Steps Of Homologous Recombination Research Articles

Efficient generation of double heterologous promoter controlled oncolytic adenovirus vectors by a single homologous recombination step in Escherichia coli

BackgroundOncolytic adenoviruses are promising agents for the multimodal treatment of cancer. However, tumor-selectivity is crucial for their applicability in patients. Recent studies by several groups demonstrated that oncolytic adenoviruses with tumor-/tissue-specific expression of the E1 and E4 genes, which are pivotal for adenoviral replication, have a specificity profile that is superior to viruses that solely target the expression of E1 or E4 genes. Presently the E1 and E4 regions are modified in a time consuming sequential fashion.ResultsBased on the widely used adenoviral cloning system AdEasy we generated a novel transfer vector that allows efficient and rapid generation of conditionally replication-competent adenovirus type 5 based vectors with the viral E1 and E4 genes under the transcriptional control of heterologous promoters. For insertion of the promoters of interest our transfer vector has two unique multiple cloning sites. Additionally, our shuttle plasmid allows encoding of a transgene within the E1A transcription unit. The modifications, including E1 mutations, are introduced into the adenoviral genome by a single homologous recombination step in Escherichia coli. Subsequently infectious viruses are rescued from plasmids. As a proof-of-concept we generated two conditionally replication-competent adenoviruses Ad.Ki•COX and Ad.COX•Ki with the promoters of the Ki-67 protein and the cyclooxygenase-2 (COX-2) driving E1 and E4 and vice versa.ConclusionWe demonstrated with our cloning system efficient generation of double heterologous promoter controlled oncolytic adenoviral vectors by a single homologous recombination step in bacteria. The generated viruses showed preferential replication in tumor cells and in a subcutaneous HT-29 colon cancer xenograft model the viruses demonstrated significant oncolytic activity comparable with dl327.

P53 Monitors Replication Fork Regression by Binding to “Chickenfoot” Intermediates

The tumor suppressor protein, p53, utilizes multiple mechanisms to ensure faithful transmission of the genome including regulation of DNA replication, repair, and recombination. Monitoring these pathways may involve direct binding of p53 to the DNA intermediates of these processes. In this study, we generated templates resembling stalled replication forks and utilized electron microscopy to examine p53 interactions with these substrates. Our results show that p53 bound with high affinity to the junction of stalled forks, whereas two cancer-derived p53 mutants showed weak binding. Additionally, some of the templates were rearranged to form "chickenfoot" structures in the presence of p53. These were mostly formed due to p53 trapping intermediates of spontaneous fork regression; however, in a small population, the protein appeared to be promoting their formation. Collectively, these results demonstrate the importance of sequence-independent binding in p53-mediated maintenance of genomic integrity.

Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme

We recently demonstrated that the RecBCD enzyme is a bipolar DNA helicase that employs two single-stranded DNA motors of opposite polarity to drive translocation and unwinding of duplex DNA. We hypothesized that this organization may explain the exceptionally high rate and processivity of DNA unwinding catalyzed by the RecBCD enzyme. Using a stopped-flow dye displacement assay for unwinding activity, we test this idea by analyzing mutant RecBCD enzymes in which either of the two helicase motors is inactivated by mutagenesis. Like the wild-type RecBCD enzyme, the two mutant proteins maintain the ability to bind tightly to blunt duplex DNA ends in the absence of ATP. However, the rate of forward translocation for the RecB motor-defective enzyme is only approximately 30% of the wild-type rate, whereas for the RecD motor-defective enzyme, it is approximately 50%. More significantly, the processivity of translocation is substantially reduced by approximately 25- and 6-fold for each mutant enzyme, respectively. Despite retaining the capacity to bind blunt dsDNA, the RecB-mutant enzyme has lost the ability to unwind DNA unless the substrate contains a short 5'-terminated single-stranded DNA overhang. The consequences of this observation for the architecture of the single-stranded DNA motors in the initiation complex are discussed.

Heteroduplex Joint Formation by a Stoichiometric Complex of Rad51 and Rad52 of Saccharomyces cerevisiae

Both Rad51 and Rad52 are required for homologous genetic recombination in Saccharomyces cerevisiae. Rad51 promotes heteroduplex joint formation, a general step in homologous recombination. Rad52 facilitates the binding of Rad51 to replication protein A (RPA)-coated single-stranded DNA. The requirement of RPA can be avoided in vitro, if the single-stranded DNA is short. Using short single-stranded DNA and homologous double-stranded DNA, in the absence of RPA, we found that Rad52 (optimal at three per Rad51) was still required for Rad51-promoted heteroduplex joint formation in vitro, as assayed by the formation of D-loops, suggesting another role for Rad52. Rad51 has to bind to the single-stranded DNA before the addition of double-stranded DNA for efficient D-loop formation. Immunoprecipitation and single-stranded DNA-bead precipitation analyses revealed the presence of the free and DNA-bound complexes of Rad51 and Rad52 at a 1 to 2 stoichiometry. In the presence of single-stranded DNA, in addition to Rad51, Rad52 was required for extensive untwisting that is an intermediate step toward D-loop formation. Thus, these results suggest that the formation of the stoichiometric complex of Rad52 with Rad51 on single-stranded DNA is required for the functional binding of the protein-single-stranded DNA complex to the double-stranded DNA to form D-loops.

The F-Box DNA helicase Fbh1 prevents Rhp51-dependent recombination without mediator proteins.

A key step in homologous recombination is the loading of Rad51 onto single-stranded DNA to form a nucleoprotein filament that promotes homologous DNA pairing and strand exchange. Mediator proteins, such as Rad52 and Rad55-Rad57, are thought to aid filament assembly by overcoming an inhibitory effect of the single-stranded-DNA-binding protein replication protein A. Here we show that mediator proteins are also required to enable fission yeast Rad51 (called Rhp51) to function in the presence of the F-box DNA helicase Fbh1. In particular, we show that the critical function of Rad22 (an orthologue of Rad52) in promoting Rhp51-dependent recombination and DNA repair can be mostly circumvented by deleting fbh1. Similarly, the reduced growth/viability and DNA damage sensitivity of an fbh1(-) mutant are variously suppressed by deletion of any one of the mediators Rad22, Rhp55, and Swi5. From these data we propose that Rhp51 action is controlled through an interplay between Fbh1 and the mediator proteins. Colocalization of Fbh1 with Rhp51 damage-induced foci suggests that this interplay occurs at the sites of nucleoprotein filament assembly. Furthermore, analysis of different fbh1 mutant alleles suggests that both the F-box and helicase activities of Fbh1 contribute to controlling Rhp51.

Differential and collaborative actions of Rad51 paralog proteins in cellular response to DNA damage

Metazoan Rad51 plays a central role in homologous DNA recombination, and its activity is controlled by a number of Rad51 cofactors. These include five Rad51 paralogs, Rad51B, Rad51C, Rad51D, XRCC2 and XRCC3. We previously hypothesized that all five paralogs participate collaboratively in repair. However, this idea was challenged by the biochemical identification of two independent complexes composed of either Rad51B/C/D/XRCC2 or Rad51C/XRCC3. To investigate if this biochemical finding is matched by genetic interactions, we made double mutants in either the same complex (rad51b/rad51d) or in both complexes (xrcc3/rad51d). In agreement with the biochemical findings the double deletion involving both complexes had an additive effect on the sensitivity to camptothecin and cisplatin. The double deletion of genes in the same complex, on the other hand, did not further increase the sensitivity to these agents. Conversely, all mutants tested displayed comparatively mild sensitivity to γ-irradiation and attenuated γ-irradiation-induced Rad51 foci formation. Thus, in accord with our previous conclusion, all paralogs appear to collaboratively facilitate Rad51 action. In conclusion, our detailed genetic study reveals a complex interplay between the five Rad51 paralogs and suggests that some of the Rad51 paralogs can separately operate in later step of homologous recombination.

Binding of Rad51 and Other Peptide Sequences to a Promiscuous, Highly Electrostatic Binding Site in p53

Homologous recombination is repressed by the binding of p53 to Rad51. We identified by fluorescence and NMR spectroscopy that peptides corresponding to residues 179-190 of Rad51 bind to the core domain of p53 in a site that overlaps with its specific DNA binding site. The p53 site is quite promiscuous, since it also binds peptides derived from 53BP1, 53BP2, Hif-1alpha, and BCL-X(L) in overlapping regions. Binding is mediated mainly by a strong, nonspecific, electrostatic component and is fine tuned by specific interactions. Competition of the different proteins with each other and with specific DNA for a single site in p53 could be a factor in regulation of its activity.

Evolution of Transposons Containing bla TEM Genes

Over 100 variants of the TEM β-lactamase, many of which display an extended-spectrum or inhibitor-resistant phenotype, have been identified (http://www.lahey.org/Studies/temtable.asp) (1, 2, 5). They are all variants of TEM-1 or TEM-2, which are encoded by three of the earliest bacterial resistance transposons to be identified, namely Tn1 (6), Tn2 (8), and Tn3 (9). The blaTEM-1a and blaTEM-1b variants, found in Tn3 and Tn2, respectively, differ by three nucleotides but encode identical TEM-1 proteins (3, 4). The blaTEM-2 variant in Tn1 differs by five or six nucleotides from the blaTEM-1 types, and TEM-2 differs from TEM-1 by a single amino acid change, Q39K (4). Though information on the genetic context of blaTEM genes would be valuable in understanding how this important resistance gene is disseminated, little or no sequence beyond the ends of the gene is available for most TEM variants. Tn3 from R1 (GenBank accession no. {type:entrez-nucleotide,attrs:{text:V00613,term_id:43710,term_text:V00613}}V00613) (7) and Tn1 from RP1/RP4 ({type:entrez-nucleotide,attrs:{text:L27758,term_id:508311,term_text:L27758}}L27758) (10) have been completely sequenced and, in addition to the blaTEM gene, they include transposase and resolvase genes, tnpA and tnpR, and a resolution site, res (Fig. ​(Fig.1A).1A). Recently, we reported the sequence of a related transposon (98 and 97% identical to Tn1 and Tn3, respectively) from a multiresistance plasmid ({type:entrez-nucleotide,attrs:{text:AY123253,term_id:40795472,term_text:AY123253}}AY123253) (11), part of which is identical to the 1.516 kb of sequence that is available for the original Tn2 from RSF1030 ({type:entrez-nucleotide,attrs:{text:X54607,term_id:21998669,term_text:X54607}}X54607) (3, 4). As the remaining sequence may also be identical, we designated this transposon Tn2* (11). FIG. 1. Mosaic nature of Tn2* and related transposons. (A) Overall structure. The boxes represent Tn1, Tn2*, and Tn3 as indicated, with vertical bars at the ends representing the 38-bp inverted repeats. The positions of the genes and the res site ... A comparison of the complete sequences of Tn1, Tn2*, and Tn3 revealed that the differences between them are not distributed randomly (Fig. ​(Fig.1A).1A). They are about 99% identical to each other over most of their 4.95-kb lengths, with most of the differences (94 of 150) confined to a region close to the res site. Between positions 2845 and 3104 (Fig. ​(Fig.1A,1A, hatched area), Tn3 shares only 83.8 and 83.1% identity with Tn1 and Tn2*, respectively. For Tn2*, the adjacent region (positions 3107 to 3493) (Fig. ​(Fig.1A,1A, shaded area) is only 87.2 and 87.5% identical to Tn1 and Tn3, respectively. The boundary between these two regions (Fig. ​(Fig.1B)1B) coincides with the AT dinucleotide in resI, at which resolvase-mediated recombination occurs (12), implicating resolution in the generation of these mosaic structures. The location of the other end of each region is less sharply defined, suggesting the involvement of a homologous recombination step. Though other explanations are possible, a simple way to generate these mosaics, for example from Tn1, is by acquisition of a short region from a hypothetical related transposon. This acquisition requires two steps, as shown for Tn2* in Fig. ​Fig.2.2. Homologous recombination between transposons on different DNA molecules, occurring at position H, would generate a single molecule containing two hybrid transposons. Subsequent resolution at position R would reseparate the DNA molecules and form Tn2* plus the reciprocal mosaic. This order is required because recombining res sites must be on the same DNA molecule (see 12). Tn3 could be generated in a similar way if the initial homologous recombination occurred on the opposite side of res. Equivalent events could produce further mosaic transposons. These findings highlight the involvement of both homologous and resolvase-mediated recombination in the evolution of transposons. FIG. 2. Scheme for generation of Tn2* by a combination of homologous and resolvase-mediated recombination. Tn1 and a hypothetical but related transposon are shown as open and shaded boxes, respectively. The vertical lines labeled H and R indicate the ...

Role of recA/RAD51 family proteins in mammals.

DNA damage causes chromosomal instability leading to oncogenesis, apoptosis, and severe failure of cell functions. The DNA repair system includes base excision repair, nucleotide excision repair, mismatch repair, translesion replication, non-homologous end-joining, and recombinational repair. Homologous recombination performs the recombinational repair. The RAD51 gene is an ortholog of Esherichia coli recA, and the gene product Rad51 protein plays a central role in the homologous recombination. In mammals, 7 recA-like genes have been identified: RAD51, RAD51L1/B, RAD51L2/C, RAD51L3/D, XRCC2, XRCC3, and DMC1. These genes, with the exception of meiosis-specific DMC1, are essential for development in mammals. Disruption of the RAD51 gene leads to cell death, whereas RAD51L1/B, RAD51L2/C, RAD51L3/D, XRCC2, and XRCC3 genes (RAD51 paralogs) are not essential for viability of cells, but these gene-deficient cells exhibit a similar defective phenotype. Yeast two-hybrid analysis, co-immunoprecipitation, mutation analysis, and domain mapping of Rad51 and Rad51 paralogs have revealed protein-protein interactions among these gene products. Recent investigations have shown that Rad51 paralogs play a role not only in an early step, but also in a late step of homologous recombination. In addition, identification of alternative transcripts of some RAD51 paralogs may reflect the complexity of the homologous recombination system.

Human Rad54 Protein Stimulates DNA Strand Exchange Activity of hRad51 Protein in the Presence of Ca2+

Rad51 and Rad54 proteins play a key role in homologous recombination in eukaryotes. Recently, we reported that Ca2+ is required in vitro for human Rad51 protein to form an active nucleoprotein filament that is important for the search of homologous DNA and for DNA strand exchange, two critical steps of homologous recombination. Here we find that Ca2+ is also required for hRad54 protein to effectively stimulate DNA strand exchange activity of hRad51 protein. This finding identifies Ca2+ as a universal cofactor of DNA strand exchange promoted by mammalian homologous recombination proteins in vitro. We further investigated the hRad54-dependent stimulation of DNA strand exchange. The mechanism of stimulation appeared to include specific interaction of hRad54 protein with the hRad51 nucleoprotein filament. Our results show that hRad54 protein significantly stimulates homology-independent coaggregation of dsDNA with the filament, which represents an essential step of the search for homologous DNA. The results obtained indicate that hRad54 protein serves as a dsDNA gateway for the hRad51-ssDNA filament, promoting binding and an ATP hydrolysis-dependent translocation of dsDNA during the search for homologous sequences.

Crystal Structures of Escherichia coli RecA in Complex with MgADP and MnAMP−PNP,

RecA catalyzes the DNA pairing and strand-exchange steps of homologous recombination, an important mechanism for repair of double-stranded DNA breaks. The binding of RecA to DNA is modulated by adenosine nucleotides. ATP increases the affinity of RecA for DNA, while ADP decreases the affinity. Previously, the crystal structures of E. coli RecA and its complex with ADP have been determined to resolutions of 2.3 and 3.0 A, respectively, but the model for the RecA-ADP complex did not include magnesium ion or side chains. Here, we have determined the crystal structures of RecA in complex with MgADP and MnAMP-PNP, a nonhydrolyzable analogue of ATP, at resolutions of 1.9 and 2.1 A, respectively. Both crystals grow in the same conditions and have RecA in a right-handed helical form with a pitch of approximately 82 A. The crystal structures show the detailed interactions of RecA with the nucleotide cofactors, including the metal ion and the gamma phosphate of AMP-PNP. There are very few conformational differences between the structures of RecA bound to ADP and AMP-PNP, which differ from uncomplexed RecA only in a slight opening of the P-loop residues 66-73 upon nucleotide binding. To interpret the functional significance of the structure of the MnAMP-PNP complex, a coprotease assay was used to compare the ability of different nucleotides to promote the active, extended conformation of RecA. Whereas ATPgammaS and ADP-AlF(4) facilitate a robust coprotease activity, ADP and AMP-PNP do not activate RecA at all. We conclude that the crystal structure of the RecA-MnAMP-PNP complex represents a preisomerization state of the RecA protein that exists after ATP has bound but before the conformational transition to the active state.

Functional analysis of the Drosophila Rad51 gene ( spn-A) in repair of DNA damage and meiotic chromosome segregation

Rad51 is a crucial enzyme in DNA repair, mediating the strand invasion and strand exchange steps of homologous recombination (HR). Mutations in the Drosophila Rad51 gene ( spn-A) disrupt somatic as well as meiotic double-strand break (DSB) repair, similar to fungal Rad51 genes. However, the sterility of spn-A mutant females prevented a thorough analysis of the role of Rad51 in meiosis. In this study, we generated transgenic animals that express spn-A dsRNA under control of an inducible promoter, and examined the effects of inhibiting expression of spn-A on DNA repair, meiotic recombination and meiotic chromosome pairing and segregation. We found that depletion of spn-A mRNA had no effect on the viability of non-mutagen-treated transgenic animals but greatly reduced the survival of larvae that were exposed to the radiomimetic drug MMS, in agreement with the MMS and X-ray sensitivity of spn-A mutant animals. We also found that increases in dose of spn-A gene enhanced larval resistance to MMS exposure, suggesting that at high damage levels, Rad51 protein levels may be limiting for DNA repair. spn-A RNAi strongly stimulated X–X nondisjunction and decreased recombination along the X in female meiosis, consistent with a requirement of Rad51 in meiotic recombination. However, neither RNAi directed against the spn-A mRNA nor homozygosity for a spn-A null mutation had any effect on male fertility or on X–Y segregation in male meiosis, indicating that Rad51 likely plays no role in male meiotic chromosome pairing. Our results support a central role for Rad51 in HR in both somatic and meiotic DSB repair, but indicate that Rad51 in Drosophila is dispensable for meiotic chromosome pairing. Our results also provide the first demonstration that RNAi can be used to inhibit the functions of meiotic genes in Drosophila.

DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1

A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5' to 3' resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.

Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity.

Human Rad51 (hRad51) protein plays a key role in homologous recombination and DNA repair. hRad51 protein forms a helical filament on single-stranded DNA (ssDNA), which performs the basic steps of homologous recombination: a search for homologous double-stranded DNA (dsDNA) and DNA strand exchange. hRad51 protein possesses DNA-dependent ATPase activity; however, the role of this activity has not been understood. Our current results show that Ca(2+) greatly stimulates DNA strand exchange activity of hRad51 protein. We found that Ca(2+) exerts its stimulatory effect by modulating the ATPase activity of hRad51 protein. Our data demonstrate that, in the presence of Mg(2+), the hRad51-ATP-ssDNA filament is quickly converted to an inactive hRad51-ADP-ssDNA form, due to relatively rapid ATP hydrolysis and slow dissociation of ADP. Ca(2+) maintains the active hRad51-ATP-ssDNA filament by reducing the ATP hydrolysis rate. These findings demonstrate a crucial role of the ATPase activity in regulation of DNA strand exchange activity of hRad51 protein. This mechanism of Rad51 protein regulation by modulating its ATPase activity is evolutionarily recent; we found no such mechanism for yeast Rad51 (yRad51) protein.

Requirement of Rrm3 helicase for repair of spontaneous DNA lesions in cells lacking Srs2 or Sgs1 helicase.

The Rrm3 DNA helicase of Saccharomyces cerevisiae interacts with proliferating cell nuclear antigen and is required for replication fork progression through ribosomal DNA repeats and subtelomeric and telomeric DNA. Here, we show that rrm3 srs2 and rrm3 sgs1 mutants, in which two different DNA helicases have been inactivated, exhibit a severe growth defect and undergo frequent cell death. Cells lacking Rrm3 and Srs2 arrest in the G(2)/M phase of the cell cycle with 2N DNA content and frequently contain only a single nucleus. The phenotypes of rrm3 srs2 and rrm3 sgs1 mutants were suppressed by disrupting early steps of homologous recombination. These observations identify Rrm3 as a new member of a network of pathways, involving Sgs1 and Srs2 helicases and Mus81 endonuclease, suggested to act during repair of stalled replication forks.

A bipolar DNA helicase gene, herA, clusters with rad50, mre11 and nurA genes in thermophilic archaea

We showed previously that rad50 and mre11 genes of thermophilic archaea are organized in an operon-like structure with a third gene (nurA) encoding a 5' to 3' exonuclease. Here, we show that the rad50, mre11 and nurA genes from the hyperthermophilic archaeon Sulfolobus acidocaldarius are co-transcribed with a fourth gene encoding a DNA helicase. This enzyme (HerA) is the prototype of a new class of DNA helicases able to utilize either 3' or 5' single-stranded DNA extensions for loading and subsequent DNA duplex unwinding. To our knowledge, DNA helicases capable of translocating along the DNA in both directions have not been identified previously. Sequence analysis of HerA shows that it is a member of the TrwB, FtsK and VirB4/VirD4 families of the PilT class NTPases. HerA homologs are found in all thermophilic archaeal species and, in all cases except one, the rad50, mre11, nurA and herA genes are grouped together. These results suggest that the archaeal Rad50-Mre11 complex might act in association with a 5' to 3' exonuclease (NurA) and a bipolar DNA helicase (HerA) indicating a probable involvement in the initiation step of homologous recombination.

Knockout of the alanine racemase gene in Lactobacillus plantarum results in septation defects and cell wall perforation

A stable mutant of Lactobacillus plantarum deficient in alanine racemase (Alr) was constructed by two successive homologous recombination steps. When the mutant was supplemented with d-alanine, growth and viability were unaffected. Surprisingly, deprivation of d-alanine during exponential growth did not result in a rapid and extensive lysis as observed in Alr-deficient strains of Escherichia coli or Bacillus subtilis. Rather, the starved mutant cells underwent a growth arrest and were gradually affected in viability with a decrease in colony forming units over 99% in less than 24 h. Additionally, fluorescent techniques demonstrated a loss of cell envelope integrity in the starved cells. Prolonged d-alanine starvation resulted in cells with an aberrant morphology. Scanning and transmission electron microscopy analyses revealed an increase in cell length, deficiencies in septum formation, thinning of the cell envelope and perforation of the cell wall in the septum region. We discuss the involvement of peptidoglycan hydrolases in these phenotypic defects in the context of the crucial role played by d-alanine in peptidoglycan biosynthesis and teichoic acids substitution.

Homologous recombination and cell cycle checkpoints: Rad51 in tumour progression and therapy resistance.

We provide an overview of the functional interrelationship between genes and proteins related to DNA repair by homologous recombination and cell cycle regulation in relation to the progression and therapy resistance of human tumours. To ensure the high-fidelity transmission of genetic information from one generation to the next, cells have evolved mechanisms to monitor genome integrity. Upon DNA damage, cells initiate complex response pathways including cell cycle arrest, activation of genes and gene products involved in DNA repair, and under some circumstances, the triggering of programmed cell death. Deregulation of this co-ordinated response leads to genetic instability and is fundamental to the aetiology of human cancer. Homologous recombination involved in DNA repair is induced by environmental damage as well as misreplication during the normal cell cycle. However, when not regulated properly, it can result in the loss of heterozygocity or genetic rearrangements, central to the process of carcinogenesis. The central step of homologous recombination is the DNA strand exchange reaction catalysed by the eukaryotic Rad51 protein. Here, we describe the recent progress in our understanding of how Rad51 is involved in the signalling and repair of DNA damage and how tumour suppressors, such as p53, ATM, BRCA1, BRCA2, BLM and FANCD2 are linked to Rad51-dependent pathways. An increased knowledge of the role of Rad51 in DNA repair by homologous recombination and its effects on cell cycle progression, tumour development and tumour resistance may provide opportunities for identifying improved diagnostic markers and developing more effective treatments for cancer.

The Recombination-deficient Mutant RPA (rfa1-t11) Is Displaced Slowly from Single-stranded DNA by Rad51 Protein

Replication protein-A (RPA) is involved in many processes of DNA metabolism, including DNA replication, repair, and recombination. Cells carrying a mutation in the largest subunit of RPA (rfa1-t11: K45E) have defects in meiotic recombination, mating-type switching, and survival after DNA damage caused by UV and methyl methanesulfonate, as well as increased genome instability; however, this mutant has no significant defect in DNA replication. We purified the RPA heterotrimer containing the rfa1-t11 substitution (RPA(rfa1-t11)). This mutant RPA binds single-stranded DNA (ssDNA) with the same site size, and the RPA(rfa1-t11).ssDNA complex shows a similar sensitivity to disruption by salt as the wild-type RPA.ssDNA complex. RPA(rfa1-t11) stimulates DNA strand exchange, provided that the Rad51 protein.ssDNA nucleoprotein complex is assembled prior to introduction of the mutant RPA. However, RPA(rfa1-t11) is displaced from ssDNA by Rad51 protein more slowly than wild-type RPA and, as a consequence, Rad51 protein-mediated DNA strand exchange is inhibited when the ssDNA is in a complex with RPA(rfa1-t11). Rad52 protein can stimulate displacement of RPA(rfa1-t11) from ssDNA by Rad51 protein, but the rate of displacement remains slow compared with wild-type RPA. These in vitro results suggest that, in vivo, RPA is bound to ssDNA prior to Rad51 protein and that RPA displacement by Rad51 protein is a critical step in homologous recombination, which is impaired in the rfa1-t11 mutation.

A Novel Function of Rad54 Protein: STABILIZATION OF THE Rad51 NUCLEOPROTEIN FILAMENT

Homologous recombination is important for the repair of double-stranded DNA breaks in all organisms. Rad51 and Rad54 proteins are two key components of the homologous recombination machinery in eukaryotes. In vitro, Rad51 protein assembles with single-stranded DNA to form the helical nucleoprotein filament that promotes DNA strand exchange, a basic step of homologous recombination. Rad54 protein interacts with this Rad51 nucleoprotein filament and stimulates its DNA pairing activity, suggesting that Rad54 protein is a component of the nucleoprotein complex involved in the DNA homology search. Here, using physical criteria, we demonstrate directly the formation of Rad54-Rad51-DNA nucleoprotein co-complexes that contain equimolar amounts of each protein. The binding of Rad54 protein significantly stabilizes the Rad51 nucleoprotein filament formed on either single-stranded DNA or double-stranded DNA. The Rad54-stabilized nucleoprotein filament is more competent in DNA strand exchange and acts over a broader range of solution conditions. Thus, the co-assembly of an interacting partner with the Rad51 nucleoprotein filament represents a novel means of stabilizing the biochemical entity central to homologous recombination, and reveals a new function of Rad54 protein.