Role of SSB protein in RecA promoted branch migration reactions

Summary The RecA protein ofEscherichia coli is essential for genetic recombination and postreplicational repair of DNA. In vitro, RecA protein promotes strand transfer reactions between full length linear duplex and single stranded circular DNA of X174 to form heteroduplex replicative form II-like structures (Cox and Lehman 1981a). In a similar way, it transfers one strand of a short duplex restriction fragment to a single stranded circle. Both reactions require RecA and single strand binding protein (SSB) in amounts sufficient to saturate the ssDNA. The rate and extent of strand transfer is enhanced considerably when SSB is added after preincubation of the DNA with RecA protein. In contrast, SSB protein is not required for RecA protein catalysed reciprocal strand exchanges between regions of duplex DNA. These results indicate that while SSB is necessary for efficient transfer between linear duplex and ssDNA to form a single heteroduplex, it is not required for branch migration reactions between duplex molecules that form two heteroduplexes.Abbreviations SSB single strand binding protein - ssDNA single stranded DNA - X phage X174 - bp base pairs - ATP[S] adenosine 5-O-(gamma-thiotriphosphate)     

A 140-Bp-Long Palindromic Sequence Induces Double-Strand Breaks during Meiosis in the Yeast Saccharomyces Cerevisiae

Palindromic sequences have the potential to form hairpin or cruciform structures, which are putative substrates for several nucleases and mismatch repair enzymes. A genetic method was developed to detect such structures in vivo in the yeast Saccharomyces cerevisiae. Using this method we previously showed that short hairpin structures are poorly repaired by the mismatch repair system in S. cerevisiae. We show here that mismatches, when present in the stem of the hairpin structure, are not processed by the repair machinery, suggesting that they are treated differently than those in the interstrand base-paired duplex DNA. A 140-bp-long palindromic sequence, on the contrary, acts as a meiotic recombination hotspot by generating a site for a double-strand break, an initiator of meiotic recombination. We suggest that long palindromic sequences undergo cruciform extrusion more readily than short ones. This cruciform structure then acts as a substrate for structure-specific nucleases resulting in the formation of a double-strand break during meiosis in yeast. In addition, we show that residual repair of the short hairpin structure occurs in an MSH2-independent pathway.     

Partial purification and characterization of a recombinase from human cells

We describe the partial purification and characterization of a human recombinase activity from RPMI 1788 B lymphoblasts. Stoichiometric amounts of recombinase carry out a strand transfer reaction between linear duplex DNA and homologous circular single-strand DNA. The product of strand transfer by the recombinase is a joint molecule composed of a single-strand circle joined to one end of the linear duplex molecule by a region of DNA heteroduplex at least 150 bp long. Formation of DNA heteroduplexes is accompanied by strand displacement. Strand invasion initiates at the ends of the linear duplex. Finally, strand displacement by human recombinase exhibits polarity and proceeds in a 3' to 5' direction. This is the first demonstration of a strand transfer activity from a high eukaryote. We discuss similarities between our recombinase and the RecA and rec1 recombination proteins from E. coli and Ustilago maydis, respectively.     

Origins of sequence selectivity in homologous genetic recombination: insights from rapid kinetic probing of RecA-mediated DNA strand exchange

Despite intense effort over the past 30 years, the molecular determinants of sequence selectivity in RecA-mediated homologous recombination have remained elusive. Here, we describe when and how sequence homology is recognized between DNA strands during recombination in the context of a kinetic model for RecA-mediated DNA strand exchange. We characterized the transient intermediates of the reaction using pre-steady-state kinetic analysis of strand exchange using oligonucleotide substrates containing a single fluorescent G analog. We observed that the reaction system was sensitive to heterology between the DNA substrates; however, such a "heterology effect" was not manifest when functional groups were added to or removed from the edges of the base-pairs facing the minor groove of the substrate duplex. Hence, RecA-mediated recombination must occur without the involvement of a triple helix, even as a transient intermediate in the process. The fastest detectable reaction phase was accelerated when the structure or stability of the substrate duplex was perturbed by internal mismatches or the replacement of G.C by I.C base-pairs. These findings indicate that the sequence specificity in recombination is achieved by Watson-Crick pairing in the context of base-pair dynamics inherent to the extended DNA structure bound by RecA during strand exchange.     

In vitro rescue of an integrated hybrid adeno-associated virus/simian virus 40 genome.

In an in vitro simian virus 40 (SV40) DNA replication assay, we have observed excision of a hybrid adeno-associated virus (AAV)/SV40 insert from a plasmid construct. The excision was dependent on the presence of the palindromic AAV terminal repeat and greatly enhanced by the addition of the SV40 T antigen to the reaction. Analysis of the excision product supports a model in which the palindromic terminal sequences of AAV form a cruciform structure (equivalent to a Holliday recombination intermediate), which is cleaved and resealed so that the excision products are linear duplex pBR322 and linear duplex AAV/SV40 insert. Both the excised linear duplex pBR322 and the excised linear duplex AAV/SV40 insert have each terminus covalently crosslinked by one copy of the palindromic region of the AAV terminal repeat region folded on itself. The excision process may be a model system for cellular homologous recombination. The process as observed was either concomitant with or subsequent to DNA replication.     

Radioprobing of a RecA-three-stranded DNA complex with iodine 125: evidence for recognition of homology in the major groove of the target duplex

A fundamental problem in homologous recombination is how homology between DNAs is recognized. In all current models, a recombination protein loads onto a single strand of DNA and scans another duplex for homology. When homology is found, a synaptic complex is formed, leading to strand exchange and a heteroduplex. A novel technique based on strand cleavage by the Auger radiodecay of iodine 125, allows us to determine the distances between (125)I on the incoming strand and the target sugars of the duplex DNA strands in an Escherichia coli RecA protein-mediated synaptic complex. Analysis of these distances shows that the complex represents a post-strand exchange intermediate in which the heteroduplex is located in the center, while the outgoing strand forms a relatively wide helix intertwined with the heteroduplex and located in its minor groove. The structure implies that homology is recognized in the major groove of the duplex.     

Nucleosome occupancy landscape and dynamics at mouse recombination hotspots

During meiosis, paternal and maternal homologous chromosomes recombine at specific recombination sites named hotspots. What renders 2% of the mammalian genomes permissive to meiotic recombination by allowing Spo11 endonuclease to initiate double‐strand breaks is largely unknown. Work in yeast has shown that chromatin accessibility seems to be important for this activity. Here, we define nucleosome profiles and dynamics at four mouse recombination hotspots by purifying highly enriched fractions of meiotic cells. We found that nucleosome occupancy is generally stable during meiosis progression. Interestingly, the cores of recombination hotspots have largely open chromatin structure, and the localization of the few nucleosomes present in these cores correlates precisely with the crossover‐free zones in recombinogenic domains. Collectively, these high‐resolution studies suggest that nucleosome occupancy seems to direct, at least in part, how meiotic recombination events are processed.     

Recombination apparatus of T4 phage

The substantial process of general DNA recombination consists of production of ssDNA, exchange of the ssDNA and its homologous strand in a duplex, and cleavage of branched DNA to maturate recombination intermediates. Ten genes of T4 phage are involved in general recombination and apparently encode all of the proteins required for its own recombination. Several proteins among them interact with each other in a highly specific manner based on a protein-protein affinity and constitute a multicomponent protein machine to create an ssDNA gap essential for production of recombinogenic ssDNA, a machine to supply recombinogenic ssDNA which has a free end, or a machine to transfer the recombinogenic single strand into a homologous duplex.     

Construction of a DNA four-way junction: Design and NMR spectroscopy

In 1964 Holliday postulated the formation of cruciform structures (four-way junctions) in duplex DNA as intermediate in genetic recombination. Since then, many biochemical and biophysical investigations were directed at solving questions concerning structural details of stable four-way junctions. Thus far, NMR spectroscopy played a minor part in these investigations on account of the minimum size of the molecule (expressed as the number of nucleotide residues) that was thought necessary to produce a stable cruciform structure. Indeed, the smallest four-way junction studied thus far by NMR methods was built from four separate DNA strands, each containing 16 nucleotides, a total of 64. Obviously, with such a large structure one runs into assignment problems. We considered the possibility of constructing a stable four-way junction from a single strand of DNA. The underlying idea was to make use of our detailed knowledge of the building principles of stable minihairpin loops. These loops, containing only two nucleotides to bridge the gap between antiparallel strands, are maximally stable in DNA sequences like 5-d(-C-TT-G-), where C and G form a normal Watson-Crick base pair and the two T residues cross the minor groove to form the minihairpin loop. Three of such miniloops could in principle cap three arms of the cruciform. The fourth arm would have an open end. The problem to be solved is to find the minimum length that is required to insure stability of the three closed arms and of the fourth open arm. We were successful with a structure that has three short stems (four base pairs each) and an open-end stem consisting of eight base pairs, a total of 46 residues. NMR experiments, carried out on this molecule in the presence of Mg²⁺, showed details of folding which have never been observed before.     

DNA strand transfer reactions catalyzed by vaccinia topoisomerase I.

Vaccinia virus DNA topoisomerase I forms a 3'-phosphoryl intermediate with duplex DNAs containing the conserved binding/cleavage motif 5'CCCTT decreases. Covalently bound enzyme is capable of transferring the incised DNA strand to a heterologous DNA acceptor containing a 5'OH terminus. Both intramolecular and intermolecular religation reactions are catalyzed. Intramolecular strand transfer occurs to the noncleaved strand of the DNA duplex and results in formation of a hairpin loop. Intermolecular religation to an exogenous DNA strand is favored over hairpin formation and requires the potential for base pairing between the acceptor and the noncleaved strand of the donor complex. As few as 4 potential base pairs are sufficient to support intermolecular transfer. These results in vitro are consistent with the proposal that vaccinia topoisomerase can catalyze sequence-specific strand transfer during genetic recombination in vivo (Shuman, S. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10104-10108.).     

Molecular cloning and sequence of the concatemer junction from vaccinia virus replicative DNA. Viral nuclease cleavage sites in cruciform structures

The concatemer junction from replicative forms of vaccinia virus DNA was cloned into plasmid vectors and shown to be a precise duplex copy of the viral terminal hairpin structure, with each strand corresponding to one of the alternative sequence isomers. The plasmids were relaxed circles with extruded cruciforms representing two copies of the vaccinia telomere hairpin structure. Head-to-head dimers containing two copies of the vaccinia virus concatemer junction were observed to contain only one set of stem-loop structures per molecule, suggesting that the initial formation of a small cruciform, and not branch migration, was the rate-limiting step in cruciform formation. The plasmids containing the concatemer junction were converted into nicked circular, linear and cross-linked linear molecules by a nuclease isolated from vaccinia virions. The region-specific cleavage near the border of the hairpin loop and the formation of DNA cross-links in some of the molecules is consistent with the nuclease acting as a nicking-closing enzyme that participates in the resolution of mature termini from replicative concatemer intermediates.     

Palindrome regeneration by template strand-switching mechanism at the origin of DNA replication of porcine circovirus via the rolling-circle melting-pot replication model

Palindromic sequences (inverted repeats) flanking the origin of DNA replication with the potential of forming single-stranded stem-loop cruciform structures have been reported to be essential for replication of the circular genomes of many prokaryotic and eukaryotic systems. In this study, mutant genomes of porcine circovirus with deletions in the origin-flanking palindrome and incapable of forming any cruciform structures invariably yielded progeny viruses containing longer and more stable palindromes. These results suggest that origin-flanking palindromes are essential for termination but not for initiation of DNA replication. Detection of template strand switching in the middle of an inverted repeat strand among the progeny viruses demonstrated that both the minus genome and a corresponding palindromic strand served as templates simultaneously during DNA biosynthesis and supports the recently proposed rolling-circle "melting-pot" replication model. The genome configuration presented by this model, a four-stranded tertiary structure, provides insights into the mechanisms of DNA replication, inverted repeat correction (or conversion), and illegitimate recombination of any circular DNA molecule with an origin-flanking palindrome.     

The structure of duplex DNA in the nucleosome core particle: An alternative model

Many studies indirectly indicate that the conformation ofin vivo duplex DNA is the double helix. The most direct view, from the X-ray analysis of the nucleosome core particle, has also been interpreted in terms of the double helix structure. However, an alternative possibility exists; that the duplex adopts a metastable side-by-side conformation which readily converts to the double helix on removal of protein. Evidence for the existence of this conformation has been obtained from a reanalysis of the electron density map for the nucleosome particle.     

Strand invasion by mixed base PNAs and a PNA-peptide chimera

Peptide nucleic acid oligomers (PNAs) have a remarkable ability to invade duplex DNA at polypurine–polypyrimidine target sequences. Applications for PNAs in medicine and biotechnology would increase if the rules governing their hybridization to mixed base sequences were also clear. Here we describe hybridization of PNAs to mixed base sequences and demonstrate that simple chemical modifications can enhance recognition. Easily synthesized and readily soluble eight and 10 base PNAs bind to plasmid DNA at an inverted repeat that is likely to form a cruciform structure, providing convenient tags for creating PNA–plasmid complexes. PNAs also bind to mixed base sequences that cannot form cruciforms, suggesting that recognition is a general phenomenon. Rates of strand invasion are temperature dependent and can be enhanced by attaching PNAs to positively charged peptides. Our results support use of PNAs to access the information within duplex DNA and demonstrate that simple chemical modifications can make PNAs even more powerful agents for strand invasion. Simple strategies for enhancing strand invasion should facilitate the use of PNAs: (i) as biophysical probes of double-stranded DNA; (ii) to target promoters to control gene expression; and (iii) to direct sequence-specific mutagenesis.     

Partial purification of an activity from human cells that promotes homologous pairing and the formation of heteroduplex DNA in the presence of ATP

Summary An activity that can promote homologous pairing and strand transfer between suitable DNA substrates has been partially purified from human skin fibroblasts and from Hela cells. The strand transfer reaction was investigated with DNA substrates consisting of single-stranded circular and duplex linear phage DNA. It requires ATP, and under optimal conditions yields heteroduplex molecules containing one strand from each parental DNA substrate. The reactions appears to be of the same general nature as those mediated by RecA proteins of Escherichia coli and the Rec1 protein of Ustilago maydis.     

A chimeric Rec-A protein that implicates non-Watson-Crick interactions in homologous pairing.

The helical filament formed by RecA protein on single-stranded DNA plays an important role in homologous recombination and pairs with a complementary single strand or homologous duplex DNA. The RecA nucleoprotein filament also recognizes an identical single strand. The chimeric protein, RecAc38, forms a nucleoprotein filament that recognizes a complementary strand but is defective in recognition of duplex DNA, and is associated with phenotypic defects in repair and recombination. As described here, RecAc38 nucleoprotein filament is also defective in recognition of an identical strand, either when the filament has within it a single strand or duplex DNA. A model that postulates three DNA binding sites rationalizes these observations and suggests that the third binding site mediates non-Watson-Crick interactions that are instrumental in recognition of homology in duplex DNA.     

Action of RecBCD enzyme on cruciform DNA

We tested the hypothesis that RecBCD enzyme of Escherichia coli resolves pre-existing Holliday recombination intermediates by examining the action of the purified enzyme on an open-ended DNA cruciform with limited ability to branch migrate. The enzyme cleaved two strands of the cruciform near its base to produce "recombinant" products, with a marked bias in the direction of cleavage. The two nicks necessary to cleave the cruciform were made separately. Cruciforms whose four termini were blocked by synthetic hairpin-shaped oligonucleotides were not detectably nicked by the enzyme. With one terminus open the enzyme made a nick at the base of the cruciform but not a double-strand cut. With two or more termini open the enzyme made double-strand cuts. We infer that RecBCD enzyme molecules must enter the termini of duplex DNA and approach the cruciform from more than one direction in order to cleave it into recombinant products. Previous results on RecBCD-mediated recombination between phage lambda and lambda dv imply that intracellular RecBCD enzyme can approach pre-existing Holliday junctions from only one direction. We infer that intracellular RecBCD enzyme cannot cleave pre-existing Holliday junctions into recombinants and suggest that the enzyme may cleave Holliday junctions in whose formation it participates.