected at the site of the break. (2) The homologous DNA pairing with an intact copy of the damaged chromatid to form a joint molecule also called displacement loop (D-loop). (3) The exchange of DNA strands and de novo DNA synthesis to restore the damaged/lost DNA. (4) The resolution of joint molecules by nucleolytic cleavage. The human tumor suppressor BRCA2 is a mediator of HR as it actively facilitates the DNA transactions of the recombination proteins RAD51 and DMC1 in a variety of ways: It stabilizes ssDNA-RAD51/DMC1 nucleoprotein filaments. It limits the assembly of RAD51 on dsDNA. It facilitates the replacement of replication protein A by RAD51. The result of these activities is a net increase of DNA strand exchange products as observed in vitro. Here, we describe some of the biochemical assays used to dissect the mediator activities of BRCA2.

Reappearance from Obscurity: Mammalian Rad52 in Homologous Recombination.

Homologous recombination (HR) plays an important role in maintaining genomic integrity. It is responsible for repair of the most harmful DNA lesions, DNA double-strand breaks and inter-strand DNA cross-links. HR function is also essential for proper segregation of homologous chromosomes in meiosis, maintenance of telomeres, and resolving stalled replication forks. Defects in HR often lead to genetic diseases and cancer. Rad52 is one of the key HR proteins, which is evolutionarily conserved from yeast to humans. In yeast, Rad52 is important for most HR events; Rad52 mutations disrupt repair of DNA double-strand breaks and targeted DNA integration. Surprisingly, in mammals, Rad52 knockouts showed no significant DNA repair or recombination phenotype. However, recent work demonstrated that mutations in human RAD52 are synthetically lethal with mutations in several other HR proteins including BRCA1 and BRCA2. These new findings indicate an important backup role for Rad52, which complements the main HR mechanism in mammals. In this review, we focus on the Rad52 activities and functions in HR and the possibility of using human RAD52 as therapeutic target in BRCA1 and BRCA2-deficient familial breast cancer and ovarian cancer.