Structural aspects of RecA-dependent homologous strand exchange involving human telomeric DNA

Telomeric DNA sequences in human cells and those of other vertebrates consist of long d(TTAGGG) repeats. In somatic cells, telomeres shorten every cell division with shortening serving as a mitotic clock that counts cell divisions and ultimately results in cellular senescence. Telomere length is principally maintained by a ribonucleoprotein, telomerase. However, a non-negligible proportion of human cells use a recombination-based mechanism for telomere maintenance, termed alternative maintenance of telomeres (ALT). Although the molecular mechanism of ALT is not known, GT-rich sequences in prokaryotes and eukaryotes display high levels of recombination relative to those of non-GT-rich DNA. We show that human telomeric strand-exchange complexes mediated by Escherichia coli RecA protein differ from those formed with nontelomeric sequences. Moreover, telomeric strand-exchange intermediates, unlike those involving nontelomeric sequences, exhibit a tendency to form higher-order nucleoprotein structures. We propose that the strong DNA unwinding activity inherent in the assembly of the RecA strand-exchange complex promotes the formation of alternative DNA structures at human telomeric loci. Organization of these noncanonical structures into higher-order complexes involving multiple DNA duplexes could facilitate the search for homology on different DNA molecules and provide a framework for understanding recombination-dependent mechanisms of telomere maintenance.     

recA protein promoted DNA strand exchange

recA protein and circular single-stranded DNA form a stable complex in the presence of single-stranded DNA binding protein (SSB), in which one recA protein monomer is bound per two nucleotides of DNA. These complexes are kinetically significant intermediates in the exchange of strands between the single-stranded DNA and an homologous linear duplex. After completion of strand exchange, the recA protein remains tightly associated with the circular duplex product of the reaction and the SSB is bound to the displaced linear single strand. Upon addition of ADP, the recA protein-duplex DNA complex dissociates. RecA protein also interacts with single-stranded DNA in the absence of SSB; however, the amount of recA protein bound is substantially reduced. These findings provide direct physical evidence for the participation of SSB in the formation of the recA protein-single-stranded DNA complexes inferred earlier from kinetic analysis. Moreover, they confirm the ability of recA protein to equilibrate between bound and free forms in the absence of SSB.     

UvsY protein of bacteriophage T4 is an accessory protein for in vitro catalysis of strand exchange

The uvsX and uvsY genes are essential to genetic recombination, recombination-dependent DNA synthesis and to the repair of DNA damage in bacteriophage T4. Purified UvsX protein has been shown to catalyze strand exchange and D-loop formation in vitro, but the role of UvsY protein has been unclear. We report that UvsY protein enhances strand exchange by UvsX protein by interacting specifically with UvsX protein: gene 32 protein (gp32) is not necessary for this effect and UvsY protein has no similar effect on the RecA protein of E. coli. UvsY protein, like UvsX protein, protects single-stranded DNA from digestion by nucleases, but, unlike UvsX protein, shows no ability to protect double-stranded DNA. UvsY protein enhances the rate of single-stranded-DNA-dependent ATP hydrolysis by UvsX protein, particularly in the presence of gp32 or high concentrations of salt, factors that otherwise reduce the ATPase activity of UvsX protein. The enhancement of ATP hydrolysis by UvsY protein is shown to result from the ability of UvsY protein to increase the affinity of UvsX protein for single-stranded DNA.     

Rad52 protein has a second stimulatory role in DNA strand exchange that complements replication protein-A function

Rad52 protein plays a central role in double strand break repair and homologous recombination in Saccharomyces cerevisiae. We have identified a new mechanism by which Rad52 protein stimulates Rad51 protein-promoted DNA strand exchange. This function of Rad52 protein is revealed when subsaturating amounts (relative to the single-stranded DNA concentration) of replication protein-A (RPA) are used. Under these conditions, Rad52 protein is needed for extensive DNA strand exchange. Interestingly, in this new role, Rad52 protein neither acts simply as a single strand DNA-binding protein per se nor, in contrast to its previously identified stimulatory roles, does it require physical interaction with RPA because it can be substituted by the Escherichia coli single strand DNA-binding protein. We propose that Rad52 protein acts by stabilizing the Rad51 presynaptic filament.     

Strand exchange protein 1 from Saccharomyces cerevisiae. A novel multifunctional protein that contains DNA strand exchange and exonuclease activities

Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae catalyzes the formation of heteroduplex DNA molecules from single-stranded circles and homologous linear duplex DNA in vitro. Previously, Sep1 was purified as a 132,000-Da species; however, DNA sequence analysis indicates that the SEP1 gene is capable of encoding a 175,000-Da protein (Tishkoff, D.X., Johnson, A.W., and Kolodner, R.D. (1991) Mol. Cell. Biol. 11, 2593-2608). The SEP1 gene was cloned into a GAL10 expression vector and expressed in a protease-deficient yeast strain. Intact Sep1, which migrated as a Mr-160,000 polypeptide during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was purified to apparent homogeneity and shown to have activities similar to those of the originally purified Mr = 132,000 fragment. We report here that, in addition to strand exchange activity, Sep1 contains an intrinsic exonuclease that is active on single- and double-stranded DNA with a severalfold preference for single-stranded DNA. The nuclease was induced in crude extracts upon induction with galactose, it co-purified with the strand exchange activity of Sep1, and the nuclease and strand exchange activities of Sep1 showed the same kinetics of heat inactivation. Sep1 nuclease, which requires Mg2+, can be functionally separated from the strand exchange activity by the substitution of Ca2+ for Mg2+. Under these conditions, the nuclease is inactive, and strand exchange activity is dependent on prior resection of the DNA ends by an exogenous exonuclease. Thus, the nuclease is necessary for synapsis but not strand exchange. Electron microscopic analysis revealed that true strand exchange products, alpha molecules and nicked double-stranded circular molecules, were formed. In addition, strand transfer proceeded to similar extents on 5'-resected and 3'-resected DNA. This result suggests that the polarity of strand transfer by Sep1 is determined by the polarity of its intrinsic nuclease.     

Identification of a novel human Rad51 variant that promotes DNA strand exchange

Rad51 plays a key role in the repair of DNA double-strand breaks through homologous recombination, which is the central process in the maintenance of genomic integrity. Five paralogs of the human Rad51 gene (hRad51) have been identified to date, including hRad51B, hRad51C, hRad51D, Xrcc2 and Xrcc3. In searches of additional hRad51 paralogs, we identified a novel hRad51 variant that lacked the sequence corresponding to exon 9 (hRad51-Δex9). The expected amino acid sequence of hRad51-Δex9 showed a frame-shift at codon 259, which resulted in a truncated C-terminus. RT-PCR analysis revealed that both hRad51 and hRad51-Δex9 were prominently expressed in the testis, but that there were subtle differences in tissue specificity. The hRad51-Δex9 protein was detected as a 31-kDa protein in the testis and localized at the nucleus. In addition, the hRad51-Δex9 protein showed a DNA-strand exchange activity comparable to that of hRad51. Taken together, these results indicate that hRad51-Δex9 promotes homologous pairing and DNA strand exchange in the nucleus, suggesting that alternative pathways in hRad51- or hRad51-Δex9-dependent manners exist for DNA recombination and repair.     

The direction of RecA protein assembly onto single strand DNA is the same as the direction of strand assimilation during strand exchange

The RecA protein of Escherichia coli optimally promotes DNA strand exchange reactions in the presence of the single strand DNA-binding protein of E. coli (SSB protein). Under these conditions, assembly of RecA protein onto single-stranded DNA (ssDNA) occurs in three steps. First, the ssDNA is rapidly covered by SSB protein. The binding of RecA protein is then initiated by nucleation of a short tract of RecA protein onto the ssDNA. Finally, cooperative polymerization of additional RecA protein accompanied by displacement of SSB protein results in a ssDNA-RecA protein filament (Griffith, J. D., Harris, L. D., and Register, J. C. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 553-559). We report here that RecA protein assembly onto circular ssDNA yields RecA protein-covered circles in which greater than 85% are completely covered by RecA protein with no remaining SSB protein-covered segments (as detected by electron microscopy). However, when linear ssDNA is used, 90% of the filaments contain a short segment at one end complexed with SSB protein. This suggests that RecA protein assembly is unidirectional. Visualization of the assembly of RecA protein onto either long ssDNA tails (containing either 5' or 3' termini) or ssDNA gaps generated in double strand DNA allowed us to determine that the RecA protein polymerizes in the 5' to 3' direction on ssDNA and preferentially nucleates at ssDNA-double strand DNA junctions containing 5' termini.     

An archaeal Rad54 protein remodels DNA and stimulates DNA strand exchange by RadA

Rad54 protein is a key member of the RAD52 epistasis group required for homologous recombination in eukaryotes. Rad54 is a duplex DNA translocase that remodels both DNA and protein–DNA complexes, and functions at multiple steps in the recombination process. Here we use biochemical criteria to demonstrate the existence of this important protein in a prokaryotic organism. The Sulfolobus solfataricus Rad54 (SsoRad54) protein is a double-strand DNA-dependent ATPase that can alter the topology of duplex DNA. Like its eukaryotic homolog, it interacts directly with the S. solfataricus Rad51 homologue, SsoRadA, to stimulate DNA strand exchange. Confirmation of this protein as an authentic Rad54 homolog establishes an essential phylogenetic bridge for identifying Rad54 homologs in the archaeal and bacterial domains.     

ATP hydrolysis and the displaced strand are two factors that determine the polarity of RecA-promoted DNA strand exchange.

When the recA protein (RecA) of Escherichia coli promotes strand exchange between single-stranded DNA (ssDNA) circles and linear double-stranded DNAs (dsDNA) with complementary 5' or 3' ends a polarity is observed. This property of RecA depends on ATP hydrolysis and the ssDNA that is displaced in the reaction since no polarity is observed in the presence of the non-hydrolyzable ATP analog, ATP gamma S, or in the presence of single-strand specific exonucleases. Based on these results a model is presented in which both the 5' and 3' complementary ends of the linear dsDNA initiate pairing with the ssDNA circle but only one end remains stably paired. According to this model, the association/dissociation of RecA in the 5' to 3' direction on the displaced strand determines the polarity of strand exchange by favoring or blocking its reinvasion into the newly formed dsDNA. Reinvasion is favored when the displaced strand is coated with RecA whereas it is blocked when it lacks RecA, remains covered by single-stranded DNA binding protein or is removed by a single-strand specific exonuclease. The requirement for ATP hydrolysis is explained if the binding of RecA to the displaced strand occurs via the dissociation and/or transfer of RecA, two functions that depend on ATP hydrolysis. The energy for strand exchange derives from the higher binding constant of RecA for the newly formed dsDNA as compared with that for ssDNA and not from ATP hydrolysis.     

A strand exchange FRET assay for DNA

A new displacement hybridisation method is reported using a single strand DNA probe, labelled with an acceptor fluorophore (oregon green 488). Detection of double stranded sample target is shown, with discrimination between the probe, duplexed during the assay, and free single stranded probe DNA achieved through the FRET from a donor grove fluorophore (Hoechst 33258). A model for the kinetics of the displacement assay is presented and the course of the assay predicted according to probe/target ratios and sequence. The modelled predictions are consistent with the experimental data showing single base pair mismatch discrimination. The pattern of response according to the mismatch/perfect complement ratio in a mixed sample is also considered with an allele-discrimination ratio lying between the homozygous gene and total mismatch case, according to ratio. The assay is shown to be tolerant of different probe concentrations and ratios and through the dual wavelength recorded signals from donor and FRET acceptor, internal baseline correction is achieved with excellent noise reduction through ratiometric measurement.     

Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange.

Two proteins encoded by bacteriophage T7, the gene 2.5 single-stranded DNA binding protein and the gene 4 helicase, mediate homologous DNA strand exchange. Gene 2.5 protein stimulates homologous base pairing of two DNA molecules containing complementary single-stranded regions. The formation of a joint molecule consisting of circular, single-stranded M13 DNA, annealed to homologous linear, duplex DNA having 3'- or 5'-single-stranded termini of approximately 100 nucleotides requires stoichiometric amounts of gene 2.5 protein. In the presence of gene 4 helicase, strand transfer proceeds at a rate of > 120 nucleotides/s in a polar 5' to 3' direction with respect to the invading strand, resulting in the production of circular duplex M13 DNA. Strand transfer is coupled to the hydrolysis of a nucleoside 5'-triphosphate. The reaction is dependent on specific interactions between gene 2.5 protein and gene 4 protein.     

DNA helicase activity of PcrA is not required for the displacement of RecA protein from DNA or inhibition of RecA-mediated strand exchange

PcrA is a conserved DNA helicase present in all gram-positive bacteria. Bacteria lacking PcrA show high levels of recombination. Lethality induced by PcrA depletion can be overcome by suppressor mutations in the recombination genes recFOR. RecFOR proteins load RecA onto single-stranded DNA during recombination. Here we test whether an essential function of PcrA is to interfere with RecA-mediated DNA recombination in vitro. We demonstrate that PcrA can inhibit the RecA-mediated DNA strand exchange reaction in vitro. Furthermore, PcrA displaced RecA from RecA nucleoprotein filaments. Interestingly, helicase mutants of PcrA also displaced RecA from DNA and inhibited RecA-mediated DNA strand exchange. Employing a novel single-pair fluorescence resonance energy transfer-based assay, we demonstrate a lengthening of double-stranded DNA upon polymerization of RecA and show that PcrA and its helicase mutants can reverse this process. Our results show that the displacement of RecA from DNA by PcrA is not dependent on its translocase activity. Further, our results show that the helicase activity of PcrA, although not essential, might play a facilitatory role in the RecA displacement reaction.     

Regulation of DNA strand exchange in homologous recombination

Homologous recombination, the exchange of DNA strands between homologous DNA molecules, is involved in repair of many structural diverse DNA lesions. This versatility stems from multiple ways in which homologous DNA strands can be rearranged. At the core of homologous recombination are recombinase proteins such as RecA and RAD51 that mediate homology recognition and DNA strand exchange through formation of a dynamic nucleoprotein filament. Four stages in the life cycle of nucleoprotein filaments are filament nucleation, filament growth, homologous DNA pairing and strand exchange, and filament dissociation. Progression through this cycle requires a sequence of recombinase-DNA and recombinase protein-protein interactions coupled to ATP binding and hydrolysis. The function of recombinases is controlled by accessory proteins that allow coordination of strand exchange with other steps of homologous recombination and that tailor to the needs of specific aberrant DNA structures undergoing recombination. Accessory proteins are also able to reverse filament formation thereby guarding against inappropriate DNA rearrangements. The dynamic instability of the recombinase-DNA interactions allows both positive and negative action of accessory proteins thereby ensuring that genome maintenance by homologous recombination is not only flexible and versatile, but also accurate.     

Interaction of a Saccharomyces cerevisiae strand exchange stimulatory factor with DNA

In the preceding paper (Norris & Kolodner, 1990), we described the purification of a Mr 33,000 polypeptide which dramatically stimulated the activity of SEP1, the yeast mitotic strand exchange protein. In this paper, we characterized this new protein, which was designated SF1, in the absence of SEP1. SF1 had a sedimentation coefficient of 1.7 S and a Stokes radius of 30 A, which was consistent with a calculated native molecular weight of 31,000, indicating that SF1 existed in solution as a monomer. Filter binding assays showed that SF1 bound preferentially to single-stranded rather than double-stranded DNA. Fluorescence spectroscopy analysis indicated that SF1 occluded approximately eight nucleotides when bound to single-stranded DNA and exhibited a dissociation constant, KD, of 2.83 x 10(-6) M. The binding of SF1 to single-stranded DNA was noncooperative and appeared to involve at least one tyrosine residue. SF1, in the absence of SEP1, stimulated the renaturation of homologous single-stranded DNA, suggesting that it might act directly in some phase of the strand exchange reaction.     

The DNA binding and pairing preferences of the archaeal RadA protein demonstrate a universal characteristic of DNA strand exchange proteins

The archaeal RadA protein is a homologue of the Escherichia coli RecA and Saccharomyces cerevisiae Rad51 proteins and possesses the same biochemical activities. Here, using in vitro selection, we show that the Sulfolobus solfataricus RadA protein displays the same preference as its homologues for binding to DNA sequences that are rich in G residues, and under-represented in A and C residues. The RadA protein also displays enhanced pairing activity with these in vitro-selected sequences. These parallels between the archaeal, eukaryal and bacterial proteins further extend the universal characteristics of DNA strand exchange proteins.     

RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand exchange: a universal step of recombinational repair

Genetic evidence suggests that the RecF, RecO, and RecR (RecFOR) proteins participate in a common step of DNA recombination and repair, yet the biochemical event requiring collaboration of all three proteins is unknown. Here, we show that the concerted action of the RecFOR complex directs the loading of RecA protein specifically onto gapped DNA that is coated with single-stranded DNA binding (SSB) protein, thereby accelerating DNA strand exchange. The RecFOR complex recognizes the junction between the ssDNA and dsDNA regions and requires a base-paired 5' terminus at the junction. Thus, the RecFOR complex is a structure-specific mediator that targets recombinational repair to ssDNA-dsDNA junctions. This reaction reconstitutes the initial steps of recombinational gapped DNA repair and uncovers an event also common to the repair of ssDNA-tailed intermediates of dsDNA-break repair. We propose that the behavior of the RecFOR proteins is mimicked by functional counterparts that exist in all organisms.     

A postsynaptic role for single-stranded DNA-binding protein in recA protein-promoted DNA strand exchange.

The single-stranded DNA-binding protein (SSB protein) is required for efficient genetic recombination in vivo. One function for SSB protein in DNA strand exchange in vitro is to remove secondary structure from single-stranded DNA (ssDNA) and thereby aid in the formation of recA protein-saturated presynaptic complexes. In the preceding paper (Lavery, P. E., and Kowalczykowski, S. C. (1992) J. Biol. Chem. 267, 9307-9314) we demonstrated that DNA strand exchange can occur in the presence of volume-occupying agents at low magnesium ion concentration, where secondary structures are reduced. Our results suggest that SSB protein is not acting during presynapsis under these conditions, yet the DNA strand exchange reaction is stimulated by the addition of SSB protein. In this study we present biochemical evidence which suggests that SSB protein stimulates DNA strand exchange by binding to the ssDNA displaced from joint molecules, thereby stabilizing them and allowing branch migration to extend the region of heteroduplex DNA. Therefore, our results indicate dual roles for SSB protein at elevated magnesium ion concentration; it functions during presynapsis, removing secondary structure from ssDNA, as indicated previously, and it also functions postsynaptically, binding to the ssDNA displaced from joint molecules.     

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.     

RecA protein reinitiates strand exchange on isolated protein-free DNA intermediates. An ADP-resistant process

Efficient homologous pairing de novo of linear duplex DNA with a circular single strand (plus strand) coated with RecA protein requires saturation and extension of the single strand by the protein. However, strand exchange, the transfer of a strand from duplex DNA to the nucleoprotein filament, which follows homologous pairing, does not require the stable binding of RecA protein to single-stranded DNA. When RecA protein was added back to isolated protein-free DNA intermediates in the presence of sufficient ADP to inhibit strongly the binding of RecA protein to single-stranded DNA, strand exchange nonetheless resumed at the original rate and went to completion. Characterization of the protein-free DNA intermediate suggested that it has a special site or region to which RecA protein binds. Part of the nascent displaced plus strand of the deproteinized intermediate was unavailable as a cofactor for the ATPase activity of RecA protein, and about 30% resisted digestion by P1 endonuclease, which acts preferentially on single-stranded DNA. At the completion of strand exchange, when the distal 5' end of the linear minus strand had been fully incorporated into heteroduplex DNA, a nucleoprotein complex remained that contained all three strands of DNA from which the nascent displaced strand dissociated only over the next 50 to 60 minutes. Deproteinization of this intermediate yielded a complex that also contained three strands of DNA in which the nascent displaced strand was partially resistant to both Escherichia coli exonuclease I and P1 endonuclease. The deproteinized complex showed a broad melting transition between 37 degrees C and temperatures high enough to melt duplex DNA. These results show that strand exchange can be subdivided into two stages: (1) the exchange of base-pairs, which creates a new heteroduplex pair in place of a parental pair; and (2) strand separation, which is the physical displacement of the unpaired strand from the nucleoprotein filament. Between the creation of new heteroduplex DNA and the eventual separation of a third strand, there exists an unusual DNA intermediate that may contain three-stranded regions of natural DNA that are several thousand bases in length.     

The RecA binding locus of RecBCD is a general domain for recruitment of DNA strand exchange proteins

RecBCD enzyme facilitates loading of RecA protein onto ssDNA produced by its helicase/nuclease activity. This process is essential for RecBCD-mediated homologous recombination. Here, we establish that the C-terminal nuclease domain of the RecB subunit (RecBnuc) forms stable complexes with RecA. Interestingly, RecBnuc also interacts with and loads noncognate DNA strand exchange proteins. Interaction is with a conserved element of the RecA-fold, but because the binding to noncognate proteins decreases in a phylogenetically consistent way, species-specific interactions are also present. RecBnuc does not impede activities of RecA that are important to DNA strand exchange, consistent with its role in targeting of RecA. Modeling predicts the interaction interface for the RecA-RecBCD complex. Because a similar interface is involved in the binding of human Rad51 to the conserved BRC repeat of BRCA2 protein, the RecB-domain may be one of several structural domains that interact with and recruit DNA strand exchange proteins to DNA.