Isolation and Characterization of Monomeric Human RAD51: A Novel Tool for Investigating Homologous Recombination in Cancer.

DNA repair protein RAD51 is a key player in the homologous recombination pathway. Upon DNA damage, RAD51 is transported into the nucleus by BRCA2, where it can repair DNA double-strand breaks. Due to the structural complexity and dynamics, researchers have not yet clarified the mechanistic details of every step of RAD51 recruitment and DNA repair. RAD51 possesses an intrinsic tendency to form oligomeric structures, which make it challenging to conduct biochemical and biophysical investigations. Here, for the first time, we report on the isolation and characterization of a human monomeric RAD51 recombinant form, obtained through a double mutation, which preserves the protein's integrity and functionality. We investigated different buffers to identify the most suitable condition needed to definitively stabilize the monomer. The monomer of human RAD51 provides the community with a unique biological tool for investigating RAD51-mediated homologous recombination, and paves the way for more reliable structural, mechanistic, and drug discovery studies.

In Vitro Assays for DNA Branch Migration.

Homologous recombination is a high-fidelity DNA double-strand break repair pathway that uses a homologous template to repair the break. Recombinases are the central enzymes that facilitate the strand invasion step of homologous recombination, which forms a DNA joint molecule. These DNA joint molecules can be moved through branch migration activity. In this chapter, we describe two assays to determine the branch migration activity and directionality of an enzyme. Monitoring the branch migration activity of an enzyme can provide insight into the roles of these factors in homologous recombination.

Homologous pairing activities of Arabidopsis thaliana RAD51 and DMC1.

In eukaryotes, homologous recombination plays a pivotal role in both genome maintenance and generation of genetic diversity. Eukaryotic RecA homologues, RAD51 and DMC1, are key proteins in homologous recombination that promote pairing between homologous DNA sequences. Arabidopsis thaliana is a prominent model plant for studying eukaryotic homologous recombination. However, A. thaliana RAD51 and DMC1 have not been biochemically characterized. In the present study, we purified A. thaliana RAD51 (AtRAD51) and DMC1 (AtDMC1). Biochemical analyses revealed that both AtRAD51 and AtDMC1 possess ATP hydrolyzing activity, filament formation activity and homologous pairing activity in vitro. We then compared the homologous pairing activities of AtRAD51 and AtDMC1 with those of the Oryza sativa and Homo sapiens RAD51 and DMC1 proteins.

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.

Determining the RAD51-DNA Nucleoprotein Filament Structure and Function by Cryo-Electron Microscopy.

Homologous recombination is a universal tool for DNA double-strand break and replication fork repair, and it is catalyzed by a highly conserved family of recombinases. In eukaryotes, Rad51 is the recombinase that catalyzes the pairing of homologous DNA molecules and the exchange of strands between the paired molecules. Rad51 assembles on single-stranded DNA (ssDNA) stemming from lesion processing to form a right-handed helical polymer that engages then samples double-stranded DNA (dsDNA) for homology. Upon matching with a homologous sequence, the Rad51-bound ssDNA invades the dsDNA, leading to the formation of a DNA joint with concomitant displacement of the strand of like polarity. The Rad51-DNA filaments are amenable to structural studies using cryo-electron microscopy (cryo-EM). In particular, recent technical breakthroughs in cryo-EM have made it possible to define the structure and function of human RAD51 at near-atomic resolution. In this chapter, we describe our cryo-EM approach to capture the human RAD51 filament structures in various stages of catalysis. The approach may also be useful for related recombinases and other helical assemblies.

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.

Approaches to Understanding the Mediator Function of Brh2 in Ustilago maydis.

Primary components of the homologous recombination pathway in eukaryotes include Rad51 whose function is to search for DNA sequence homology and promote strand exchange, its mediator BRCA2, and Dss1, a key regulator of BRCA2. We seek to understand the role of BRCA2 in governing the activity of Rad51 and to learn how BRCA2 function is regulated by Dss1. We use the microbe Ustilago maydis as a model system for experimentation because it has a well-conserved BRCA2-homolog, Brh2, and is amenable to biochemical and molecular genetic manipulations and analysis. The powerful attributes of this system open the way for gaining insight into BRCA2's molecular mechanism through avenues not immediately approachable in the vertebrate systems. Here we provide protocols for preparing Brh2, Dss1, and Rad51 as reagents for use in biochemical assays to monitor function and present methods for transposon-based mutational analysis of Brh2 for use in genetic dissection of function.

Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase.

The protozoan parasite responsible for human amoebiasis is Entamoeba histolytica. An important facet of the life cycle of E. histolytica involves the conversion of the mature trophozoite to a cyst. This transition is thought to involve homologous recombination (HR), which is dependent upon the Rad51 recombinase. Here, a biochemical characterization of highly purified ehRad51 protein is presented. The ehRad51 protein preferentially binds ssDNA, forms a presynaptic filament and possesses ATP hydrolysis activity that is stimulated by the presence of DNA. Evidence is provided that ehRad51 catalyzes robust DNA strand exchange over at least 5.4 kilobase pairs. Although the homologous DNA pairing activity of ehRad51 is weak, it is strongly enhanced by the presence of two HR accessory cofactors, calcium and Hop2-Mnd1. The biochemical system described herein was used to demonstrate the potential for targeting ehRad51 with two small molecule inhibitors of human RAD51. We show that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited ehRad51 by interfering with DNA binding and attenuated encystation in Entamoeba invadens, while B02 had no effect on ehRad51 strand exchange activity. These results provide insight into the underlying mechanism of homology-directed DNA repair in E. histolytica.

Purification of the human SMN-GEMIN2 complex and assessment of its stimulation of RAD51-mediated DNA recombination reactions.

A deficiency in the SMN gene product causes the motor neuron degenerative disease spinal muscular atrophy. GEMIN2 was identified as an SMN-interacting protein, and the SMN-GEMIN2 complex constitutes part of the large SMN complex, which promotes the assembly of the spliceosomal small nuclear ribonucleoprotein (snRNP). In addition to its splicing function, we previously found that GEMIN2 alone stimulates RAD51-mediated recombination in vitro, and functions in DNA double-strand-break (DSB) repair through homologous recombination in vivo. However, the function of SMN in homologous recombination has not been reported. In the present study, we successfully purified the SMN-GEMIN2 complex as a fusion protein. The SMN-GEMIN2 fusion protein complemented the growth-defective phenotype of GEMIN2-knockout cells. The purified SMN-GEMIN2 fusion protein enhanced the RAD51-mediated homologous pairing much more efficiently than GEMIN2 alone. SMN-GEMIN2 possessed DNA-binding activity, which was not observed with the GEMIN2 protein, and significantly stimulated the secondary duplex DNA capture by the RAD51-single-stranded DNA complex during homologous pairing. These results provide the first evidence that the SMN-GEMIN2 complex plays a role in homologous recombination, in addition to spliceosomal snRNP assembly.

Visualizing RAD51-mediated joint molecules: implications for recombination mechanism and the effect of sequence heterology.

The defining event in homologous recombination is the exchange of base-paired partners between a single-stranded (ss) DNA and a homologous duplex driven by recombinase proteins, such as human RAD51. To understand the mechanism of this essential genome maintenance event, we analyzed the structure of RAD51-DNA complexes representing strand exchange intermediates at nanometer resolution by scanning force microscopy. Joint molecules were formed between substrates with a defined ssDNA segment and homologous region on a double-stranded (ds) partner. We discovered and quantified several notable architectural features of RAD51 joint molecules. Each end of the RAD51-bound joints had a distinct structure. Using linear substrates, a 10-nt region of mispaired bases blocked extension of joint molecules in all examples observed, whereas 4 nt of heterology only partially blocked joint molecule extension. Joint molecules, including 10 nt of heterology, had paired DNA on either side of the heterologous substitution, indicating that pairing could initiate from the free 3'end of ssDNA or from a region adjacent to the ss-ds junction. RAD51 filaments covering joint ss-dsDNA regions were more stable to disassembly than filaments covering dsDNA. We discuss how distinct structural features of RAD51-bound DNA joints can play important roles as recognition sites for proteins that facilitate and control strand exchange.

Homologous recombination properties of OsRad51, a recombinase from rice.

cDNA corresponding to OsRad51 protein was isolated from cDNA library of rice flowers (Oryza sativa, Indica cultivar group) and cloned in to pET28a expression vector. The protein was over expressed in E. coli BL21 (DE3) and purified. Purified OsRad51 could bind single and double stranded DNA, however it showed higher affinity for single stranded DNA. Transmission Electron Microscopy (TEM) studies of OsRad51-DNA complexes showed that this protein formed ring like structures and bound DNA forming filaments. OsRad51 protein promoted renaturation of complementary single strands in to duplex DNA molecules and also showed ATPase activity, which was stimulated by single strand DNA. Fluorescence resonance energy transfer (FRET) assays revealed that OsRad51 promoted homology dependent renaturation as well as strand exchange reactions. Renaturation activity was ATP dependent; however strand exchange activity was ATP independent. This is the first report on in vitro characterization of Rad51 protein from crop plants.

Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics.

Rad51 and Rad54 are key proteins that collaborate during homologous recombination. Rad51 forms a presynaptic filament with ATP and ssDNA active in homology search and DNA strand exchange, but the precise role of its ATPase activity is poorly understood. Rad54 is an ATP-dependent dsDNA motor protein that can dissociate Rad51 from dsDNA, the product complex of DNA strand exchange. Kinetic analysis of the budding yeast proteins revealed that the catalytic efficiency of the Rad54 ATPase was stimulated by partial filaments of wild-type and Rad51-K191R mutant protein on dsDNA, unambiguously demonstrating that the Rad54 ATPase activity is stimulated under these conditions. Experiments with Rad51-K191R as well as with wild-type Rad51-dsDNA filaments formed in the presence of ATP, ADP or ATP-gamma-S showed that efficient Rad51 turnover from dsDNA requires both the Rad51 ATPase and the Rad54 ATPase activities. The results with Rad51-K191R mutant protein also revealed an unexpected defect in binding to DNA. Once formed, Rad51-K191R-DNA filaments appeared normal upon electron microscopic inspection, but displayed significantly increased stability. These biochemical defects in the Rad51-K191R protein could lead to deficiencies in presynapsis (filament formation) and postsynapsis (filament disassembly) in vivo.

Purification and assays of Saccharomyces cerevisiae homologous recombination proteins.

Homologous recombination is an important means of eliminating DNA double strand breaks from chromosomes. The homologous recombination reaction is mediated by the Rad51 recombinase, which requires a number of ancillary factors for maximal efficiency. The development of purification procedures and biochemical assays for yeast Rad51 and other yeast recombination proteins has allowed investigators to begin dissecting the hierarchy of physical and functional interactions among these protein factors that govern the integrity of the homologous recombination machinery. The biochemical studies done with yeast recombination factors have helped formulate conceptual frameworks to guide similar endeavors in other eukaryotes, including humans. Continuing efforts with reconstituted systems that comprise yeast factors will undoubtedly continue to provide insights into the mechanistic intricacy of the homologous recombination machinery.