Physical analyses of E. coli heteroduplex recombination products in vivo: on the prevalence of 5' and 3' patches

Background

Homologous recombination in Escherichia coli creates patches (non-crossovers) or splices (half crossovers), each of which may have associated heteroduplex DNA. Heteroduplex patches have recombinant DNA in one strand of the duplex, with parental flanking markers. Which DNA strand is exchanged in heteroduplex patches reflects the molecular mechanism of recombination. Several models for the mechanism of E. coli RecBCD-mediated recombinational double-strand-end (DSE) repair specify that only the 3′-ending strand invades the homologous DNA, forming heteroduplex in that strand. There is, however, in vivo evidence that patches are found in both strands.

Methodology/Principle Findings

This paper re-examines heteroduplex-patch-strand polarity using phage λ and the λdv plasmid as DNA substrates recombined via the E. coli RecBCD system in vivo. These DNAs are mutant for λ recombination functions, including orf and rap, which were functional in previous studies. Heteroduplexes are isolated, separated on polyacrylamide gels, and quantified using Southern blots for heteroduplex analysis. This method reveals that heteroduplexes are still found in either 5′ or 3′ DNA strands in approximately equal amounts, even in the absence of orf and rap. Also observed is an independence of the RuvC Holliday-junction endonuclease on patch formation, and a slight but statistically significant alteration of patch polarity by recD mutation.

Conclusions/Significance

These results indicate that orf and rap did not contribute to the presence of patches, and imply that patches occurring in both DNA strands reflects the molecular mechanism of recombination in E. coli. Most importantly, the lack of a requirement for RuvC implies that endonucleolytic resolution of Holliday junctions is not necessary for heteroduplex-patch formation, contrary to predictions of all of the major previous models. This implies that patches are not an alternative resolution of the same intermediate that produces splices, and do not bear on models for splice formation. We consider two mechanisms that use DNA replication instead of endonucleolytic resolution for formation of heteroduplex patches in either DNA strand: synthesis-dependent-strand annealing and a strand-assimilation mechanism.     

Effects of DNA sequence non-homology on formation of bacteriophage lambda recombinants.

The presence of DNA sequence non-homologies limits the parental material contribution to the genomes of unduplicated bacteriophage λ recombinants. Crosses involving closely linked markers within the lacZ region of λ plac5 have been carried out under conditions severely limiting DNA synthesis. The presence of density labels distinguishing the parental phage permits an assessment of their material contribution to the lacZ recombinant phage that emerge.When both parents harbor point mutations, the recombinants exhibit a broad range of relative parental DNA contributions, providing support for the proposal that the lacZ⁺ recombinants that are detected under these conditions result from mismatch repair processes acting on heterozygous sites within the long regions of heteroduplex structure present in the products of recombination. The presence of a region of non-homology, either a deletion or an insertion sequence, in one of the parents results in a limitation in the material contribution of that parent to the recombinant products. When both parents carry regions of non-homology, the relative parental contributions to recombinant products are further limited and are confined to the density range expected of the products of double-stranded breakage and joining events within the region separating the two mutational sites. These observations suggest that regions of non-homology are excluded from heteroduplex structures and provide support for a role of branch migration of a Holliday (1964) structure in the formation of the heteroduplex regions that are present in the products of recombination.     

Intermediates of yeast meiotic recombination contain heteroduplex DNA

The formation of heteroduplex DNA features prominently in all models for homologous recombination. A central intermediate in the current double-strand break repair model contains two Holliday junctions flanking a region of heteroduplex DNA. Studies of yeast meiosis have identified such intermediates but failed to detect associated heteroduplex DNA. We show here that these intermediates contain heteroduplex DNA, providing an important validation of the double-strand break repair model. However, we also detect intermediates where both Holliday junctions are to one side of the initiating DSB site, while the intervening region shows no evidence of heteroduplex DNA. Such structures are not easily accommodated by the canonical version of the double-strand break repair model.     

DNA base sequence homology between coliphages T7 and phiII and between T3 and phiII as determined by heteroduplex mapping in the electron microscope

The genetic relatedness of the similar coliphages T7, T3 and øII was investigated by electron microscopic observations of the DNA heteroduplexes formed with one DNA strand from coliphage T7 (or T3) and the complementary strand from øII. The T7-øII heteroduplex is almost invariant with denaturing conditions, below the melting temperature of T7 DNA. This observation indicates that the DNAs of T7 and øII have regions of complete homology and regions of complete non-homology. In direct contrast, the T3-øII heteroduplex varies with denaturing conditions. The fraction of the T3-øII heteroduplex observed as double-stranded decreases with an increase in denaturing conditions below the melting temperature. This observation indicates that the DNAs of T3 and øII have extensive sequences of partial homology.     

The roles of RecBCD, Ssb and RecA proteins in the formation of heteroduplexes from linear-duplex DNA in vitro

Summary The formation of heteroduplexes from linear duplex DNA, where one molecule possesses a DNA doublestrand break, was assayed by agarose gel electrophoresis. Using unlabeled whole-length linear duplex DNA and ³H-labeled half-length linear duplex DNA (obtained from plasmid pACYC184), the appearance of ³H-labeled DNA that migrated as whole-length linear DNA was taken as evidence for formation of heteroduplex DNA. When the DNA mixtures were incubated with RecA, RecBCD, or Ssb proteins, or any double or triple combination of these proteins under a variety of reaction conditions, no heteroduplex DNA was detected. However, heteroduplex DNA was detected when the DNA mixtures were first incubated briefly with the RecBCD and Ssb proteins under reaction conditions that allow unwinding to proceed, and then the MgCl₂ concentration was raised such that renaturation could proceed. The inclusion of the RecBCD and Ssb proteins was sufficient to catalyze the slow formation of heteroduplex DNA, but the presence of RecA protein greatly increased the kinetics. The roles of the RecBCD, Ssb and RecA proteins in heteroduplex formation in vitro are discussed.     

RAD54 controls access to the invading 3′-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae

Rad51 is a key protein in homologous recombination performing homology search and DNA strand invasion. After DNA strand exchange Rad51 protein is stuck on the double-stranded heteroduplex DNA product of DNA strand invasion. This is a problem, because DNA polymerase requires access to the invading 3′-OH end to initiate DNA synthesis. Here we show that, the Saccharomyces cerevisiae dsDNA motor protein Rad54 solves this problem by dissociating yeast Rad51 protein bound to the heteroduplex DNA after DNA strand invasion. The reaction required species-specific interaction between both proteins and the ATPase activity of Rad54 protein. This mechanism rationalizes the in vivo requirement of Rad54 protein for the turnover of Rad51 foci and explains the observed dependence of the transition from homologous pairing to DNA synthesis on Rad54 protein in vegetative and meiotic yeast cells.     

Characterization of the ColE1-Km plasmids pCR1 and pCR11 by electron microscope and restriction endonuclease mapping.

The ColE1-Km plasmids pCR1 and pCR11 have been characterized by electron microscope techniques. Their sizes have been determined to be 13.1 and 9.2 kb, respectively, and heteroduplex studies show that the plasmids differ in the presence of a 3.9 kb deletion in the ColE1 region of pCR11. Both contain a nontandem inverted duplication of a 1.06 kb sequence. The single HindIII site, 3.5 kb from the EcoR1 site, lies in the 0.97 kb of DNA between the inverted repeat sequences. Since DNA insertions at the HindIII site destroy kanamycin resistance, it can be concluded that the kanamycin phosphotransferase gene is contained in this region. Electron microscopy of self-annealed plasmids treated with restriction endonucleases shows that each inverted duplication sequence contains one HindII site and at least two SmaI sites.     

Steady-state ATPase activity of E. coli MutS modulated by its dissociation from heteroduplex DNA

The ability of MutS to recognize mismatched DNA is required to initiate a mismatch repair (MMR) system. ATP binding and hydrolysis are essential in this process, but their role in MMR is still not fully understood. In this study, steady-state ATPase activities of MutS from Escherichia coli were investigated using the spectrophotometric method with a double end-blocked heteroduplex containing gapped bases. The ATPase activities of MutS increased as the number of gapped bases increased in a double end-blocked heteroduplex with 2-8 gapped bases in the chain, indicating that MutS dissociates from DNA when it reaches a scission during movement along the DNA. Since movement of MutS along the chain does not require extensive ATP hydrolysis and the ATPase activity is only enhanced when MutS dissociates from a heteroduplex, these results support the sliding clamp model in which ATP binding by MutS induces the formation of a hydrolysis-independent sliding clamp.     

Formation of heteroduplex DNA promoted by the combined activities of Escherichia coli recA and recBCD proteins

We have established an in vitro reaction in which heteroduplex DNA formation is dependent on the concerted actions of recA and recBCD proteins, the major components of the recBCD pathway of genetic recombination in vivo. We find that heteroduplex DNA formation requires three distinct enzymatic functions: first, the helicase activity of recBCD enzyme initiates heteroduplex DNA formation by unwinding the linear double-stranded DNA molecule to transiently form single-stranded DNA (ssDNA); second, recA protein traps this ssDNA before it reanneals; third, recA protein catalyzes the pairing of this ssDNA molecule with another homologous ssDNA molecule, followed by the renaturation of these molecules to form heteroduplex DNA. The first two functions should be important to all in vitro reactions involving recA and recBCD proteins, whereas the third will be specific to the DNA substrates used. The rate-limiting step of heteroduplex DNA formation is the trapping by recA protein of the ssDNA produced by recBCD enzyme. A model for this reaction is described.     

RecA-mediated joint molecule formation between O-methylated RNA/DNA hairpins and single-stranded targets

Chimeric oligonucleotides consisting of one DNA strand paired with an O-methylated RNA strand interrupted by six DNA residues have been used in gene targeting experiments. Here we demonstrate that these hairpins can form a heteroduplex (or joint molecule) with single-stranded DNA targets in a reaction mediated by the E. coli RecA protein. One end of the double hairpin may unwind to form a 14-base-RecA filament which initiates the reaction. Chimeric oligonucleotides containing only O-methylated RNA residues on one strand or truncated hairpins lacking this 14-base segment did not participate in RecA-driven heteroduplex formation under these reaction conditions. The results presented here represent a first step in studying one facet of a strategy which uses O-methylated RNA residues as participants in homologous pairing events. Received: 2 June 1997 / Accepted: 29 September 1997     

Gene conversion in Escherichia coli. Resolution of heteroallelic mismatched nucleotides by co-repair

We have constructed heteroduplex plasmid DNA that is similar in structure to the heteroduplex DNA expected to be produced during genetic recombination of plasmids, and studied its repair after transformation into different Escherichia coli strains. The heteroduplex DNA was constructed using two different parental plasmids, each of which contained a different ten-nucleotide insertion mutation. The effect of different defined states of dam-methylation on repair was also examined. We found that heteroduplex DNA repair occurred prior to the replication of the substrate DNA 60 to 80% of the time, regardless of the state of DNA methylation. Most excision/synthesis tracts covered two markers separated by 1243 base-pairs, and this process has been termed co-repair. The most efficient co-repair pathway was the Dam-instructed repair pathway that required the mutH, mutL, mutS and uvrD gene products and preferentially used the methylated strand as the template for DNA synthesis. If there was no methylation asymmetry, mismatch nucleotide repair occurred with a similar frequency; however, no strand bias was observed. Co-repair of symmetrically methylated heteroduplex DNA required the mutS and uvrD gene products, while repair of unmethylated heteroduplex DNA also required the mutL and mutH gene products.     

An analysis of repeated sequence heterogeneity

High-resolution thermal denaturation was used to measure the heterogeneity within repeated DNA sequences. An analysis of combined denaturation/redenaturation experiments on mouse satellite DNA suggests the existence of two minor components, one of which does not appear in the prepared EcoRII monomer. The resolving power of the denaturation/redenaturation experiment is estimated and contrasted with that of the reassociation experiment, often used to estimate repeated sequence heterogeneity. A mathematical model of the redenaturation experiment was developed and applied to mouse satellite data; the results suggest that only one-fourth of the mismatched base pairs are energetically significant in the reduction of heteroduplex stability.     

Characterization of bacteriophage lambda reverse as an Escherichia coli phage carrying a unique set of host-derived recombination functions

λ reverse (λrev) (Zissler et al., 1971a) is a recombination proficient derivative of a A phage which had lost the phage recombination genes by deletion. In this work, phages with the Rev phenotype have been obtained by a method of selection different from that of Zissler et al. (1971a). Comparison of DNA from two of our isolates and one of Zissler's by electron microscopic heteroduplex mapping shows that all three phages carry substitutions of non-λ DNA which are indistinguishable in extent, location and base sequence. Genetic and biochemical characterization of λrev strongly suggests that the substituted DNA codes for recombination functions different from the λ recombination functions which are deleted. These substituted genes apparently derive from the host chromosome or a prophage, and may be the same as the genes responsible for the SbcA and Rac phenotypes in the host.     

Molecular cloning and characterization of the chicken gene homologous to the transforming gene of rous sarcoma virus

Four molecular clones containing DNA homologous to the Rous sarcoma virus transforming gene (src) have been isolated from a random library of normal chicken DNA. The four clones are distinct overlapping isolates, which together span approximately 33 kb of cellular DNA. The cloned locus appears to represent the major region of chicken DNA homologous to src, since src-containing restriction fragments of this locus account for the fragments detected by hybridization of src-specific probe to restriction digests of total chicken DNA. Analysis of the cloned chicken src locus by restriction and heteroduplex mapping indicates that the locus contains 1.6-1.9 kb of DNA homologous to the viral src gene. The chicken DNA sequences homologous to viral src are interrupted by five or six nonhomologous regions, totaling approximately 6 kb, which presumably represent introns in the cellular src gene.     

Heteroduplex DNA Formation Is Associated with Replication and Recombination in Poxvirus-Infected Cells

Poxviruses are large DNA viruses that replicate in the cytoplasm of infected cells and recombine at high frequencies. Calcium phosphate precipitates were used to cotransfect Shope fibroma virus-infected cells with different DNA substrates and the recombinant products assayed by genetic and biochemical methods. We have shown previously that bacteriophage lambda DNAs can be used as substrates in these experiments and recombinants assayed on Escherichia coli following DNA recovery and in vitro packaging. Using this assay it was observed that 2-3% of the phage recovered from crosses between point mutants retained heteroduplex at at least one of the mutant sites. The reliability of this genetic analysis was confirmed using DNA substrates that permitted the direct detection of heteroduplex molecules by denaturant gel electrophoresis and Southern blotting. It was further noted that heteroduplex formation coincided with the onset of both replication and recombination suggesting that poxviruses, like certain bacteriophage, make no clear biochemical distinction between these three processes. The fraction of heteroduplex molecules peaked about 12-hr postinfection then declined later in the infection. This decline was probably due to DNA replication rather than mismatch repair because, while high levels of induced DNA polymerase persisted beyond the time of maximal heteroduplex recovery, we were unable to detect any type of mismatch repair activity in cytoplasmic extracts. These results suggest that, although heteroduplex molecules are formed during the progress of poxviral infection, gene conversion through mismatch repair probably does not produce most of the recombinants. The significance of these observations are discussed considering some of the unique properties of poxviral biology.     

The relatedness and evolution of repeated nucleotide sequences in the genomes of some gramineae species

Reassociation kinetics of sheared denatured DNAs from wheat, barley, rye, and oats at 60 C in 0.18 cm Na⁺ indicate that approximately 80% of these genomes consist of repeated nucleotide sequences, using hydroxylapatite chromatography to detect reannealed DNA. The remaining DNA appears to consist of sequences present in only one or a few copies per haploid genome. Studies on heterologous duplexes formed in vitro between the repeated sequence DNA fractions from each of the species in turn indicate that many of the families of repeated sequences in these cereals evolved from common ancestral sequences. The extent of heteroduplex formation and duplex thermal stabilities suggest a scheme for the evolution of these species which agrees with taxonomic and genetic evidence. Further analyses of these parameters indicate that many quantitative changes in the chromosomal complement of repeated sequences have occurred during divergence of these species.     

DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

The formation of heteroduplex DNA is a central step in the exchange of DNA sequences via homologous recombination, and in the accurate repair of broken chromosomes via homology-directed repair pathways. In cells, heteroduplex DNA largely arises through the activities of recombination proteins that promote DNA-pairing and annealing reactions. Classes of proteins involved in pairing and annealing include RecA-family DNA-pairing proteins, single-stranded DNA (ssDNA)-binding proteins, recombination mediator proteins, annealing proteins, and nucleases. This review explores the properties of these pairing and annealing proteins, and highlights their roles in complex recombination processes including the double Holliday junction (DhJ) formation, synthesis-dependent strand annealing, and single-strand annealing pathways—DNA transactions that are critical both for genome stability in individual organisms and for the evolution of species.A central step in the process of homologous recombination is the formation of heteroduplex DNA. In this article, heteroduplex DNA is defined as double-stranded DNA that arose from recombination, in which the two strands are derived from different parental DNA molecules or regions. The two strands of the heteroduplex may be fully complementary in sequence, or may contain small regions of noncomplementarity embedded within their otherwise complementary sequences. In either case, Watson-Crick base pairs must stabilize the heteroduplex to the extent that it can exist as free DNA following the dissociation of the recombination proteins that promoted its formation.The ability to form heteroduplex DNA using strands from two different parental DNA molecules lies at the heart of fundamental biological processes that control genome stability in individual organisms, inheritance of genetic information by their progeny, and genetic diversity within the resulting populations (Amunugama and Fishel 2012). During meiosis, the formation of heteroduplex DNA facilitates crossing-over and allelic exchange between homologous chromosomes; this process ensures that progeny are not identical clones of their parents and that sexual reproduction between individuals will result in a genetically diverse population (see Lam and Keeney 2015; Zickler and Kleckner 2015). Heteroduplex DNA generated by meiotic COs also ensures proper segregation of homologous chromosomes, so that each gamete receives a complete but genetically distinct set of chromosomes (Bascom-Slack et al. 1997; Gerton and Hawley 2005). In mitotic cells, heteroduplex DNA formation between sister chromatids is essential for homology-directed repair (HR) of DNA double-strand breaks (DSBs), stalled replication forks, and other lesions (Maher et al. 2011; Amunugama and Fishel 2012; Mehta and Haber 2014). Prokaryotic organisms also generate heteroduplex DNA to perform HR transactions, and to promote genetic exchanges, such as occur during bacterial conjugation (Cox 1999; Thomas and Nielsen 2005).Fundamentally, heteroduplex DNA generation involves the formation of tracts of Watson-Crick base pairs between strands of DNA derived from two different progenitor (parental) DNA molecules. Mechanistically, the DNA transactions giving rise to heteroduplex may involve two, three, or four strands of DNA (Fig. 1). DNA annealing refers to heteroduplex formation from two complementary (or nearly complementary) molecules or regions of single-stranded DNA (ssDNA) (Fig. 1A). DNA annealing may occur spontaneously, but it is promoted in vivo by certain classes of annealing proteins. Three-stranded reactions yielding heteroduplex DNA proceed by a different mechanism referred to as DNA pairing, strand invasion, or strand exchange. These reactions involve the invasion of a duplex DNA molecule by homologous (or nearly homologous) ssDNA. The invading DNA may be completely single stranded, as is often the case in in vitro assays for DNA-pairing activity (Fig. 1B) (Cox and Lehman 1981). Under physiological conditions, however, the invading ssDNA is contained as a single-stranded tail or gap within a duplex (Fig. 1C,D). DNA-pairing reactions are promoted by DNA-pairing proteins of the RecA family (Bianco et al. 1998), and proceed via the formation of D-loop or joint molecule intermediates that contain the heteroduplex DNA (Fig. 1B–D). Three-stranded reactions may also be promoted by exonuclease/annealing protein complexes found in certain viruses. Four-stranded reactions generating heteroduplex DNA involve branch migration of a Holliday junction (Fig. 1D). In practice, a four-stranded reaction must be initiated by a three-stranded pairing reaction catalyzed by a DNA-pairing protein, after which the heteroduplex is extended into duplex regions through the action of the DNA-pairing protein or of an associated DNA helicase/translocase (Das Gupta et al. 1981; Kim et al. 1992; Tsaneva et al. 1992).Open in a separate windowFigure 1.Common DNA annealing and pairing reactions. (A) Simple annealing between two complementary molecules of single-stranded DNA to form a heteroduplex. (B) Three-stranded DNA-pairing reaction of the type used for in vitro assays of RecA-family DNA-pairing proteins. The single-stranded circle is homologous to the linear duplex. Formation of heteroduplex (red strand base-paired to black) requires protein-promoted invasion of the duplex by the ssDNA to form a joint molecule or D-loop (i). The length of the heteroduplex may be extended by branch migration (ii). (C) Three-stranded DNA-pairing reaction of the type used for high-fidelity repair of DNA DSBs in vivo. The invading strand is the ssDNA tail of a resected DSB. The 3′ end of the invading strand is incorporated into the heteroduplex within the D-loop intermediate. (D) Example of a four-stranded DNA-pairing transaction that is initiated by a three-stranded pairing event and extended by branch migration. The ssDNA in a gapped duplex serves as the invading strand to generate a joint molecule (i), reminiscent of the reaction shown in panel B. Protein-directed branch migration may proceed into the duplex region adjacent to the original gap, generating α-structure intermediates (ii), or eventually a complete exchange of strands (iii).     

Optimized PCR fragments for heteroduplex analysis of the whole human mitochondrial genome with denaturing HPLC

Denaturing high pressure liquid chromatography (dHPLC) is an efficient method for discovery of unknown mutations by heteroduplex analysis of PCR fragments. For comprehensive mutation scanning of the whole 16.569 bp human mitochondrial genome, we developed a set of 67 primer pairs defining overlapping PCR fragments that are well suited for heteroduplex analysis. The aim of our optimization efforts was to ensure that point mutations are detectable at every nucleotide position of each amplicon. Some GC-rich regions of mitochondrial DNA (mtDNA) were found to have unfavourable melting profiles in all possible amplicons, therefore requiring GC-clamps at the end of one or both oligonucleotide PCR primers. Following detection of a heteroduplex pattern by dHPLC, our primers can also be employed for DNA sequencing to identify the underlying mutation. In case of heteroplasmic mutations with a low proportion of mutant mtDNA, a fragment collector is useful to recover the heteroduplex peak, which contains mutant and wildtype DNA molecules in a 1:1 ratio.     

Asymmetric repair of bacteriophage T7 heteroduplex DNA

Summary Heteroduplex DNA molecules were prepared in vitro using one strand of DNA carrying a point mutation and one strand of the corresponding wild-type DNA. The heteroduplex DNA was transfected into competent bacteria and the progeny genotypes in the resulting infective centers were determined. From the results were conclude that about 80% of all transfected DNA molecules are repaired before DNA replication starts. This fraction of repaired DNA is independent of the location of the mismatched nucleotide pair. However, mismatch correction occurs preferentially on the H strand of the heteroduplex DNA.The repair does not depend on a known phage coded function but requires the active bacterial genes mut U, mut H, mut S and probably mut L.