Architecture of recombination intermediates visualized by in-gel FRET of lambda integrase-Holliday junction-arm DNA complexes

Lambda integrase (Int) mediates recombination between attachment sites on phage and Escherichia coli DNA. Int is assisted by accessory protein-induced DNA loops in bridging pairs of distinct "arm-type" and "core-type" DNA sites to form synapsed recombination complexes that subsequently recombine by means of a Holliday junction (HJ) intermediate. An in-gel FRET assay was developed and used to measure 15 distances between six points in two Int-HJ complexes containing arm-DNA oligonucleotides, and 3D maps of these complexes were derived by distance-geometry calculations. The maps reveal unexpected positions for the arm-type DNAs relative to core sites on the HJ and a new Int conformation in the HJ tetramer. The results show how the position of arm DNAs determines the bias of catalytic activities responsible for directional resolution, provide insights into the organization of Int higher-order complexes, and lead to models of the structure of the full HJ recombination intermediates.     

Holliday junction-containing DNA structures persist in cells lacking Sgs1 or Top3 following exposure to DNA damage

The Sgs1-Rmi1-Top3 "dissolvasome" is required for the maintenance of genome stability and has been implicated in the processing of various types of DNA structures arising during DNA replication. Previous investigations have revealed that unprocessed (X-shaped) homologous recombination repair (HRR) intermediates persist when S-phase is perturbed by using methyl methanesulfonate (MMS) in Saccharomyces cerevisiae cells with impaired Sgs1 or Top3. However, the precise nature of these persistent DNA structures remains poorly characterized. Here, we report that ectopic expression of either of two heterologous and structurally unrelated Holliday junction (HJ) resolvases, Escherichia coli RusA or human GEN1(1-527), promotes the removal of these X-structures in vivo. Moreover, other types of DNA replication intermediates, including stalled replication forks and non-HRR-dependent X-structures, are refractory to RusA or GEN1(1-527), demonstrating specificity of these HJ resolvases for MMS-induced X-structures in vivo. These data suggest that the X-structures persisting in cells with impaired Sgs1 or Top3 contain HJs. Furthermore, we demonstrate that Sgs1 directly promotes X-structure removal, because the persistent structures arising in Sgs1-deficient strains are eliminated when Sgs1 is reactivated in vivo. We propose that HJ resolvases and Sgs1-Top3-Rmi1 comprise two independent processes to deal with HJ-containing DNA intermediates arising during HRR in S-phase.     

Holliday junction dynamics and branch migration: single-molecule analysis

The Holliday junction (HJ) is a central intermediate in various genetic processes including homologous and site-specific recombination and DNA replication. Branch migration allows the exchange between homologous DNA regions, but the detailed mechanism for this key step of DNA recombination is unidentified. Here, we report direct real-time detection of branch migration in individual molecules. Using appropriately designed HJ constructs we were able to follow junction branch migration at the single-molecule level. Branch migration is detected as a stepwise random process with the overall kinetics dependent on Mg2+ concentration. We developed a theoretical approach to analyze the mechanism of HJ branch migration. The data show steps in which the junction flips between conformations favorable to branch migration and conformations unfavorable to it. In the favorable conformation (the extended HJ geometry), the branch can migrate over several base pairs detected, usually as a single large step. Mg2+ cations stabilize folded conformations and stall branch migration for a period considerably longer than the hopping step. The conformational flip and the variable base pair hopping step provide insights into the regulatory mechanism of genetic processes involving HJs.     

Nucleoprotein architectures regulating the directionality of viral integration and excision

The virally encoded site-specific recombinase Int collaborates with its accessory DNA bending proteins IHF, Xis, and Fis to assemble two distinct, very large, nucleoprotein complexes that carry out either integrative or excisive recombination along regulated and essentially unidirectional pathways. The core of each complex consists of a tetramer of Integrase protein (Int), which is a heterobivalent DNA binding protein that binds and bridges a core-type DNA site (where strand cleavage and ligation are executed), and a distal arm-type site, that is brought within range by one or more DNA bending proteins. The recent determination of the patterns of these Int bridges has made it possible to think realistically about the global architecture of the recombinogenic complexes. Here, we combined the previously determined Int bridging patterns with in-gel FRET experiments and in silico modeling to characterize and differentiate the two 400-kDa multiprotein Holiday junction recombination intermediates formed during λ integration and excision. The results lead to architectural models that explain how integration and excision are regulated in λ site-specific recombination. Our confidence in the basic features of these architectures is based on the redundancy and self-consistency of the underlying data from two very different experimental approaches to establish bridging interactions, a set of strategic intracomplex distances from FRET experiments, and the model’s ability to explain key aspects of the integrative and excisive recombination pathways, such as topological changes, the mechanism of capturing attB, and the features of asymmetry and flexibility within the complexes.High-precision DNA transactions responsible for a variety of fundamental processes are typically promoted and modulated by large multiprotein machines that use cooperative interactions and involve DNA bending and/or wrapping. One well-studied example is the tightly regulated and highly directional site-specific recombination by which bacteriophage λ inserts and excises its DNA into and out of the Escherichia coli host chromosome, using the phage attP and the bacterial attB DNA sequences for integration and the resulting junction sequences, attL and attR, for excision. These reactions are catalyzed by the phage-encoded Integrase protein (Int), the founding member of the tyrosine recombinase family of site-specific recombinases (1). In addition to mediating integration and excision of viral genomes, members of this family function in a variety of other cellular processes including chromosome segregation, gene regulation, and conjugative transposition (2).Int has three well-characterized domains: an N-terminal DNA-binding domain (NTD), a central core-binding domain (CB), and a C-terminal catalytic domain (CAT). The CB and CAT domains (referred to here as the CTD) are together responsible for binding to the core-type DNA sequences where strand exchange and ligation take place. The NTD binds with high affinity to arm-type sites that are located 40–120 bp from the core sites. As summarized in Fig. 1, Int is a heterobivalent DNA-binding protein that depends on three accessory DNA bending proteins (IHF, integration host factor; Xis, excision factor; and Fis, factor for inversion stimulation) to bridge between distinct DNA-binding elements and thereby execute the regulated directionality of integrative and excisive recombination. Some tyrosine recombinases, such as Cre and Flp, have only the CB and CAT domains. These enzymes carry out unregulated recombination between 34-bp core sites using a strand exchange mechanism that is shared by Int and other family members. The key intermediate in this pathway is a four-way Holliday junction (HJ) that is formed following cleavage and exchange of one pair of strands between the two recombining sites. Resolution of the HJ intermediate by cleavage and exchange of the second pair of strands completes the recombination reaction (3, 4).Open in a separate windowFig. 1.Holliday junction intermediates, Int bridges, and dye positions. The attL and attR substrates used to trap HJ intermediates contain changes in the 7-bp overlap sequence (in lowercase). The unpaired bases at positions −1 and −2 become paired on HJ formation. At positions +2 and +4, the sequence differences between the two partners disfavors resolution of the junction. The resulting trapped excisive HJ with a fully base paired overlap region is shown with labeled color-coded boxes representing the DNA binding sequences for the NTD of Int arm-type sites (green); the CTDs of Int core-type sites (purple); IHF (gray); Xis (tan); and Fis (pink). The unfilled boxes are used in integrative but not excisive recombination. The excisive HJ shows the relative positions of the fluorescent dyes (Fig. 2 and Fig. S1) and the three pairs of Int-mediated arm–core bridges (ref. 16). The integrative HJ shows the four pairs of Int-mediated arm–core bridges in integrative recombination (ref. 16).The structure of Int and the details of its interactions with DNA have been determined from a variety of NMR and crystal structures (5–8). In addition, NMR and/or crystal structures of IHF (9), Xis (10–13), and Fis (11, 14, 15) have provided structural models for the accessory proteins and their corresponding DNA complexes. When combined with the large body of genetic and biochemical analyses that have been performed in these systems, the structural data have revealed how DNA bending by the accessory proteins could facilitate Int bridging between the core- and arm-type DNA binding sites. However, despite these efforts, there are still no structural models available for the recombinogenic complexes formed in either pathway that are able to explain the vast body of data that exists for the λ Integrase system. The primary limitation has been a lack of understanding of which core-type sites are bridged to which arm-type sites by Int. In the companion paper (16), we identified these Int bridges for both the integrative and excisive recombination pathways (Fig. 1). Here, we combined the Int bridging data with in-gel FRET experiments and in silico modeling to characterize and differentiate the two 400-kDa multiprotein HJ recombination intermediates formed during λ integration and excision. The results lead to architectural models that explain how integration and excision are regulated in λ site-specific recombination.     

The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA

The Holliday junction (HJ), a cross-shaped structure that physically links the two DNA helices, is a key intermediate in homologous recombination, DNA repair, and replication. Several helicase-like proteins are known to bind HJs and promote their branch migration (BM) by translocating along DNA at the expense of ATP hydrolysis. Surprisingly, the bacterial recombinase protein RecA and its eukaryotic homologue Rad51 also promote BM of HJs despite the fact they do not bind HJs preferentially and do not translocate along DNA. RecA/Rad51 plays a key role in DNA double-stranded break repair and homologous recombination. RecA/Rad51 binds to ssDNA and forms contiguous filaments that promote the search for homologous DNA sequences and DNA strand exchange. The mechanism of BM promoted by RecA/RAD51 is unknown. Here, we demonstrate that cycles of RecA/Rad51 polymerization and dissociation coupled with ATP hydrolysis drives the BM of HJs.     

The Holliday junction in an inverted repeat DNA sequence: sequence effects on the structure of four-way junctions

Holliday junctions are important structural intermediates in recombination, viral integration, and DNA repair. We present here the single-crystal structure of the inverted repeat sequence d(CCGGTACCGG) as a Holliday junction at the nominal resolution of 2. 1 A. Unlike the previous crystal structures, this DNA junction has B-DNA arms with all standard Watson-Crick base pairs; it therefore represents the intermediate proposed by Holliday as being involved in homologous recombination. The junction is in the stacked-X conformation, with two interconnected duplexes formed by coaxially stacked arms, and is crossed at an angle of 41.4 degrees as a right-handed X. A sequence comparison with previous B-DNA and junction crystal structures shows that an ACC trinucleotide forms the core of a stable junction in this system. The 3'-C x G base pair of this ACC core forms direct and water-mediated hydrogen bonds to the phosphates at the crossover strands. Interactions within this core define the conformation of the Holliday junction, including the angle relating the stacked duplexes and how the base pairs are stacked in the stable form of the junction.     

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.     

From the Cover: Capturing reaction paths and intermediates in Cre-loxP recombination using single-molecule fluorescence

Site-specific recombination plays key roles in microbe biology and is exploited extensively to manipulate the genomes of higher organisms. Cre is a well studied site-specific recombinase, responsible for establishment and maintenance of the P1 bacteriophage genome in bacteria. During recombination, Cre forms a synaptic complex between two 34-bp DNA sequences called loxP after which a pair of strand exchanges forms a Holliday junction (HJ) intermediate; HJ isomerization then allows a second pair of strand exchanges and thus formation of the final recombinant product. Despite extensive work on the Cre-loxP system, many of its mechanisms have remained unclear, mainly due to the transient nature of complexes formed and the ensemble averaging inherent to most biochemical work. Here, we address these limitations by introducing tethered fluorophore motion (TFM), a method that monitors large-scale DNA motions through reports of the diffusional freedom of a single fluorophore. We combine TFM with Förster resonance energy transfer (FRET) and simultaneously observe both large- and small-scale conformational changes within single DNA molecules. Using TFM–FRET, we observed individual recombination reactions in real time and analyzed their kinetics. Recombination was initiated predominantly by exchange of the “bottom-strands” of the DNA substrate. In productive complexes we used FRET distributions to infer rapid isomerization of the HJ intermediates and that a rate-limiting step occurs after this isomerization. We also observed two nonproductive synaptic complexes, one of which was structurally distinct from conformations in crystals. After recombination, the product synaptic complex was extremely stable and refractory to subsequent rounds of recombination.     

The catalytic domain of λ site-specific recombinase

The Escherichia coli phage λ integrase protein (Int) belongs to the large Int family of site-specific recombinases. It is a heterobivalent DNA binding protein that makes use of a high energy covalent phosphotyrosine intermediate to catalyze integrative and excisive recombination at specific chromosomal sites (att sites). A 293-amino acid carboxy-terminal fragment of Int (C65) has been cloned, characterized, and used to further dissect the protein. From this we have cloned and characterized a 188-amino acid, protease-resistant, carboxy-terminal fragment (C170) that we believe is the minimal catalytically competent domain of Int. C170 has topoisomerase activity and converts att suicide substrates to the covalent phosphotyrosine complexes characteristic of recombination intermediates. However, it does not show efficient binding to att site DNA in a native gel shift assay. We propose that λ Int consists of three functional and structural domains: residues 1–64 specify recognition of “arm-type” DNA sequences distant from the region of strand exchange; residues 65–169 contribute to specific recognition of “core-type” sequences at the sites of strand exchange and possibly to protein–protein interactions; and residues 170–356 carry out the chemistry of DNA cleavage and ligation. The finding that the active site nucleophile Tyr-342 is in a uniquely protease-sensitive region complements and reinforces the recently solved C170 crystal structure, which places Tyr-342 at the center of a 17-amino acid flexible loop. It is proposed that C170 is likely to represent a generic Int family domain that thus affords a specific route to studying the chemistry of DNA cleavage and ligation in these recombinases.     

Mapping the λ Integrase bridges in the nucleoprotein Holliday junction intermediates of viral integrative and excisive recombination

The site-specific recombinase encoded by bacteriophage λ [λ Integrase (Int)] is responsible for integrating and excising the viral chromosome into and out of the chromosome of its Escherichia coli host. In contrast to the other well-studied and highly exploited tyrosine recombinase family members, such as Cre and Flp, Int carries out a reaction that is highly directional, tightly regulated, and depends on an ensemble of accessory DNA bending proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental signals. Int recombinase is a heterobivalent DNA binding protein that binds via its small amino-terminal domain to high affinity arm-type DNA sites and via its large, compound carboxyl-terminal domain to core-type DNA sites, where DNA cleavage and ligation are executed. Each of the four Int protomers, within a multiprotein 400-kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm- and one core-type DNA site. Despite a wealth of genetic, biochemical, and functional information generated by many laboratories over the last 50 y, it has not been possible to decipher the patterns of Int bridges, an essential step in understanding the architectures responsible for regulated directionality of recombination. We used site-directed chemical cross-linking of Int in trapped Holliday junction recombination intermediates and recombination reactions with chimeric recombinases, to identify the unique and monogamous patterns of Int bridges for integrative and excisive recombination.The tyrosine recombinase family, which includes the well-studied and highly exploited Cre, Flp, and λ Integrase (Int) recombinases, is responsible for such diverse functions as chromosome segregation, chromosome copy number control, gene expression, conjugative transposition, gene dissemination, and viral integration and excision [for reviews, see Mobile DNA II (1) and the in preparation Mobile DNA III]. The virally encoded λ Int recombinase is responsible for integrating and excising the λ chromosome into and out of the chromosome of its Escherichia coli host in response to a variety of physiological and environmental signals (2). Although all members of this family use the same isoenergetic chemistry and strand exchange mechanisms to execute DNA rearrangements, Int (in contrast to Cre and Flp) depends on an ensemble of accessory DNA bending proteins and carries out a recombination, between att site target DNAs, that is highly directional and tightly regulated (3–8).Int is a heterobivalent DNA binding protein that binds to high-affinity “arm-type” DNA sites via its small amino-terminal domain (NTD), and to “core-type” DNA sites, where DNA cleavage and ligation takes place, via a central core binding domain (CB) and a C-terminal catalytic domain (CAT); the latter two domains are referred to here as the CTD. Each of the four Int protomers, within a multiprotein 400-kDa recombinogenic complex, is thought to bind and bridge one arm- and one core-type DNA site; the bridging interactions are facilitated by accessory DNA bending proteins IHF, Xis, and Fis. Differential occupancy of the 16 DNA protein binding sites (encoded by 240 bp of att site DNA) generates two overlapping ensembles that differentiate integrative from excisive recombination, as diagrammed in Fig. 1.Open in a separate windowFig. 1.The overlapping ensembles of protein binding sites that comprise att site DNA and the DNA modifications used for cross-linking. Integrative recombination between supercoiled attP and linear attB requires the virally encoded Integrase (Int) (29) and the host-encoded accessory DNA bending protein IHF (30, 31) and gives rise to an integrated phage chromosome bounded by attL and attR. Excisive recombination between attL and attR to regenerate attP and attB additionally requires the phage-encoded Xis protein (which inhibits integrative recombination) (32) and is stimulated by the host-encoded Fis protein (33). Both reactions proceed through a Holliday junction intermediate that is first generated and then resolved by single-strand exchanges on the left and right side of the 7-bp overlap region, respectively. The two reactions proceed with the same order of sequential strand exchanges (not the reverse order) and use different subsets of protein binding sites in the P and P′ arms, as indicated by the filled boxes: Int arm-type P1, P2, P′1, P′2, and P′3 (green); IHF, H1, H2, and H′ (gray); Xis, X1, X1.5, and X2 (gold); and Fis (pink). The four core-type Int binding sites, C, C′, B, and B′ (blue boxes) are each bound in a C-clamp fashion by the CB and CAT domains, referred to here as the CTD. These core sites are where Int executes isoenergetic DNA strand cleavages and ligations via a high energy covalent 3′-phospho-tyrosine intermediate. The CTD of Int and the tetrameric Int complex surrounding the two overlap regions are functionally and structurally similar to the Cre, Flp, and XerC/D proteins. The bottom line shows the locations of the cystamine modifications (S) within the core- and arm-type consensus sequences (see main text and Tables S1 and S2). (Right) DNA sequence changes made in the 7-bp overlap regions to trap Holliday junction intermediates (lowercase letters). Following the first pair of Int cleavages (via the active site Tyr) on one side of the overlap regions (arranged here in antiparallel orientation) the “top” strands are swapped to form the HJ; this simultaneously converts the unpaired (bubble) bases to duplex DNA. On the other side, the sequence differences between the two overlap regions strongly disfavor the second (“bottom”) strand swap that would resolve the HJ, because this would generate unpaired bubbles in the product (11, 12). This diagram applies to both integrative and excisive recombination (even though the labels refer to integrative recombination).Despite a wealth of genetic, biochemical, functional, and structural information, generated by many laboratories over the last 50 y, it has not been possible to determine which of the five arm-type DNA sites is paired via an Int bridge with which of the four core-type sites. This deficiency has been the major obstacle in understanding the architectures responsible for regulated directionality of integrative and excisive recombination. In this report, we address the need for a direct determination of the λ Int bridging patterns using site-directed chemical cross-linking of Int to trapped Holliday junction recombination intermediates, and we confirm the observed Int bridges in recombination reactions with chimeric recombinases.     

Two structural features of lambda integrase that are critical for DNA cleavage by multimers but not by monomers

Despite many years of genetic and biochemical studies on the lambda integrase (Int) recombination system, it is still not known whether the Int protein is competent for DNA cleavage as a monomer. We have addressed this question, as part of a larger study of Int functions critical for the formation of higher-order complexes, by isolating "multimer-specific" mutants. We identify a pair of oppositely charged residues, E153 and R169, that comprise an intermolecular salt bridge within a functional Int multimer. Mutation of either of these residues significantly reduces both the cleavage of full-att sites and the resolution of Holliday junctions without compromising the cleavage of half-att site substrates. Allele-specific suppressor mutations were generated at these residues. Their interaction with wild-type Int on preformed Holliday junctions indicates that the mutated residues comprise an intermolecular salt bridge. We have also shown that the most C-terminal seven residues of Int, which comprise another previously identified subunit interface, inhibit DNA cleavage by monomeric but not multimeric Int. Taken together, our results lead us to conclude that Int can cleave DNA as a monomer. We also identify and discuss unique structural features of Int that act negatively to reduce its activity as a monomer and other features that act positively to enhance its activity as a multimer.     

Bacterial-type DNA holliday junction resolvases in eukaryotic viruses

Homologous DNA recombination promotes genetic diversity and the maintenance of genome integrity, yet no enzymes with specificity for the Holliday junction (HJ)-a key DNA recombination intermediate-have been purified and characterized from metazoa or their viruses. Here we identify critical structural elements of RuvC, a bacterial HJ resolvase, in uncharacterized open reading frames from poxviruses and an iridovirus. The putative vaccinia virus resolvase was expressed as a recombinant protein, affinity purified, and shown to specifically bind and cleave a synthetic HJ to yield nicked duplex molecules. Mutation of either of two conserved acidic amino acids abrogated the catalytic activity of the A22R protein without affecting HJ binding. The presence of bacterial-type enzymes in metazoan viruses raises evolutionary questions.     

Bacillus subtilis RecU protein cleaves Holliday junctions and anneals single-stranded DNA

Bacillus subtilis RecU protein is involved in homologous recombination, DNA repair, and chromosome segregation. Purified RecU binds preferentially to three- and four-strand junctions when compared to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) ( approximately 10- and approximately 40-fold lower efficiency, respectively). RecU cleaves mobile four-way junctions but fails to cleave a linear dsDNA with a putative cognate site, a finding consistent with a similar genetic defect observed for genes classified within the epsilon epistatic group (namely ruvA, recD, and recU). In the presence of Mg(2+), RecU also anneals a circular ssDNA and a homologous linear dsDNA with a ssDNA tail and a linear ssDNA and a homologous supercoiled dsDNA substrate. These results suggest that RecU, which cleaves recombination intermediates with high specificity, might also help in their assembly.     

Homology-associated nonhomologous recombination in mammalian gene targeting.

Nonhomologous (illegitimate) recombination of DNA underlies many changes in the genome. It involves no or little homology between recombining DNAs and has been considered unrelated with homologous recombination, which requires long homology. In mouse cells, however, we found recombination products whose sequences suggest that homologous interaction between DNAs caused nonhomologous recombination with another DNA. The intermediates of homologous recombination were apparently trapped at various stages and shunted to nonhomologous recombination. In one product, the nonhomologous recombination disrupted gene conversion. In another, it took place exactly at the end of long homology shared between two DNAs. This finding explains why gene targeting needs long uninterrupted homology and why mammalian homologous recombination is often nonconservative. We discuss possible consequences and roles of this type of homology-driven gene destruction mechanism.     

Chromosomal instability syndromes are sensitive to poly ADP-ribose polymerase inhibitors

Poly ADP-ribose polymerase inhibitors have been shown to target cells with homologous recombination DNA repair defects. We report that poly ADP-ribose polymerase inhibitors induces apoptosis in cells deficient in other key DNA repair components. Chromosomal instability disorders, Fanconi Anemia and Bloom's syndrome have dysfunctional DNA repair and an increased likelihood of leukemic transformation. PI addition to Fanconi Anemia and Bloom's syndrome cells resulted in significant apoptosis. Furthermore, poly ADP-ribose polymerase inhibitors induced apoptosis in DNA repair signaling defective ATM(-/-) and NBS(-/-) fibroblasts. Immunocytochemistry showed homologous recombination was abrogated in NBS(-/-) and ATM(-/-) fibroblasts, compromised in Fanconi anemia and normal in Bloom's syndrome cells in response to poly ADP-ribose polymerase inhibitors. Strikingly, poly ADP-ribose polymerase inhibitors increases non-homologous end joining repair activity, whilst non-homologous end joining deficient cells are extremely sensitive to poly ADP-ribose polymerase inhibitors. These data suggest poly ADP-ribose polymerase inhibitors target cells with DNA repair and signaling defects rather than solely defects in homologous recombination improving the potential of poly ADP-ribose polymerase inhibitors therapy in a wider range of cancers.     

Homologous pairing in genetic recombination: complexes of recA protein and DNA.

recA protein, which is essential for general genetic recombination in Escherichia coli, promotes the homologous pairing of single-stranded DNA with double-stranded DNA to form a D loop. The amount of recA protein required for the reaction was directly proportional to the amount of single stranded DNA and was unaffected by similar variations in the amount of double-stranded DNA. The ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), which was not rapidly hydrolyzed by recA protein, blocked the formation of D loops but promoted the formation of stable complexes of recA protein and single-stranded DNA. These complexes, in turn, bound homologous or heterologous double-stranded DNA and partially unwound it. Because ATP gamma S competitively inhibited the ATPase activity of recA protein (Km/Ki approximately 300), we infer that ATP gamma S binds at a site that overlaps the site for ATP and that the functional complexes formed in the presence of the analog probably represent partial steps in the overall reaction. If the complexes formed in the presence of ATP gamma S reflect natural intermediates in the formation of D loops, recA protein must promote homologous pairing either by moving juxtaposed single-stranded and double-stranded DNA relative to one another or by forming and dissociating complexes reiteratively until a homologous match occurs.     

Double-strand gap repair results in homologous recombination in mouse L cells.

Previous studies have demonstrated that the presence of double-strand breaks or double-strand gaps increases the frequency of homologous recombination between two cotransferred DNAs when they are introduced into cultured mammalian cells. Here we demonstrate that the repair of these double-strand gaps is a major mechanism for homologous recombination between exogenous DNAs. In particular, when a plasmid DNA containing a 104-base-pair (bp) gap in its tk gene (herpes simplex virus gene for thymidine kinase) undergoes recombination in mouse L cells to generate an intact gene, the majority of events result from direct repair of the double-strand gap using a cotransferred DNA as the template. We analyzed the recombination events by comparing the frequency of tk+ colonies, Southern blotting of cloned tk+ cell lines, and cloning recombined functional tk genes by plasmid rescue. In addition, by creating double-strand breaks within or adjacent to heterologous insertions in a mutant tk gene, we estimate that the L cell can generate a double-strand gap of between 152 and 248 bp and then can repair the gap to create a functional tk gene. We conclude that double-strand breaks and double-strand gaps are recombinogenic in transferred plasmid DNAs because they serve as intermediates in homologous recombination by double-strand gap repair, a nonreciprocal exchange of DNA or gene conversion event.     

Resolution of Holliday junctions in genetic recombination: RuvC protein nicks DNA at the point of strand exchange.

The RuvC protein of Escherichia coli catalyzes the resolution of recombination intermediates during genetic recombination and the recombinational repair of damaged DNA. Resolution involves specific recognition of the Holliday structure to form a complex that exhibits twofold symmetry with the DNA in an open configuration. Cleavage occurs when strands of like polarity are nicked at the sequence 5'-WTT decreases S-3' (where W is A or T and S is G or C). To determine whether the cleavage site needs to be located at, or close to, the point at which DNA strands exchange partners, Holliday structures were constructed with the junction points at defined sites within this sequence. We found that the efficiency of resolution was optimal when the cleavage site was coincident with the position of DNA strand exchange. In these studies, junction targeting was achieved by incorporating uncharged methyl phosphonates into the DNA backbone, providing further evidence for the importance of charge-charge repulsions in determining DNA structure.     

Nucleosomes on linear duplex DNA allow homologous pairing but prevent strand exchange promoted by RecA protein.

To understand the molecular basis of gene targeting, we have studied interactions of nucleoprotein filaments comprised of single-stranded DNA and RecA protein with chromatin templates reconstituted from linear duplex DNA and histones. We observed that for the chromatin templates with histone/DNA mass ratios of 0.8 and 1.6, the efficiency of homologous pairing was indistinguishable from that of naked duplex DNA but strand exchange was repressed. In contrast, the chromatin templates with a histone/DNA mass ratio of 9.0 supported neither homologous pairing nor strand exchange. The addition of histone H1, in stoichiometric amounts, to chromatin templates quells homologous pairing. The pairing of chromatin templates with nucleoprotein filaments of RecA protein-single-stranded DNA proceeded without the production of detectable networks of DNA, suggesting that coaggregates are unlikely to be the intermediates in homologous pairing. The application of these observations to strategies for gene targeting and their implications for models of genetic recombination are discussed.