Roots: The history of the DNA heteroduplex

possible explanations for a number of other phenom- ena.. .pairing between homologous chromosomes at meiosis may depend on pairing between specific bases. We shall discuss these ideas in detail elsewhere.’ This passage is ambiguous, as everything depends on the meaning of the words ‘specific bases’. Does this imply complementary base pairing, or not? What is clear is that the authors did not publish anything further on the topic of recombination. Perhaps, having looked at the current literature on the subject, they decided to steer clear of the considerable confusion that existed.

In 1954, Levinthal published a paper in Genetics(3) in which he discussed the properties of bacteriophage T2 heterozygotes. (Phage heterozygotes contain wild-type and mutant information from both parents, and the two alleles segregate in progeny from a single virus par- ticle.) In the discussion section he included diagrams of two interpretations based on chromosome structures of the phage genome, and two based on DNA structures. The latter show with complete clarity the heteroduplex overlapping structure (later referred to by Stahl(4) as a ‘splice’) and the insertion heterozygote, where a part of the polynucleotide chain of one parent is replaced by that from the other parent (referred to by Stahl as a ‘patch’). In fact, almost all T2 heterozygotes segregate because they carry terminal redundancies; nevertheless, Levinthal’s insight into the molecular structure of heterozygotes was correct in many contexts (see be-

Another topic which was discussed a great deal in the early days was the problem of unwinding the intertwined Watson and Crick strands during the replication of the genetic material. Platt(’) published an interesting proposal in 1955, in which he considered the special properties of palindromes, or inverted repeats in DNA. He pointed out that they could form side arm loops, and the pulling out of the loops resulted in the untwisting of the linear DNA molecule. Platt therefore suggested that if DNA contained many large palindromes, the problem of the unwinding of DNA during replication would be overcome. His figure to illustrate the structure also shows clearly for the first time the 4-way cruciform junction, which was later shown to be an essential intermediate in recombination.

low).

Introduction Many scientists involved in day to day experimental work are not too concerned about the history of their subject and prefer to get on with the job in hand. Nevertheless, if they are to assess the current situation in their field and to think about future advances, it can be helpful to know how the present position in research was reached. In this article I will discuss the history of ideas in genetic recombination, starting in 1953 when the double helix structure of DNA was revealed by Watson and Crick. Although the account will be accurate, it will not attempt to achieve historical objectivity, because it is primarily a record of my own involvement in the field.

In October 1952, when I went up to Cambridge University to study biological sciences, I was not to know that Watson and Crick were deeply involved in unravelling the structure of DNA. Their famous paper was published in Nature in the spring of 1953(l), and it is well known that it caused considerable excitement, particularly amongst geneticists. Nevertheless, under- graduates in biology and biochemistry were not told about the momentous discovery, which in itself pro- vides significant comment on the Cambridge University teaching system at the time. In my own case, I first heard about the structure in October 1954, about 18 months after its publication. This was in the first of a series of lectures on microbial genetics by Harold Whitehouse in my last undergraduate year. Up to that time I had been interested in genetics but by no means committed to it. In the space of one hour, the principle of genetic replication, the concept of coded information in the sequential base pairs and the likely mechanism of gene mutation were all revealed. That, more than anything else, made me decide that I wanted to special- ize in genetics.

Early Insights It is interesting to look back to those early years and see to what extent genetic recombination was discussed in the light of the DNA structure. Watson and Crick in their second paper in 1953(’), which discussed the genetic implications of the structure described in the first paper, wrote the following: ‘Our model suggests

The Structure of the Gene At that time the gene was considered to be the basic unit of function which could mutate to stable new alleles. Genes were arranged in a linear array along the chromosome, and cross overs occurred between genes, not within them. Exceptions to this were sometimes seen, especially in Drosophilu. For example, from crosses between two non-complementing mutant eye colour alleles, wild-type progeny were occasionally observed. Results such as this gave rise to the concepts of pseudo alleles and complex loci. An elaborate theory was developed which proposed that genes with related functions were clustered or closely linked, and that a

mutation in only one of the genes disrupted the function. This also depended on a cis-trans position effect, because two mutants in clusters on different homologous chromosomes gave a mutant phenotype, and crossing over between them gave a wild-type chromosome and one with two mutations. Pontecorvo realised that in most instances this interpretation was nonsense (several publications reviewed in ref. 6 ) . He understood the special properties of DNA molecules and in particular that mutation should occur at many sites in one functional unit. The Drosophila results and similar data from plants could be explained on this simple basis provided recombination could occur in DNA itself within the gene. By the mid 1950's it was well established that bacteriophage genomes consisted of DNA and that recombination could occur in such genomes. Pontecorvo and his colleagues set out to test this molecular interpretation using the fungus Aspergillus nidulans. Mutants with the same phenotype which did not complement in heterokaryons or diploids should be due to different base changes in one gene. Since the mutants were auxotrophs, rare wild-type recombinants (prototrophs) would be readily selected. The first convincing linear map of the ad8 ene in Aspergillus

Benzer was working on the rII genes of bacteriopha e

and later went on to publish much more detailed analyses of gene structure. The term 'genetic fine structure' was introduced by these studies and the concept of genes and chromosomes as 'beads on a string' became obsolete.

The splitting of the gene had enormous implications for the further study of recombination mechanisms, but this was not appreciated by most geneticists at the time. In fact, complications arose in the experiments by Pritchard and others which were discussed at great length, but later proved to be something of a red herring. In his analysis of the ad8 locus, Pritchard used genetic markers on each side of the gene. He discovered that when prototrophs were selected they were not always associated with recombination of outside markers. Frequently, the prototrophs had parental outside markers, or even the recombined markers expected from a triple cross over. The term negative interference was introduced to describe the situation where one exchange (or cross over) greatly increased the probability of additional exchanges in the same short region of the chromosome. Negative interference was also demonstrated in other organisms, such as Neurospora and bacteriophages.

was published by Pritchard in 1955( 8 . At the same time

T4. He published his first gene maps in the same year 6)

Gene Conversion Many fungi have the special property of producing tetrads or octads after meiosis. The isolation of the products of a single meiotic event made it possible to obtain completely new information about recombination. Lindegren, who had been a pioneer of Neuro-

spora genetics, later realized the great potential of the yeast Saccharornyces as an experimental organism. He analysed tetrads from multifactor crosses and published evidence for gene conversion in 1953(9). A normal Mendelian segregation in a tetrad is 2:2, and gene conversion is an exception to this, as the segregation ratio is 3: 1 or 1:3. (In a fungus with eight spores, the corresponding ratios are 6:2 and 2:6, since the eight spores arise by one further mitotic division after meiosis.) At that time there was some argument amongst yeast geneticists about the interpretation of abnormal segregation ratios, and Lindegren's result was not generally accepted. Gene conversion became respec- table when it was rediscovered in 1955 by Mitchell in Neurospora, an organism with impeccable genetic cre- dentials("). Subsequent studies clearly showed that gene conversion was frequently associated with the crossing over of outside markers. Since gene conversion alone is formally equivalent to a double exchange in one of the members of the tetrad, an associated cross over could easily generate the triple exchange class seen by Pritchard and others. Essentially all the negative interference data could be accommodated in any recombination scheme including gene conversion plus a single cross over.

The first copy choice model was proposed by Leder- berg(") to explain transduction data in bacteria, but it was also used by several fungal geneticists to explain gene conversion and crossing over. The model was based on the conservative replication of DNA in which parental DNA molecules are templates for the synthesis of daughter molecules(12). It was proposed that the synthesis of daughters could switch between templates during pre-meiotic DNA synthesis. Miscopying could produce gene conversion and reciprocal switching would produce crossing over. It is remarkable that the models were so widely discussed, if not accepted, in the late 1950's, since they bore little relationship to the actual structure of DNA and its semi-conservative replication, which had by that time been established in bacteria by the experiments of Meselson and Stahl, and also in the chromosomes of higher organisms by Taylor. The products of replication are not new daughter strands, but half parental and half daughter strands. Another failing of copy choice models was their in- ability to account for the accuracy of recombination. Innumerable studies have demonstrated that recombination was not associated with the gain or loss of genetic information, but could, for example, reconstitute one functional wild-type gene from a cross between two different mutations. In terms of DNA, this meant that the cross over had to be at exactly homologous positions in interacting DNA molecules. There was nothing in copy choice models to suggest how this might occur.

Heteroduplex DNA in Fungi The aim of my PhD project was to study the relationship between fine structure mapping and recombination

in the smut fungus Ustilago maydis. In the event, I was involved mainly in the isolation of mutants and the development of a genetic system. At the end of my statutory three year period of research (1955-58), my supervisor Harold Whitehouse considered that I had enough data for a thesis, although I was disappointed that I had gained no new insights into recombination. Before I left Cambridge I was thinking hard about alternatives to copy choice. It seemed to me that molecular synapsis and accuracy in recombination could be achieved if the process depended on the unravelling of DNA strands and their annealing, by complementary base pairing, with opposite partners. The points where strands exchanged pairing partners could be the sites of breakage and reunion of polynucleotide chains and therefore of whole DNA molecules. If the re-annealed region of DNA crossed a heterozygous site (i.e. where parental molecules differed by one base change), then mismatched bases would be produced. To explain conversion, one had to suppose that the mismatched spaces were corrected in some way to produce wild-type or mutant DNA. Since I knew about phage heterozygotes and DNA overlaps, I remember searching the literature to find out whether any one had suggested that heterozygotes might be eliminated by the correction of mismatched bases, but I found no such discussion. I also went to see Francis Crick who at that time was housed in a temporary building outside the Cavendish Laboratory. I wanted to know if there was any reason why mismatched base pairs might be intrinsically unstable - perhaps there might be unspecified exchange reactions, which could replace one base with another. Although Crick was genuinely encouraging, he also explained to me the intrinsic stability of covalent bonds. With characteristic insight, he also pointed out that it would not be unreasonable to suppose that there might be repair mechanisms which could recognize a mismatch and restore normal base pairing.

My first post-doctoral position was at the John Innes Institute, which at that time (1958) was at Bayfordbury, Hertford. My initial task was to write my PhD thesis, but this took much longer than it should have done, for the simple reason that I was still struggling with recombination mechanisms. During this period I ac- cumulated a pile of drawings or diagrams elaborating models of varying complexity. I built one of the simplest out of paper (Fig. 1) and I remember discussing this with two new colleagues, Peter Day and Len LaCour. The recombination intermediate had no free ends, so could not possibly be generated with strict preservation of right handed helices (see below), but it did demonstrate the symmetry of the 4-way junction. It was also evident that the breakage and reformation of the hydrogen bonds between complementary base pairs would result in the movement of the exchange point along the molecule, provided the helices rotated around their long axis. This was later called branch migration. In the structure in Fig. 1, the two exchange points could

5'

i m t m

3'5' 5'3' Fig. 1. A recombination intermediate, first formulated in 1959, involving the formation of reciprocal regions of hybrid or heteroduplex DNA. If such regions include a heterozygous marker, then mismatched bases will occur. Such mismatches might be repaired, to produce gene conversion, or separate at the next round of DNA synthesis to produce post meiotic segregation, including aberrant 4: 4 s. The arrows indicate points where polynucleotide chains of like polarity could break and rejoin to produce crossing over or non- crossing over of outside markers with equal frequency. The equivalence of the strands at the exchange points can be easily demonstrated with a simple three-dimensional model consisting of a paper cylinder, on the surface of which the DNA is represented by hydrogen-bonded linear polynucleotide chains. With this model (the 1959 original of which still exists), the strands do not 'cross over', as in the Figure shown here, but exchange pairing partners in a completely symmetrical mode (see also ref. 33). The structure shown has been proposed as a recombination intermediate in various contexts. For instance, it can be generated following breakage and reunion of polynucleotide ~ h a i n d ~ ~ . ~ , ~ ~ ) . It could be produced by topoisomerase activity("), or it could be the result of synapsis to form a paranemic joint, with both right and left handed (Z DNA) helical regions, which can sub- sequent1 be stabilized by topoisomerase to form a plectonemic joint(56-&,

move up or down in concert , without any change in the length of the heteroduplex region. When my thesis was fully completed in 1959, it contained no discussion of possible molecular mechanisms of recombination. My external examiner was John Fincham and after the oral examination at lunch I clearly remember explaining to him my ideas about crossing over and gene conversion with diagrams on a table napkin. On this visit to Cambridge and another during the same period, I also discussed these ideas with David Hopwood and Harold Whitehouse.

In 1960 there was an important symposium on microbial genetics organized by the Society for General Microbio1ogy(l3) and held in the imposing amphitheatre of the Royal Institution in London. Major contributors or discussants included Crick, Lederberg, Jacob, Hayes, Pritchard, Catcheside and others leading the field at that time. I had prepared one slide, which

outlined my simple scheme for crossing over and gene conversion, and I hoped to present it in a suitable discussion period. However, I had essentially no experi- ence in speaking before a large audience, let alone one full of distinguished scientists, and my courage failed me. It was particularly unfortunate because Pritchard at that meeting explicitly discussed intragenic recombination and gene conversion and in relation to negative interference and interpreted the results on the basis of the copy choice model (see ref. 13). However, I was soon greatly encouraged when a prediction of the heteroduplex model was confirmed by the first reports of post-meiotic segregation in fungi with 8 spores. It was observed that pairs of spores which should have been genetically identical occasionally consisted of one mutant and one wild-type spore. This would be expected if a DNA heteroduplex with a mismatched base pair was not repaired, but segregated out at the next mitotic division.

In the field of yeast genetics, Herschel Roman had been doing experiments which were closely related to those in Aspergillus. He showed that many different non-complementing alleles could be isolated at distinct genes controlling adenine biosynthesis and he coined the term ‘heteroalleles’(14). Heteroalleles and diploids produced wild-type recombinants spontaneously and at enormously higher frequency when the cells were treated with UV light. I had continued with studies of U. maydis in my post-doctoral work and had developed a mitotic recombination system. Both haploids and diploids produce yeast-like cells, and recombination could be studied in the latter. Thus there were strong connections between the work going on in Roman’s laboratory in the University of Washington, Seattle, and my own, and it therefore seemed appropriate to spend a year as a post-doctoral associate with him, from 1962-63. Before I left for the U S A . I had written up some results on UV-induced mutation and repair in U. maydis, and included in the discussion a brief summary of my model of heteroduplex DNA and ene conver-

year in Seattle there was considerable discussion of recombination mechanisms, and I even did some experiments on gene conversion in yeast. In particular, I wondered if the three mutant alleles in a 1: 3 conversion tetrad were really the same. If they had different base pairs at the same site they might have distinguishable properties, for example, their rate of back mutation to wild-type. Donald Hawthorne provided the tetrads and I did some back mutation experiments, and the totally equivocal results were reported in the Microbial Gen- etics Bulletin. (Later on Fogel and Mortimer(16), using an ingenious genetic test, demonstrated the molecular fidelity of gene conversion in yeast.) With regard to heteroduplex DNA recombination models, I would say that the general idea was treated with interested scepticism, with one exception. Stirling Emerson was spend- ing a few months in the laboratory to give a lecture course on recombination in Neurospora and related

sion by correction of mismatched bases(15 P . During the

topics. He was genuinely interested in my ideas and encouraging in discussions with him. He invited me to visit the California Institute of Technology and give a seminar. This was entirely about Ustilago experiments, not mechanisms of recombination, but nevertheless there was discussion about recombination at other times and I distinctly remember Robert Edgar pointing out the need for enzymes in any recombination process. Geneticists tended to think in formal rather than in biochemical or molecular terms (as the devotees of copy choice models illustrated): DNA was a physical structure which could mutate and recombine, and it was not necessary to worry at this stage about the underlying biochemistry.

In 1963 I returned to Europe in time for the Inter- national Congress of Genetics at The Hague, and I soon met Lewis Frost on the promenade. He had been a demonstrator in botany at Cambridge and therefore a junior colleague of Harold Whitehouse, and he told me that Harold had a brand new model for recombination which explained ‘everything’. Moreover, he had been given a special opportunity at the Congress, arranged at the last minute, to present his ideas. His model, which I thought unnecessarily complicated, was based on the formation of hybrid DNA and incorporated mismatch base repair, but it was primarily designed to explain crossing over rather than gene conversion. A conversion event, without crossing over of outside markers, arose from two reciprocal exchanges. .

The 1964 Model I was extremely put out, to say the least, by White- house’s apparent achievement of priority, and on my return to the John Innes Institute decided to write up in full my own ideas, especially as Nature soon published Whitehouse’s model(17). Amongst the alternative schemes I had at hand, I definitely invoked Occam’s Razor and decided to concentrate on the simplest molecular mechanism I could envisage which might explain all published data. Amongst current studies, the significance of the new organism Ascobolus immersus soon became apparent. This was being used in Rizet’s laboratory at Orsay, and one of the younger members of his group, Jean-Luc Rossignol, subsequently exploited spore colour mutants in a brilliant series of experiments with various colleagues, which provide some of the best experimental evidence for the formation of heteroduplex DNA at meiosis and the correction of mismatched bases. In the early work polarized gene conversion was discovered, and the word ‘polaron’ was coined(’’). It was evident that amongst the series of non-complementing mutants in a gene controlling spore colour, gene conversion was most common at one end of the gene and then decreased in frequency to the other end. Polarity in the behaviour of outside markers had also been demonstrated in Aspergillus and Neuro- spora, in analyses which did not depend on

These studies strongly indicated that

events without crossing over of outside markers, i.e. conversions, were polarized in a given gene. It therefore seemed reasonable to suppose that recombination was initiated at preferred sites and that hybrid or heteroduplex DNA extended from that site for a variable distance. The closer a mutant was to the initiation site the greater the chance of gene conversion by mismatch repair. The main problem in formulating a realistic heteroduplex model, was the awkward and undisputed fact that a series of mutant alleles could be arranged in a linear array - a fine structure map. The problem was as follows: if two mutant sites are close together then they are both likely to be included in a single stretch of heteroduplex DNA. By invoking random repair of the two mismatched base pairs (random with respect to which base of each mismatch is corrected), it follows that wild-type genes should be produced, at least in 25 % of cases. Thus mutants close together should recombine frequently - perhaps almost as frequently - as those further apart in the gene. This difficulty was probably the main reason for not writing up my model a few years earlier than I did. In 1963 after The Hague Congress I grappled with the problem. I went to see Crick again to ask him if he thought that mismatched base pairs might interfere with the formation of heteroduplex DNA, especially if they were close together. Again he was generally encouraging about my ideas, but professed his ignorance of the problem of genetic recombination. I also scanned the literature to search for anomalies in fine structure maps. In this I was partly successful. It was certainly well established that fine structure maps were linear, but there was a common discrepancy in the additivity of recombination frequencies. I had come across the phenomenon of ‘map expansion’ in published maps and also in some unpublished ones in Schizosaccharomyces which I had obtained from Urs Leupold. If the length of the map is defined by the sum of the smallest intervals, then mutant sites several intervals apart gave a significantly greater frequency of recombination greater than the sum of all the intervening sites. This seemed to be an effect of markers on the recombination frequency and I seized upon it in my paper. Fine structure maps and conversion by mismatch repair could be reconciled if mismatches close together did indeed inhibit the formation of heteroduplex DNA and this inhibition disap- peared as the distance between the mutant sites increased. Much of my final manuscript was therefore devoted to a discussion of map expansion and marker effects on recombination.

Since Whitehouse had published his model in Nature(”), and had not referred to my earlier published idea of mismatch correction and gene conversion(15), I submitted my manuscript to the same journal. It was rather long, but as I pointed out to the Editor, Nature had from time to time published articles of greater length. I received a polite reply saying that the manuscript could be accepted, but only if it was cut to a third or half its length. However, I felt that the discussion of

map expansion was essential to the argument, so I did not take up the offer. I sent it instead to Genetics, as I knew that the editor David Perkins (whom I had met earlier in the same year) was interested in recombination mechanisms. The manuscript was again returned with the comment that it was not the editorial policy of Genetics to publish theoretical papers. This made little sense as Genetics frequently published papers on popu- lation genetics or quantitative genetics. I therefore sent my manuscript to Genetical Research, which had previously Published my Ustilago papers, and it was accepted 21). In the interim, I unexpectedly received a kind letter from Max Delbruck offering to publish my paper in Molecular and General Genetics (MGG), but by that time it was already in press. Delbruck was one of the new editors in the conversion of the Zeitschriftfiir Vererbungslehre, the oldest genetical journal, into its new international format and title. Perkins must have liked my ideas on recombination, because he either told Delbruck about them or perhaps showed him a copy of my manuscript. In his letter Delbruck suggested I should include discussion of the recently discovered excision repair of pyrimidine dimers induced by UV light. I was aware of these two landmark papers published by Setlow and Carrier(22) and Boyce and Howard Flanders(”) in 1964, but had not recognized their direct relevance to the repair of mismatched bases. This was made much clearer to me soon after- wards, when there was an International Congress of Botany at Edinburgh and I was invited to contribute to a symposium on Genetic Recombination. Pritchard summed up the symposium and in doing so he pointed out that the repair of mismatched bases might be similar to the excision repair of pyrimidine dimers. The re- moval of a mismatched base might involve the formation of a single-stranded gap which would be filled by repair synthesis. Thus, if the initial incision step was slow (which the existence of post-meiotic segregation indicated) and the other events fast, then two mismatched bases close together would be repaired by one event, and one or other parental genotype would be produced. This was later called co-conversion. At a stroke, Pritchard’s suggestion removed the problem of reconciling fine structure maps with mismatch correction, because mutants close together would not generate wild-type progeny, unless the region of heteroduplex DNA ended between them.

In 1967, I was invited to contribute to an international symposium on The Replication and Recombination of Genetic Material held at Canberra, Australia. In my contribution, I discussed for the first time the likely role of DNases or other enzymes in recombination. I also re- interpreted map expansion in relation to the excision repair of mismatched bases(24). It was evident that non- additivity due to map expansion could be explained if the excision repair track was always at least of a certain length, whereas sites a little further apart might be repaired independently. Maps of suppressor tRNA loci in Schizosaccharomyces did not show map expansion,

and this suggested that the minimum length of the excision repair track was about 100 nucleotides, which was in agreement with another indirect estimate. I initially thought that the 'Pritchard model' eliminated the need to invoke marker effects to explain map expansion, but in the discussion after my paper, Mesel- son made it clear, with a simple blackboard equation, that map expansion must necessarily be due to a marker effect, whatever the underlying molecular mechanism. Later on, John Fincham and I extended the quantitative analysis of map expansion, based on the excision repair model, and we published a paper about it(25).

1964 was a vintage year for recombination studies. I have mentioned the discovery of excision repair. This soon led to the elucidation of another repair pathway which depended on recombination between daughter DNA molecules(26). This work would not have been done if mutants defective in recombination had not previously been isolated in E. coli. Clark and Margulies were characterizing the first recA mutants in 1964 and the results were published in 1965(27). The connections between recombination and repair were at that time becoming increasingly obvious, and in 1964 I set out to find repair deficient strains of U. maydis, using a simple replicating technique. They proved to be very easy to obtain and the first report of the isolation of radiation- sensitive mutants in any eukaryotic organism was published in 1965(28). Two of these mutants, later renamed recl and rec2, proved to be severely abnormal in mitotic recombination and m e i ~ s i s ( ~ ~ - ~ ~ ) . 1964 also marked the publication of the definitive proof by Meselson that recombination could occur by breakage and reunion of DNA molecules(32). He used density-labelled A DNA and showed that recombinants could consist of DNA inherited from both parents in the absence of genome replication. This experimental system was later exploited in much more detailed analyses of A recombination by Stahl and his colleague^(^).

Structure of the 4-way Junction The underlying symmetry of the strand exchange point in the 1964 model was evident from the beginning, although not explicit1 illustrated. This was done later by Stirling Emersod3 ). The first accurate atomic model was built by Sigal and A l b e r t ~ ( ~ ~ ) , and this was an important advance, because it was clear that normal hydrogen bonding between bases could be preserved on either side of the exchange point. The model also demonstrated that the exchange point could diffuse up and down by the process of branch migration with maintenance of hydrogen bonding. At the Mill Hill MRC Institute, a new Genetics Division was being set up and when a PhD student, Penny Jeggo, arrived before any laboratory space was available, I suggested to her supervisor, Geoff Banks, that they build a recombination intermediate using the Dore atomic models which were available at the Institute. The model

Y

also showed that complete hydrogen bonding could be preserved across the exchange point, but this conclusion was not published, because the paper by Sigal and Alberts appeared soon after the Mill Hill model was assembled.

Sigal and Alberts stated in their paper that the strands which exchange pairing partners on the inside of the structure could easily swap places with those on the outside and vice versa. The legend to the appropriate figure reads: 'Molecular models demonstrate that by simple rotation of helical sections (see text), the original pair of outside strands in the exchange become bridging strands, while simultaneously the original pair of bridging strands become outside strands.' This turned out to be incorrect. The X-ray crystallographer Henry Sobell had become interested in recombination and he proposed a new model based in part on the bacteriophage T4 branched recombination structures seen by electron microscopy(35336). In the model, the initiation sites for a recombination were small palindromes in DNA, but the essential intermediate was the recombination structure shown in Fig. 1. (Because this was discussed at length by Sobell at various meetings, it is still sometimes referred to as the 'Sobell bubble'.) Sobell also built atomic models of the cross over structure with parallel arms to ether with the cruciform with arms separated by 90"' ). He coined the word isomerization for the switching of the arms on the inside to the outside, and vice versa, in the parallel structure. This involved movement of the arms first through 180" to form the cruciform and then another 180" rotation (Fig. 2) . There was subsequently much discussion of the role of isomerization in recombination (see below).

The Sigal-Alberts parallel structure was generally accepted, but recently controversy has arisen about the proposal that the true structure is anti-parallel. Instead of all the strands extending from the top to the bottom, two of them are turned through a sharp angle (approxi- mately 180") at the exchange point (see Fig. 2). Evi- dence that the anti-parallel structure might exist came from studies with immobile 4-way junctions, in which branch migration cannot occur because no pairs of arms have homologous Anti-parallel structures can isomerize more easily than the Sigal-Alberts structure, but since they lack homology they are arti- ficial constructs, possibly of little relevance to real recombination intermediates. In particular, it is easy to see that branch migration would not readily occur in any anti-parallel structure between homologous DNA molecules. Instead of bases merely transferring from one base pairing position to the other in a horizontal plane, the bases would have to traverse the kink and flip over through a 180". Moreover, this would be happen- ing in opposite directions at the same time in the two strands at the anti-parallel exchange point. It is therefore hard to see how the structure could be preserved during branch migration. It is much more likely that it would adopt the cruciform configuration, where the central 'hole' demonstrated by Sobell's would

6

A D

A B 5' 3' 3 5'

3' 5' 5'3' C D II

D C 5' 3' 3' 5'

3' 5' 5'3' A B

Fig. 2. The two isomeric forms of the Sigal and Alberts structure("' are shown left and right. S~belI(~') demonstrated that the isomerization reaction requires 180" rotation of arms to form a cruciform intermediate, followed by a further 180" rotation. When the double helices are homologous, all these structures can branch migrate by the breaking and reforming of hydrogen bonds. It has been proposed that immobile junctions which cannot branch migrate(38' may form the structures shown at the top and bottom("'. Isomerization of these involves simple opening or closing of arms. It is unlikely that these structures are formed during recombination between homologous DNA molecules, because they are essentially static configurations, which lack the mobility required for branch migration of recombination intermediates (see Text).

not impede base movement, or perhaps a similar symmetrical structure where the four arms extend to the corners of a tetrahedron.

Possible recombination intermediates had been visu- alized b electron microscopy in linear and circular

structures seen really represented the exchange of DNA strands at points of homology. More convincing studies utilized circular plasmids which could be cut at just one site with a restriction enzyme. A figure of eight recombination intermediate was thereby converted to a four-armed intermediate (Fig. 3), referred to by Potter and Dressler as a x structure, in which each pair of arms is of equal length(42). Moreover, partial denaturation of the arms demonstrates single-stranded regions at identical places in the pairs of arms, as well as opening out the cruciform to produce a visible hole in the centre of the x. Potter and Dressler interpreted their EM structures on the basis of the 1964 model and this certainly helped persuade many that it had underlying reality.

phaged4 a: 41). However, it was never certain that the

Fig. 3. Recombination between circular plasmids produces figure 8 structures which are cut with a restriction enzyme specific for one site in each circle. This generates a x form (Potter and Dressler, ref. 42), and the one shown here has been prepared for the electron microscope in the presence of a high concentration of formamide. Under these conditions denaturation to single stranded regions occurs, as can be seen at the centre of the cruciform structure where a space is bounded by single strand DNA. As expected from the DNA model, the homologous arms are opposite each other. (Photograph courtesy of Dr. Huntington Potter.)

EMBO Workshops on Recombination Studies of genetic recombination were greatly stimu- lated by a series of workshops supported by EMBO. The first was held at the Villa Serbelloni, Bellagio, in 1971 and the number of participants was limited to about two dozen. Nevertheless, a start was made in bringing together those working on recombination in fungi or other eukaryotes and those who used bacteriophage or bacteria, and this tradition was maintained in all subsequent workshops. The second workshop was held at Aviemore, Scotland in 1973 and seven further workshops at the village of Nethybridge nearby, from 1975 to 1988, organized primarily by Neville Symonds. During this period many detailed studies of crossing over, gene conversion and post-meiotic segregation were carried out, particularly in Ascobolus and yeast. Co-conversion had been established by tetrad analysis, and strong evidence was obtained that genetic markers (mutants) differed in their pattern of conversion or post-meiotic segregation. There was much discussion of reciprocal versus non-reciprocal events, and whether gene conversion could arise by mechanisms other than correction of mismatched bases. A notable landmark was the formulation of a model during the course of the second workshop by Meselson and Radding, sometimes referred to as the Aviemore model, which incorporated known biochemical features of recombination in pro- karyotes and was published two years l a t e ~ - ( ~ ~ ) . It was proposed that the initial recombination event was non- reciprocal and that isomerization of DNA could produce reciprocal regions of heteroduplex DNA (Fig. 4).

H rc.ch

1 H

rcL.h

n 1 H

Fig. 4. The model for recombination devised by Meselson and R a d d i ~ ~ g ‘ ~ ~ ) during the course of an EMBO Workshop on Recombi- nation at Aviemore, Scotland, in 1973. Recombination is initiated (top left) by a single strand break and a strand is displaced by a DNA polymerase. This strand pairs with its complementary sequence in another molecule and a second single strand break is induced in the recipient. Concerted strand displacement and assimilation are driven by the combined action of a polymerase and exonuclease, a process called asymmetric strand transfer. The structure can isomerize (see Fig. 2) to that shown bottom left, bringing the single strand ends (3’ and 5 ’ ) into juxtaposition. The exchange point can move right (bottom) or left by rotary diffusion (branch migration). This generates reciprocal or symmetric regions of hybrid DNA. The action of endonuclease and ligase can resolve the recombination intermediate to form products with asymmetric and symmetric regions of heteroduplex DNA (H), with either parental or recombinant flanking DNA.

There was much agonizing during this period in trying to explain tetrad data in terms of particular molecular models. It seemed to me that discussants nearly always thought about recombination in terms of static models, where heteroduplex DNA, whether reciprocal or non- reciprocal, had fixed position and dimensions. In fact, branch migration allows for a more dynamic recombination intermediate which can be found at one location and subsequently move and be resolved at another(u). In this way one can explain tetrads, for example, where a gene conversion is separated from a classical cross over by a marker with normal Mendelian segregation. Models with fixed regions of heteroduplex DNA have some difficulty in explaining such tetrads.

Models discussed during the period of the EMBO Workshops had many common features, and differed primarily in the mode of initiation of the events. Initiation was by reciprocal strand exchange in my 1964

model, by single strand invasion in the Aviemore model and by double strand break in the later model of Szostak et The latter also proposed that gene conversion could occur by filling in a gap extended from the double strand break. There are many reasons for believing that gap filling cannot be a primary explanation of gene conversion at meiosis (reviewed in ref. 46), but this does not invalidate the suggestion that a double strand break is the initiating event in recombination, and that heteroduplex DNA extends from there in one or both directions. The repair of double strand breaks in DNA has been known for many years in yeast, and also in organisms such as U. maydis and Deinococcus radiodurans, which are particularly resist- ant to ionizing radiation. An earlier model had proposed that this type of repair depended on recombination with an intact sister chromatid(47).

Recombination Enzymes As I mentioned, geneticists thinking about recombination in the early days did not concern themselves about the enzymes and proteins that might be required. This changed with the isolation of mutants that had strong effects on recombination. RecA mutants of E. coli abolish several recombination pathways involving homologous DNA. It took some time for the wild-type protein to be identified and purified, but subsequently large amounts could be obtained and the protein was shown to be capable of a variety of synapsis reactions in the presence of ATP (reviewed in ref. 48). These included the formation of the cruciform structure from appropriate substrated4’). Another breakthrough was the discovery that enz mes exist which can specifically cleave such structures6’). The integration of bacteriophage A also involves the formation of a 4-way junction, and this reaction, together with the excision of A are probably understood in more detail than any others in recombination (reviewed in ref. 51). There were also surprises in the study of other enzymes which act on DNA. Topoisomerases can cut, untwist and rejoin DNA molecules. As first pointed out by Champoux(’2), this makes it possible to generate recombination intermediates such as that in Fig. 1 without the formation of free ends. It had also been proposed that DNA might form 4-stranded helices(53), and this also makes it possible to envisage the formation of recombination intermediates without breakage of polynucleotide chains(54). Another surprise has been the discovery that alternating purine and pyrimidine strands of DNA could adopt a left-handed Z-DNA structure(55). This raises the possibility that the initial synapsis of DNA could be between intact molecules. The experimental evidence for this has come mainly from studies of the recl protein of U. maydis, which catalyses the synapsis of homologous DNA molecules (reviewed in ref. 56). The protein takes its name from the r e d mutants of U. maydis, which have pleiotropic effects on the genetic system of the organism(30), including aberrant mitotic

recombination, and lack recl protein activity. Recl protein was found to bind more tightly to the Z form of DNA than the right-handed B form, which suggests that Z-DNA might be an intermediate in the synapsis reaction. This conclusion has been supported by the finding that antibody to Z-DNA combines with synapsed molecules. The results suggested that a paranemic joint is an intermediate in recombination. Such a structure has equal numbers of left- and right-handed twists in DNA, and it can be formed and pulled apart without any intertwining of DNA strands. The paranemic joint can be converted to a plectonemic joint by the combined activity of recl protein and a topoisomerase, without any formation of free ends of DNA(56). (A plectonemic joint has only right-hand helices with intertwined strands.) The plectonemic bubble with two regions of heteroduplex shown in Fig. 1 can be resolved to a cross over or a non-cross over by the enzyme which cuts 4-way junctions. The structure has been invoked as a recombination intermediate in so many different contexts(35’44’45’52,54,56-58), that one almost begins to believe in its reality.

Recombination Models The process of recombination has always been an intellectual challenge to geneticists, and probably as many different models as there are years since 1953 have been proposed and published. Many of these were designed to explain a particular set of data, so did not have general application; others were supposed to be of general validity but simply ignored data which was not explained by the model in question. This breaks a cardinal rule of model building: a model should be well adapted to the environment of published data. If it cannot explain such data, it is, in effect, selected against and plays no serious part in further discussion. Or, as the environment changes - by the publication of new data - a model may become better adapted, by modifi- cation or revision, so that it still survives.

A new recombination model should make it possible to think about data in a new way and, of course, suggest new experiments. However, it is certainly not true that scientists have open minds, and welcome new ideas with enthusiasm. Nor do they have closed minds; instead they usually view novel interpretations with suspicion, scepticism or caution. Model builders are often guilty of excessive propaganda for their new proposal and are disappointed when their presentations at conferences are met with criticism or silence, rather than enthusiastic acceptance. What is important is to publish the model in a reputable journal, which need not be a prestigious one, and let future research workers assess its validity. In the case of the paper describing the 1964 model, it was cited infrequently for about 12 years, and thereafter with increasing frequency. As I mentioned, it was the actual visualization of cruciform structure, and other molecular studies carried out during the same period, which began to

persuade geneticists that this recombination intermediate might be real.

Acknowledgements This article is based on a lecture at the FASEB Conference on Genetic Recombination and Genome Re- arrangements, Copper Mountain, Colorado, July 9th-14th 1989. I would like to thank the organisers John Wilson and Richard Kolodner for the invitation to present this lecture on the 25th anniversary of the publication of my model for recombination in Genetical Research. I would also like to thank James German, who invited me to deliver the 6th L. C. Dunn lecture with the same title at the New York Blood Centre on October 7th 1986.

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45 SZOSTAK, J. w. , ORR-WEAVER, T. L. , ROTHSTEIN, R. B. A N D STAHL, F. w.

International Union of Biochemistry Fellowships

for attendance at

THE 15TH INTERNATIONAL CONGRESS OF BIOCHEMISTRY

Jerusalem, Israel, August 4-9, 1991

The International Union of Biochemistry and the Organizing Committee of the 15th IUB Congress will, together, make available fellowship awards to younger biochemists who wish to attend the Congress. Preference will be given to residents of countries where biochemical research is in the early stages of development.

Fellowships will provide partial support of travel (normally up to a maximum of one-half of the economy air fare or 70 % of the Apex fare, whichever is lower and subsistence during the Congress.) The Organizing Committee will waive the registration fee for all Fellows.

Application forms can be obtained from Dr. R. L. Hill, Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 U.S. A. Completed applications should be received as early as possible and no later than August 25, 1990. It is anticipated that decisions will be made by December 1, 1990.

Documents

Roots: The history of the DNA heteroduplex