Homologous DNA Molecules Research Articles

Effects of tumor-associated mutations on Rad54 functions.

Yeast RAD54 gene, a member of the RAD52 epistasis group, plays an important role in homologous recombination and DNA double strand break repair. Rad54 belongs to the Snf2/Swi2 protein family, and it possesses a robust DNA-dependent ATPase activity, uses free energy from ATP hydrolysis to supercoil DNA, and cooperates with the Rad51 recombinase in DNA joint formation. There are two RAD54-homologous genes in human cells, hRAD54 and RAD54B. Mutations in these human genes have been found in tumors. These tumor-associated mutations map to conserved regions of the hRad54 and hRad54B proteins. Here we introduced the equivalent mutations into the Saccharomyces cerevisiae RAD54 gene in an effort to examine the functional consequences of these gene changes. One mutant, rad54 G484R, showed sensitivity to DNA-damaging agents and reduced homologous recombination rates, indicating a loss of function. Even though the purified rad54 G484R mutant protein retained the ability to bind DNA and interact with Rad51, it was nearly devoid of ATPase activity and was similarly defective in DNA supercoiling and D-loop formation. Two other mutants, rad54 N616S and rad54 D442Y, were not sensitive to genotoxic agents and behaved like the wild type allele in homologous recombination assays. Consistent with the mild phenotype associated with the rad54 N616S allele, its encoded protein was similar to wild type Rad54 protein in biochemical attributes. Because dysfunctional homologous recombination gives rise to genome instability, our results are consistent with the premise that tumor-associated mutations in hRad54 and Rad54B could contribute to the tumor phenotype or enhance the genome instability seen in tumor cells.

RecA mediated initial alignment of homologous DNA molecules displays apparent first order kinetics with little effect of heterology

The mechanism and determinants of RecA mediated initial alignment of homologous DNA molecules were studied by performing Monte Carlo simulations of the dynamics of DNA molecules. The simulation procedure was used to assess the effect of heterologous DNA and dilution on the rate of formation and yield of homologous alignments. The results show that the apparent first order kinetic behavior and the impact of heterologous DNA, reported in literature [J. Biol. Chem. 261 (1986) 1025], can be observed even if the conversion of the initially aligned molecules into a stable joint is not rate-determining. The present study is the first step towards developing rigorous computational models to describe the process of homologous recombination, and theoretical frameworks to retrieve biophysical parameters of strand pairing and exchange proteins from in vitro assays of joint molecule formation.

Rad54, a Jack of all trades in homologous recombination

Homologous recombination mediates the transfer or exchange of genetic information between homologous DNA molecules. It plays important roles in central processes in the cell such as genome duplication and DNA damage repair. Recent experiments reveal the surprising versatility of one of its central actors, the Rad54 protein.

ADP stabilizes the human Rad51-single stranded DNA complex and promotes its DNA annealing activity.

Human Rad51 protein (HsRad51) is a homologue of Escherichia coli RecA protein, and involved in homologous recombination. These eukaryotic and bacterial proteins catalyse strand exchange between two homologous DNA molecules, each forming a complex with single-stranded DNA (ssDNA) and ATP as the initial step. Both proteins hydrolyse ATP; however, the role of ATP hydrolysis appears to vary between the two proteins. Measurements using the fluorescence ssDNA analogue, poly(1,N6-etheno-deoxyadenosine), indicate that ATP affects the HsRad51-ssDNA complex, promoting two conformational states: one transient, rather rigid transition state and a final more flexible state. While ADP lowers the affinity of RecA protein to ssDNA, it is found to rather stabilize the HsRad51-ssDNA complex. ADP does not activate the strand exchange by HsRad51 but instead stimulates annealing between complementary ssDNAs. The hydrolysis of ATP promotes a transition of the HsRad51-ssDNA complex from a stiff state to less stiff state. The first state may be important for the strand separation of dsDNA in the initial step of strand exchange, while the second state may be important for annealing in the next step. However, hydrolysis does not dissociate HsRad51 from DNA as a component step of its recycling.

Elucidating a key intermediate in homologous DNA strand exchange: structural characterization of the RecA-triple-stranded DNA complex using fluorescence resonance energy transfer.

The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA-tsDNA complex), has been reported. We present a systematic characterization of the helical geometries of the three DNA strands of the RecA-tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions. FRET donor and acceptor dyes were used to label different DNA strands, and the interfluorophore distances were inferred from energy transfer efficiencies measured as a function of the base-pair separation between the two dyes. The energy transfer efficiencies were first measured on a control RecA-dsDNA complex, and the calculated helical parameters (h approximately 5 A, Omega(h) approximately 20 degrees ) were consistent with structural conclusions derived from electron microscopy (EM) and other classic biochemical methods. Measurements of the helical parameters for the RecA-tsDNA complex revealed that all three DNA strands adopt extended and unwound conformations similar to those of RecA-bound dsDNA. The structural data are consistent with the hypothesis that this complex is a late, post-strand-exchange intermediate with the outgoing strand shifted by about three base-pairs with respect to its registry with the incoming and complementary strands. Furthermore, the bases of the incoming and complementary strands are displaced away from the helix axis toward the minor groove of the heteroduplex, and the bases of the outgoing strand lie in the major groove of the heteroduplex. We present a model for the strand exchange intermediate in which homologous contacts preceding strand exchange arise in the minor groove of the substrate dsDNA.

Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage.

Recombination between homologous DNA molecules is essential for the proper maintenance and duplication of the genome, and for the repair of exogenously induced DNA damage such as double-strand breaks. Homologous recombination requires the RAD52 group proteins, including Rad51, Rad52 and Rad54. Upon treatment of mammalian cells with ionizing radiation, these proteins accumulate into foci at sites of DNA damage induction. We show that these foci are dynamic structures of which Rad51 is a stably associated core component, whereas Rad52 and Rad54 rapidly and reversibly interact with the structure. Furthermore, we show that the majority of the proteins are not part of the same multi-protein complex in the absence of DNA damage. Executing DNA transactions through dynamic multi-protein complexes, rather than stable holo-complexes, allows flexibility. In the case of DNA repair, for example, it will facilitate cross-talk between different DNA repair pathways and coupling to other DNA transactions, such as replication.

The stretched DNA geometry of recombination and repair nucleoprotein filaments.

The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA x tsDNA complex), has been elucidated by classical methods. Here we review the systematic characterization of the helical geometries of the three DNA strands of the RecA x tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions. Measurements of the helical parameters for the RecA x tsDNA complex are consistent with the hypothesis that this complex is a late, poststrand-exchange intermediate with the outgoing strand shifted by about three base pairs with respect to its registry with the incoming and complementary strands. All three strands in the RecA x tsDNA complex adopt extended and unwound conformations similar to those of RecA-bound ssDNA and dsDNA.

The architecture of the human Rad54-DNA complex provides evidence for protein translocation along DNA.

Proper maintenance and duplication of the genome require accurate recombination between homologous DNA molecules. In eukaryotic cells, the Rad51 protein mediates pairing between homologous DNA molecules. This reaction is assisted by the Rad54 protein. To gain insight into how Rad54 functions, we studied the interaction of the human Rad54 (hRad54) protein with double-stranded DNA. We have recently shown that binding of hRad54 to DNA induces a change in DNA topology. To determine whether this change was caused by a protein-constrained change in twist, a protein-constrained change in writhe, or the introduction of unconstrained plectonemic supercoils, we investigated the hRad54--DNA complex by scanning force microscopy. The architecture of the observed complexes suggests that movement of the hRad54 protein complex along the DNA helix generates unconstrained plectonemic supercoils. We discuss how hRad54-induced superhelical stress in the target DNA may function to facilitate homologous DNA pairing by the hRad51 protein directly. In addition, the induction of supercoiling by hRad54 could stimulate recombination indirectly by displacing histones and/or other proteins packaging the DNA into chromatin. This function of DNA translocating motors might be of general importance in chromatin metabolism.

Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA.

Both the bacterial RecA protein and the eukaryotic Rad51 protein form helical nucleoprotein filaments on DNA that catalyze strand transfer between two homologous DNA molecules. However, only the ATP-binding cores of these proteins have been conserved, and this same core is also found within helicases and the F1-ATPase. The C-terminal domain of the RecA protein forms lobes within the helical RecA filament. However, the Rad51 proteins do not have the C-terminal domain found in RecA, but have an N-terminal extension that is absent in the RecA protein. Both the RecA C-terminal domain and the Rad51 N-terminal domain bind DNA. We have used electron microscopy to show that the lobes of the yeast and human Rad51 filaments appear to be formed by N-terminal domains. These lobes are conformationally flexible in both RecA and Rad51. Within RecA filaments, the change between the "active" and "inactive" states appears to mainly involve a large movement of the C-terminal lobe. The N-terminal domain of Rad51 and the C-terminal domain of RecA may have arisen from convergent evolution to play similar roles in the filaments.

Mismatch repair: praying for genome stability

Over the past years additional functions have emerged for mismatch repair proteins. The first was the involvement of mismatch repair proteins with homologous DNA recombination, one of the pathways that repair DNA double-strand breaks. A key intermediate in this repair process is heteroduplex joint formation between the ends of the broken molecule and an intact, homologous partner (Fig. 3Fig. 3). During meiosis, the specialized MutS-homologous dimer MSH4–MSH5 stimulates the induction of crossovers at joints between homologous autosomes, in conjunction with the MutL homologues (Fig. 3Fig. 3a). Polymorphisms, commonly present in homologous autosomes, are apparent as mismatches within the joints. MSH2–MSH6, together with the MutL homologues, corrects these mismatches in a process called gene conversion (Fig. 3Fig. 3a,b). Double strand breaks in somatic cells, such as those induced by X-rays, may result in translocations or in loss of genetic information when homologous recombination takes place between nonidentical sequences, such as pseudogenes or homologous autosomes, rather than between identical sister chromatids. In these cells, mismatch repair generally does not perform gene conversion, but instead antagonizes mismatch-containing heteroduplex joints in a process called anti-recombination (Fig. 3Fig. 3c). Absence of mismatch repair, therefore, allows crossover between diverged sequences. As an example, crosses between E. coli and mismatch-repair-deficient Salmonella typhimurium cells result in interspecies hybrids, because crossovers between the 20% diverged genomes now proceed unrestrained.Fig. 3Roles of mismatch repair in determining the outcome of homologous DNA recombination. Top: both ends of each strand of a broken, gapped, DNA molecule (gray backbone) form a heteroduplex junction with a homologous DNA molecule (orange backbone), each consisting of two heteroduplex joints. Note that this represents a simplification of the Holliday model. Sequence divergences between the recombining molecules are apparent as mismatches (M). Mismatch repair proteins can determine the outcome of recombination in three ways: (a) crossover, resulting in an exchange of flanking DNA. This generally is accompanied by repair of the mismatch (‘gene conversion’, indicated by CM). (b) Gene conversion without crossover. (c) Anti-recombination by mismatch repair proteins in somatic cells results in reversion of mismatch-containing heteroduplexes.View Large Image | View Hi-Res Image | Download PowerPoint SlideIn mammalian cells, mismatch repair is involved in triggering apoptosis induced by certain DNA damaging agents. The best examples are agents that methylate the O6 position of guanosine residues, including certain carcinostatic drugs. Wild-type cells are extremely sensitive to these drugs, as incorporation of either C or T opposite O6Me–G residues triggers mismatch-repair-dependent excision. However, resynthesis introduces the same ‘mismatch’, resulting in futile cycles of mismatch repair that ultimately result in apoptosis. An alternative explanation for the mismatch repair dependence of the toxicity of these agents is provided by the similarity of MSH2–MSH6 to Ras nucleotide exchange factors that are active in a GTP-bound form and inactive in a GDP-bound form. MSH2–MSH6, binding to the methylated mispair, exchanges ATP for ADP and might directly signal to downstream effectors of apoptosis. Supporting this model is the detection of mismatch repair proteins in the so-called BRCA1-associated genome surveillance complex, implicated in signaling of DNA damage to cell cycle regulators.Mismatch repair proteins are implicated in several other DNA repair processes, including repair of oxidative nucleotide damage in the transcribed strand of genes, in repair of DNA crosslinks, and in the processing of DNA ends during the single-strand annealing pathway of double strand break repair. All these seemingly diverse activities justify the assignment to mismatch repair of the honorary title ‘Caretaker of our Genome’.

RecA-mediated strand exchange traverses substitutional heterologies more easily than deletions or insertions.

RecA protein in bacteria and its eukaryotic homolog Rad51 protein are responsible for initiation of strand exchange between homologous DNA molecules. This process is crucial for homologous recombination, the repair of certain types of DNA damage and for the reinitiation of DNA replication on collapsed replication forks. We show here, using two different types of in vitro assays, that in the absence of ATP hydrolysis RecA-mediated strand exchange traverses small substitutional heterologies between the interacting DNAs, whereas small deletions or insertions block the ongoing strand exchange. We discuss evolutionary implications of RecA selectivity against insertions and deletions and propose a molecular mechanism by which RecA can exert this selectivity.

Basis for Avid Homologous DNA Strand Exchange by Human Rad51 and RPA

Human Rad51 (hRad51), a member of a conserved family of general recombinases, is shown here to have an avid capability to make DNA joints between homologous DNA molecules and promote highly efficient DNA strand exchange of the paired molecules over at least 5.4 kilobase pairs. Furthermore, maximal efficiency of homologous DNA pairing and strand exchange is strongly dependent on the heterotrimeric single-stranded DNA binding factor hRPA and requires conditions that lessen interactions of the homologous duplex with the hRad51-single-stranded DNA nucleoprotein filament. The homologous DNA pairing and strand exchange system described should be valuable for dissecting the action mechanism of hRad51 and for deciphering its functional interactions with other recombination factors.

Homologous recombination: from model organisms to human disease.

Recent experiments show that properly controlled recombination between homologous DNA molecules is essential for the maintenance of genome stability and for the prevention of tumorigenesis.

RecA protein promotes strand exchange with DNA substrates containing isoguanine and 5-methyl isocytosine.

The Escherichia coli RecA protein pairs homologous DNA molecules and promotes DNA strand exchange in vitro. We have examined DNA strand exchange between a 70 nucleotide ssDNA fragment and a 40 bp duplex, in which all G and C residues (at 18 positions distributed throughout the 40 bp exchanged region) were replaced with the nonstandard nucleosides 2'-deoxyisoguanosine (iG) and 2'-deoxy-5-methylisocytidine (MiC), respectively. We demonstrate that the nonstandard oligonucleotides are substrates for the RecA protein, permitting DNA strand exchange in vitro at a rate and efficiency comparable to exchange with normal DNA substrates. This observation provides an expanded experimental basis for discussions of potential roles for iG and MiC in a genetic code. Experiments of this type also provide another avenue for exploring RecA-facilitated DNA pairing mechanisms.

Rad51/RecA protein families and the associated proteins in eukaryotes

Searching for homologous DNA molecules, pairing of the molecules and exchange of the strand with one of a counterpart duplex molecule are a central process for homologous recombination. RecA protein in Esherichia coli has been known to play a pivotal role in the processes. The recA gene was identified by A.J. Clark and his colleagues more than w x 30 years ago 1 and the first biochemical activities of the protein were described in 1978 by Roberts et w x w x al. 2 and Ogawa et al. 3 independently. Since then, the RecA protein has been the main focus in w x the field studying recombination 4,5 . The RecA Ž . protein binds to single-stranded DNA ssDNA in the presence of a high-energy nucleotide cofactor such as ATP and forms a typical right-handed helical

A synthetic holliday junction is sandwiched between two tetrameric Mycobacterium leprae RuvA structures in solution: new insights from neutron scattering contrast variation and modelling

The interaction between homologous DNA molecules in recombination and DNA repair leads to the formation of crossover intermediates known as Holliday junctions. Their enzymatic processing by the RuvABC system in bacteria involves the formation of a complex between RuvA and the Holliday junction. To study the solution structure of this complex, contrast variation by neutron scattering was applied to Mycobacterium leprae RuvA (MleRuvA), a synthetic analogue of a Holliday junction with 16 base-pairs in each arm, and their stable complex. Unbound MleRuvA was octameric in solution, and formed an octameric complex with the DNA junction. The radii of gyration at infinite contrast were determined to be 3.65 nm, 2.74 nm and 4.15 nm for MleRuvA, DNA junction and their complex, respectively, showing that the complex was structurally more extended than MleRuvA. No difference was observed in the presence or absence of Mg2+. The large difference in RG values for the free and complexed protein in 65% 2H2O, where the DNA component is “invisible”, showed that a substantial structural change had occurred in complexed MleRuvA. The slopes of the Stuhrmann plots for MleRuvA and the complex were 19 and 15 or less (× 10−5), respectively, indicating that DNA passed through the centre of the complex. Automated constrained molecular modelling based on the Escherichia coli RuvA crystal structure demonstrated that the scattering curve of octameric MleRuvA in 65% and 100% 2H2O is explained by a face-to-face association of two MleRuvA tetramers stabilised by salt-bridges. The corresponding modelling of the complex in 65% 2H2O showed that the two tetramers are separated by a void space of about 1–2 nm, which can accommodate the width of B-form DNA. Minor conformational changes between unbound and complexed MleRuvA may occur. These observations show that RuvA plays a more complex role in homologous recombination than previously thought.

Overexpression of Rad51 protein stimulates homologous recombination and increases resistance of mammalian cells to ionizing radiation.

Rad51 proteins share both structural and functional homologies with the bacterial recombinase RecA. The human Rad51 (HsRad51) is able to catalyse strand exchange between homologous DNA molecules in vitro . However the biological functions of Rad51 in mammals are largely unknown. In order to address this question, we have cloned hamster Rad51 cDNA and overexpressed the corresponding protein in CHO cells. We found that 2-3-fold overexpression of the protein stimulated the homologous recombination between integrated genes by 20-fold indicating that Rad51 is a functional and key enzyme of an intrachromosomal recombination pathway. Cells overexpressing Rad51 were resistant to ionizing radiation when irradiated in late S/G2phase of the cell cycle. This suggests that Rad51 participate in the repair of double-strand breaks most likely by homologous recombination involving sister chromatids formed after the S phase.

The Role of Negative Superhelicity and Length of Homology in the Formation of Paranemic Joints Promoted by RecA Protein

Escherichia coli RecA protein pairs homologous DNA molecules to form paranemic joints when there is an absence of a free end in the region of homologous contact. Paranemic joints are a key intermediate in homologous recombination and are important in understanding the mechanism for a search of homology. The efficiency of paranemic joint formation depended on the length of homology and the topological forms of the duplex DNA. The presence of negative superhelicity increased the pairing efficiency and reduced the minimal length of homology required for paranemic joint formation. Negative superhelicity stimulated joint formation by favoring the initial unwinding of duplex DNA that occurred during the homology search and was not essential in the maintenance of the paired structure. Regardless of length of homology, formation of paranemic joints using circular duplex DNA required the presence of more than six negative supercoils. Above six negative turns, an increasing degree of negative superhelicity resulted in a linear increase in the pairing efficiency. These results support a model of two distinct kinds of DNA unwinding occurring in paranemic joint formation: an initial unwinding caused by heterologous contacts during synapsis and a later one during pairing of the homologous molecules.

Regulation of Rad51 Function by c-Abl in Response to DNA Damage

The Rad51 protein, a homolog of bacterial RecA, functions in DNA double-strand break repair and genetic recombination. Whereas Rad51 catalyzes ATP-dependent pairing and strand exchange between homologous DNA molecules, regulation of this function is unknown. The c-Abl tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. Here we demonstrate that c-Abl interacts constitutively with Rad51. We show that c-Abl phosphorylates Rad51 on Tyr-54 in vitro. The results also show that treatment of cells with ionizing radiation induces c-Abl-dependent phosphorylation of Rad51. Phosphorylation of Rad51 by c-Abl inhibits the binding of Rad51 to DNA and the function of Rad51 in ATP-dependent DNA strand exchange reactions. These findings represent the first demonstration that Rad51 is regulated by phosphorylation and support a functional role for c-Abl in regulating Rad51-dependent recombination in the response to DNA damage.

Stimulation by Rad52 of yeast Rad51-mediated recombination.

In Saccharomyces cerevisiae, the RAD51 and RAD52 genes are involved in recombination and in repair of damaged DNA. The RAD51 gene is a structural and functional homologue of the recA gene and the gene product participates in strand exchange and single-stranded-DNA-dependent ATP hydrolysis by means of nucleoprotein filament formation. The RAD52 gene is important in RAD51-mediated recombination. Binding of this protein to Rad51 suggests that they cooperate in recombination. Homologues of both Rad51 and Rad52 are conserved from yeast to humans, suggesting that the mechanisms used for pairing homologous DNA molecules during recombination may be universal in eukaryotes. Here we show that Rad52 protein stimulates Rad51 reactions and that binding to Rad51 is necessary for this stimulatory effect. We conclude that this binding is crucial in recombination and that it facilitates the formation of Rad51 nucleoprotein filaments.