Homologous Recombination Intermediates Research Articles

Genotoxic profile of inhibitors of topoisomerases I (camptothecin) and II (etoposide) in a mitotic recombination and sex-chromosome loss somatic eye assay of Drosophila melanogaster

Genotoxic carcinogens which interact with DNA may produce double-strand breaks as normal intermediates of homologous mitotic recombination, and may give rise to structural chromosome aberrations and inter-chromosomal deletion-recombination. The genotoxic profile of two inhibitors of DNA topoisomerases were evaluated using an in vivo somatic w/w + eye assay of Drosophila melanogaster for the detection of loss of heterozygosity (LOH) by homologous mitotic recombination, intra-chromosomal recombination and structural chromosomal aberrations. We studied camptothecin (CPT) as a topoisomerase-I-interactive agent and etoposide (ETOP) as a topoisomerase II inhibitor. These drugs act by stabilizing a ternary complex consisting of topoisomerases covalently linked to DNA at single-strand or at double-strand breaks, thereby preventing the relegation step of the breakage/rejoining reaction mediated by the enzyme. The genotoxic profiles were determined from the appearance of eye tissue in adult flies, in which LOH and expression of the reporter gene white produced light clones. The results demonstrated that both compounds were significantly genotoxic, with CPT being more effective than ETOP. Inter-chromosomal mitotic recombination was the major mechanism responsible for the induction of light spots by both compounds in XX females. Loss of the ring X chromosome (rX), was significantly enhanced by CPT, and this topoisomerase blocker also produced intra-chromosomal recombination (XY males).

Human DNA Polymerase η Promotes DNA Synthesis from Strand Invasion Intermediates of Homologous Recombination

Stalled replication forks pose a serious threat to genome integrity. To overcome the catastrophic consequences associated with fork demise, translesion synthesis (TLS) polymerases such as poleta promote DNA synthesis past lesions. Alternatively, a stalled fork may collapse and undergo repair by homologous recombination. By using fractionated cell extracts and purified recombinant proteins, we show that poleta extends DNA synthesis from D loop recombination intermediates in which an invading strand serves as the primer. Extracts from XP-V cells, which are defective in poleta, exhibit severely reduced D loop extension activity. The D loop extension activity of poleta is unusual, as this reaction cannot be promoted by the replicative DNA polymerase delta or by other TLS polymerases such as poliota. Moreover, we find that poleta interacts with RAD51 recombinase and RAD51 stimulates poleta-mediated D loop extension. Our results indicate a dual function for poleta at stalled replication forks: the promotion of translesion synthesis and the reinitiation of DNA synthesis by homologous recombination repair.

The HRDC domain of BLM is required for the dissolution of double Holliday junctions

Bloom's syndrome is a hereditary cancer-predisposition disorder resulting from mutations in the BLM gene. In humans, BLM encodes one of five members of the RecQ helicase family. One function of BLM is to act in concert with topoisomerase IIIalpha (TOPO IIIalpha) to resolve recombination intermediates containing double Holliday junctions by a process called double Holliday junction dissolution, herein termed dissolution. Here, we show that dissolution is highly specific for BLM among human RecQ helicases and critically depends upon a functional HRDC domain in BLM. We show that the HRDC domain confers DNA structure specificity, and is required for the efficient binding to and unwinding of double Holliday junctions, but not for the unwinding of a simple partial duplex substrate. Furthermore, we show that lysine-1270 of BLM, which resides in the HRDC domain and is predicted to play a role in mediating interactions with DNA, is required for efficient dissolution.

Holliday junction-binding peptides inhibit distinct junction-processing enzymes

Holliday junctions (HJ) are the central intermediates in both homologous recombination and site-specific recombination performed by tyrosine recombinases such as the bacteriophage lambda Integrase (Int) protein. Previously, our lab identified peptide inhibitors of Int-mediated recombination that prevent the resolution of HJ intermediates. We now show that two of these inhibitors bind HJ DNA in the square-planar conformation even in the absence of Int protein. The peptides prevent unwinding of branched DNA substrates by the RecG helicase of Escherichia coli and interfere with the resolution of HJ substrates by the RuvABC complex. Our results suggest that these peptides target all proteins that process HJ in the square-planar conformation. These inhibitors should be extremely useful for dissecting homologous recombination and recombination-dependent repair in vitro and in vivo.

Characterization of the interaction of wheat HMGa with linear and four-way junction DNAs.

Wheat HMGa protein is a typical member of the plant HMGA family. It has four AT hooks and a histone H1-like region. A panel of deletion mutants of HMGa was generated to study the role of different regions of HMGa in its binding to 4H (a synthetic DNA that mimics the in vivo structure of intermediates of homologous recombination and DNA repair) and linear DNAs. Although the histone H1-like region of HMGa does not bind to 4H or linear DNAs, it does enhance the binding. Mutants with any two adjacent AT hooks show specific binding to both 4H and linear P268 (and P31) with different binding affinities, which is partly due to the flanking regions between AT hooks. Conformational studies indicate that the alpha-helical content of HMGa increases significantly when it binds to 4H compared to that after binding to P31, linear DNA. In contrast, linear DNA, but not 4H, undergoes substantial conformational change when it binds to HMGa, indicating that linear DNA is relatively more flexible than 4H. A more significant difference in the affinities of binding of the mutants of HMGa to 4H was observed compared to their affinities of binding to linear DNA, P31. These differences could be due to the rigidity of the DNA and the characters of the AT hook regions in the mutants.

Structure of DNA-RecA protein complex, intermediate of homologous recombination, determined by polarised-light spectroscopy.

The structure of long filament of DNA-RecA protein complex, active in homologous recombination, has been assessed using linear dichroism (LD) spectroscopy. The angular orientation of the DNA nucleobases relative to the fibre axis was estimated to be 70 degrees from the unique LD band of etheno-modified poly(dA) at 320 nm. Angular orientation data for 2 tryptophan and 7 tyrosine residues of RecA were deduced from differential LD of wild type RecA versus modified proteins which were engineered to attenuate the UV absorption of selected residues. The results revealed a rotation by some 40 degrees of the RecA subunits relative to the arrangement in crystal without DNA. Conformational changes are also indicated for tyrosine residues assigned to be involved in DNA binding or in RecA-RecA contacts.

XRCC3 Controls the Fidelity of Homologous Recombination: Roles for XRCC3 in Late Stages of Recombination

XRCC3 is a RAD51 paralog that functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). XRCC3 mutation causes severe chromosome instability. We find that XRCC3 mutant cells display radically altered HR product spectra, with increased gene conversion tract lengths, increased frequencies of discontinuous tracts, and frequent local rearrangements associated with HR. These results indicate that XRCC3 function is not limited to HR initiation, but extends to later stages in formation and resolution of HR intermediates, possibly by stabilizing heteroduplex DNA. The results further demonstrate that HR defects can promote genomic instability not only through failure to initiate HR (leading to nonhomologous repair) but also through aberrant processing of HR intermediates. Both mechanisms may contribute to carcinogenesis in HR-deficient cells.

RadA protein from Archaeoglobus fulgidus forms rings, nucleoprotein filaments and catalyses homologous recombination.

Proteins that catalyse homologous recombination have been identified in all living organisms and are essential for the repair of damaged DNA as well as for the generation of genetic diversity. In bacteria homologous recombination is performed by the RecA protein, whereas in the eukarya a related protein called Rad51 is required to catalyse recombination and repair. More recently, archaeal homologues of RecA/Rad51 (RadA) have been identified and isolated. In this work we have cloned and purified the RadA protein from the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus and characterised its in vitro activities. We show that (i) RadA protein forms ring structures in solution and binds single- but not double-stranded DNA to form nucleoprotein filaments, (ii) RadA is a single-stranded DNA-dependent ATPase at elevated temperatures, and (iii) RadA catalyses efficient D-loop formation and strand exchange at temperatures of 60-70 degrees C. Finally, we have used electron microscopy to visualise RadA-mediated joint molecules, the intermediates of homologous recombination. Intriguingly, RadA shares properties of both the bacterial RecA and eukaryotic Rad51 recombinases.

Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: a possible advantage of DNA over RNA as genomic material.

Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes. A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins. In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology. Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2' methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and single-stranded DNA during the formation of heteroduplex joints. The pivotal role of the 2' methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes. Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling). These properties may give an extensive evolutionary advantage to DNA.

Biochemical characterization of the hjc holliday junction resolvase of Pyrococcus furiosus.

The Hjc protein of Pyrococcus furiosus is an endonuclease that resolves Holliday junctions, the intermediates in homologous recombination. The amino acid sequence of Hjc is conserved in Archaea, however, it is not similar to any of the well-characterized Holliday junction resolvases. In order to investigate the similarity and diversity of the enzymatic properties of Hjc as a Holliday junction resolvase, highly purified Hjc produced in recombinant Escherichia coli was used for detailed biochemical characterizations. Hjc has specific binding activity to the Holliday-structured DNA, with an apparent dissociation constant (K:(d)) of 60 nM. The dimeric form of Hjc binds to the substrate DNA. The optimal reaction conditions were determined using a synthetic Holliday junction as substrate. Hjc required a divalent cation for cleavage activity and Mg(2+) at 5-10 mM was optimal. Mn(2+) could substitute for Mg(2+), but it was much less efficient than Mg(2+) as the cofactor. The cleavage reaction was stimulated by alkaline pH and KCl at approximately 200 mM. In addition to the high specific activity, Hjc was found to be extremely heat stable. In contrast to the case of SULFOLOBUS:, the Holliday junction resolving activity detected in P. furiosus cell extract thus far is only derived from Hjc.

Analysis of conserved basic residues associated with DNA binding (Arg69) and catalysis (Lys76) by the RusA holliday junction resolvase

Holliday junctions are key intermediates in both homologous recombination and DNA repair, and are also formed from replication forks stalled at lesions in the template strands. Their resolution is critical for chromosome segregation and cell viability, and is mediated by a class of small, homodimeric endonucleases that bind the structure and cleave the DNA. All the enzymes studied require divalent metal ions for strand cleavage and their active centres are characterised by conserved aspartate/glutamate residues that provide ligands for metal binding. Sequence alignments reveal that they also contain a number of conserved basic residues. We used site-directed mutagenesis to investigate such residues in the RusA resolvase. RusA is a 120 amino acid residue polypeptide that can be activated in Escherichia coli to promote recombination and repair in the absence of the Ruv proteins. The RuvA, RuvB and RuvC proteins form a complex on Holliday junction DNA that drives coupled branch migration (RuvAB) and resolution (RuvC) reactions. In contrast to RuvC, the RusA resolvase does not interact directly with a branch migration motor, which simplifies analysis of its resolution activity. Catalysis depends on three highly conserved acidic residues (Asp70, Asp72 and Asp91) that define the catalytic centre. We show that Lys76, which is invariant in RusA sequences, is essential for catalysis, but not for DNA binding, and that an invariant asparagine residue (Asn73) is required for optimal activity. Analysis of DNA binding revealed that RusA may interact with one face of an open junction before manipulating its conformation in the presence of Mg 2+ as part of the catalytic process. A well-conserved arginine residue (Arg69) is linked with this critical stage. These findings provide the first insights into the roles played by basic residues in DNA binding and catalysis by a Holliday junction resolvase.

Modulation of RuvB function by the mobile domain III of the holliday junction recognition protein RuvA

In prokaryotes, RuvA-RuvB complexes play a crucial role in the migration of the Holliday junction, which is a key intermediate of homologous recombination. RuvA binds to the Holliday junction and enhances the ATPase activity of RuvB required for branch migration. RuvA adopts a unique domain structure, which assembles into a tetrameric molecule. The previous mutational and proteolytic analyses suggested that mutations in a carboxyl-terminal domain (domain III) impair binding of RuvA to RuvB. In order to clarify the functional role of each domain in vitro, we established the recombinant expression systems, which allow us to analyze structural and biochemical properties of each domain separately. A small-angle X-ray scattering solution study, combined with X-ray crystallographic analyses, was applied to the tetrameric full-length RuvA and its tetrameric NH2 region (domains I and II) lacking the domain III. These results demonstrated that domain III can be completely separate from the tetrameric major core of the NH2 region and freely mobile in solution, through a remarkably flexible loop. Biochemical analyses indicated that domain III not only interacts with RuvB, but also modulates its ATPase activity. This modulation may facilitate the dynamic coupling between RuvA and RuvB during branch migration.

A Holliday junction resolvase from Pyrococcus furiosus: functional similarity to Escherichia coli RuvC provides evidence for conserved mechanism of homologous recombination in Bacteria, Eukarya, and Archaea.

The Holliday junction is an essential intermediate of homologous recombination. RecA of Bacteria, Rad51 of Eukarya, and RadA of Archaea are structural and functional homologs. These proteins play a pivotal role in the formation of Holliday junctions from two homologous DNA duplexes. RuvC is a specific endonuclease that resolves Holliday junctions in Bacteria. A Holliday junction-resolving activity has been found in both yeast and mammalian cells. To examine whether the paradigm of homologous recombination apply to Archaea, we assayed and found the activity to resolve a synthetic Holliday junction in crude extract of Pyrococcus furiosus cells. The gene, hjc (Holliday junction cleavage), encodes a protein composed of 123 amino acids, whose sequence is not similar to that of any proteins with known function. However, all four archaea, whose total genome sequences have been published, have the homologous genes. The purified Hjc protein cleaved the recombination intermediates formed by RecA in vitro. These results support the notion that the formation and resolution of Holliday junction is the common mechanism of homologous recombination in the three domains of life.

Novel properties of the Thermus thermophilus RuvB protein, which promotes branch migration of Holliday junctions.

Branch migration of Holliday junctions, which are central DNA intermediates in homologous recombination, is promoted by the RuvA-RuvB protein complex, and the junctions are resolved by the action of the RuvC protein in Escherichia coli. We report here the cloning of the ruvB gene from a thermophilic eubacterium, Thermus thermophilus HB8 (Tth), and the biochemical characterization of the gene product expressed in E. coli. The Tth ruvB gene could not complement the UV sensitivity of an E. coli ruvB deletion mutant and made the wild-type strain more sensitive to UV. In contrast to E. coli RuvB, whose ATPase activity is strongly enhanced by supercoiled DNA but only weakly enhanced by linear duplex DNA, the ATPase activity of Tth RuvB was efficiently and equally enhanced by supercoiled and linear duplex DNA. Tth RuvB hydrolyzed a broader range of nucleoside triphosphates than E. coli RuvB. In addition, Tth RuvB, in the absence of RuvA protein, promoted branch migration of a synthetic Holliday junction at 60 degrees C in an ATP-dependent manner. The protein, as judged by its ATPase activity, required ATP for thermostability. Since a RuvA protein has not yet been identified in T. thermophilus, we used E. coli RuvA to examine the effects of RuvA on the activities of Tth RuvB. E. coli RuvA greatly enhanced the ability of Tth RuvB to hydrolyze ATP in the presence of DNA and to promote branch migration of a synthetic Holliday junction at 37 degrees C. These results indicate the conservation of the RuvA-RuvB interaction in different bacterial species, and suggest the existence of a ruvA homolog in T. thermophilus. Although GTP and dGTP were efficiently hydrolyzed by Tth RuvB, these nucleoside triphosphates could not be utilized for branch migration in vitro, implying that the conformational change in RuvB brought about by ATP hydrolysis, which is necessary for driving the Holliday junction branch migration, cannot be accomplished by the hydrolysis of these nucleoside triphosphates.

Recognition of DNA alterations by the mismatch repair system

Misincorporation of non-complementary bases by DNA polymerases is a major source of the occurrence of promutagenic base-pairing errors during DNA replication or repair. Base–base mismatches or loops of extra bases can arise which, if left unrepaired, will generate point or frameshift mutations respectively. To counteract this mutagenic potential, organisms have developed a number of elaborate surveillance and repair strategies which co-operate to maintain the integrity of their genomes. An important replication-associated correction function is provided by the post-replicative mismatch repair system. This system is highly conserved among species and appears to be the major pathway for strand-specific elimination of base–base mispairs and short insertion/deletion loops (IDLs), not only during DNA replication, but also in intermediates of homologous recombination. The efficiency of repair of different base-pairing errors in the DNA varies, and appears to depend on multiple factors, such as the physical structure of the mismatch and sequence context effects. These structural aspects of mismatch repair are poorly understood. In contrast, remarkable progress in understanding the biochemical role of error-recognition proteins has been made in the recent past. In eukaryotes, two heterodimers consisting of MutS-homologous proteins have been shown to share the function of mismatch recognition in vivo and in vitro. A first MutS homologue, MSH2, is present in both heterodimers, and the specificity for mismatch recognition is dictated by its association with either of two other MutS homologues: MSH6 for recognition of base–base mismatches and small IDLs, or MSH3 for recognition of IDLs only. Mismatch repair deficiency in cells can arise through mutation, transcriptional silencing or as a result of imbalanced expression of these genes.

Recognition of DNA alterations by the mismatch repair system

Misincorporation of non-complementary bases by DNA polymerases is a major source of the occurrence of promutagenic base-pairing errors during DNA replication or repair. Base-base mismatches or loops of extra bases can arise which, if left unrepaired, will generate point or frameshift mutations respectively. To counteract this mutagenic potential, organisms have developed a number of elaborate surveillance and repair strategies which co-operate to maintain the integrity of their genomes. An important replication-associated correction function is provided by the post-replicative mismatch repair system. This system is highly conserved among species and appears to be the major pathway for strand-specific elimination of base-base mispairs and short insertion/deletion loops (IDLs), not only during DNA replication, but also in intermediates of homologous recombination. The efficiency of repair of different base-pairing errors in the DNA varies, and appears to depend on multiple factors, such as the physical structure of the mismatch and sequence context effects. These structural aspects of mismatch repair are poorly understood. In contrast, remarkable progress in understanding the biochemical role of error-recognition proteins has been made in the recent past. In eukaryotes, two heterodimers consisting of MutS-homologous proteins have been shown to share the function of mismatch recognition in vivo and in vitro. A first MutS homologue, MSH2, is present in both heterodimers, and the specificity for mismatch recognition is dictated by its association with either of two other MutS homologues: MSH6 for recognition of base-base mismatches and small IDLs, or MSH3 for recognition of IDLs only. Mismatch repair deficiency in cells can arise through mutation, transcriptional silencing or as a result of imbalanced expression of these genes.

Crystal Structure of an Octameric RuvA–Holliday Junction Complex

Holliday junctions occur as intermediates in homologous recombination and DNA repair. In bacteria, resolution of Holliday junctions is accomplished by the RuvABC system, consisting of a junction-specific helicase complex RuvAB, which promotes branch migration, and a junction-specific endonuclease RuvC, which nicks two strands. The crystal structure of a complex between the RuvA protein of M. leprae and a synthetic four-way junction has now been determined. Rather than binding on the open surface of a RuvA tetramer as previously suggested, the DNA is sandwiched between two RuvA tetramers, which form a closed octameric shell, stabilized by a conserved tetramer–tetramer interface. Interactions between the DNA backbone and helix-hairpin-helix motifs from both tetramers suggest a mechanism for strand separation promoted by RuvA.

Structural Biology Paper elert

The Holliday junction is a well-known intermediate of homologous recombination. Recently, the proteins involved in the correct processing of the Holliday structure into mature recombinant molecules, namely RuvA, RuvB, RuvC and RecG have been isolated and characterized. This has culminated in a model for their synergistic mechanism of action and the solving of the RuvC crystal structure.

Structure and dynamics of supercoil-stabilized DNA cruciforms

Understanding DNA function requires knowledge of the structure of local, sequence-dependent conformations that can be dramatically different from the B-form helix. One alternative DNA conformation is the cruciform, which has been shown to have a critical role in the initiation of DNA replication and the regulation of transcription in certain systems. In addition, cruciforms provide a model system for structural studies of Holliday junctions, intermediates in homologous DNA recombination. Cruciforms are not thermodynamically stable in linear DNA due to branch point migration, which makes their study using many biophysical techniques problematic. Atomic Force Microscopy (AFM) was applied to visualize cruciforms in negatively supercoiled plasmid DNA. Cruciforms are seen as clear-cut extrusions on the DNA filament with the lengths of the arms consistent with the size of the hairpins expected from a 106 bp inverted repeat. The cruciform exists in two different conformations, an extended one with the angle of ca. 180° between the hairpin arms and a compact, X-type conformation, with acute angles between the hairpin arms and the main DNA strands. The ratio of molecules with the different conformations of cruciforms depends on ionic conditions. In the presence of high salt or Mg cations, a compact, X-type conformation is highly preferable. Remarkably, the X-conformation was highly mobile allowing the cruciform arms to adopt a parallel orientation. The structure observed is consistent with a model of the Holliday junction with a parallel orientation of the exchanging strands.

Mismatch repair by efficient nick-directed, and less efficient mismatch-specific, mechanisms in homologous recombination intermediates in Chinese hamster ovary cells.

Repair of single-base mismatches formed in recombination intermediates in vivo was investigated in Chinese hamster ovary cells. Extrachromosomal recombination was stimulated by double-strand breaks (DSBs) introduced into regions of shared homology in pairs of plasmid substrates heteroallelic at 11 phenotypically silent mutations. Recombination was expected to occur primarily by single-strand annealing, yielding predicted heteroduplex DNA (hDNA) regions with three to nine mismatches. Product spectra were consistent with hDNA only occurring between DSBs. Nicks were predicted on opposite strands flanking hDNA at positions corresponding to original DSB sites. Most products had continuous marker patterns, and observed conversion gradients closely matched predicted gradients for repair initiated at nicks, consistent with an efficient nick-directed, excision-based mismatch repair system. Discontinuous patterns, seen in approximately 10% of products, and deviations from predicted gradients provided evidence for less efficient mismatch-specific repair, including G-A-->G-C specific repair that may reflect processing by a homologue of Escherichia coli MutY. Mismatch repair was > 80% efficient, which is higher than seen previously with covalently closed, artificial hDNA substrates. Products were found in which all mismatches were repaired in a single tract initiated from one or the other nick. We also observed products resulting from two tracts of intermediate length initiated from two nicks.