Proc. Nat. Acad. Sci. USA

Vol. 72, No. 1, pp. 279-283, January 1975

A Mechanism to Activate Branch Migration Between Homologous DNA Molecules in Genetic Recombination (synapsis/unwinding protein/protein-nucleic acid symmetry/gene conversion)

HENRY M. SOBELL Department of Chemistry, The University of Rochester, River Campus Station, Rochester, New York 14627, and Department of Radiation Biology and Biophysics, The University of Rochester, School of Medicine and Dentistry, Rochester, New York 14620

Communicated by Allan Campbell, October 29, 1974 ABSTRACT A mechanism to activate branch migration between homologous DNA molecules is described that leads to synapsis in genetic recombination. The model involves a restriction-like endonucleolytic enzyme that first nicks DNA (to produce single-strand breaks) on strands of opposite polarity at symmetrically arranged nucleotide sequences (located at ends of genes or operons). This is followed by local denaturation of the region, promoted by a single-strand-specific DNA binding protein (i.e., an unwinding protein). Hydrogen-bonding between homologous DNA molecules can then be initiated and this allows for subsequent propagation of hybrid DNA in the pathway to formation of the synapton structure.

Several years ago, I outlined a mechanism for genetic recombination that was specifically addressed to explaining the eukaryotic meiotic gene conversion data and the associated correlation of crossing-over (1-3). The model provides a working framework to understand both polarity in the conversion process and crossing-over within the gene, and can account for the scarcity of two-strand double exchanges in the yeast unselected tetrad data (4, 5). In addition, I have found the model useful in understanding much of the viral recombination data (6-10) and certain aspects of the prokaryotic data (11, 12). A fundamental feature of the model is the formation of a synaptic structure at the ends of genes or operons that can migrate into the structural genome and be resolved by a series of endo- and exonucleolytic steps to effect genetic recombination. This synaptic structure (denoted a "synapton") consists of two Holliday-type crossed strand exchanges and is formed by the action of polynucleotide ligase on a Broker-Lehman type structural intermediate. First this latter structure arises by endonucleolytic nicking of branch migratable junctions (postulated to be at ends of genes or operons) on strands of opposite polarity at or near the same point on homologous DNA molecules. These junctions can next be opened up to initiate hydrogen-bonding between homologous DNA molecules and allow subsequent propagation of hybrid DNA through branch migration. In many respects, my model synthesizes key concepts in the Broker-Lehman and Holliday models for genetic recombination (8, 13, 14), while allowing for enzyme-catalyzed branch migration of the kind envisioned by Radding (15). The latter process results in the asymmetric formation of hybrid DNA in recombinant chromatids, and it is this feature that can explain the scarcity of two-strand double exchanges in the yeast data (for further discussion, see ref. 5). A similar mechanism may operate in fl viral recombination to result in a bias with respect to the type of parent and recombinant obtained from a single recombinational event. This has been discussed by Zinder and his colleagues

(6, 7). 279

The purpose of this paper is to discuss in further detail a possible mechanism to activate branch migration between homologous DNA molecules that leads to synapsis in genetic recombination. The model involves a restriction-like endonucleolytic enzyme that first nicks DNA (to produce singlestrand breaks) on strands of opposite polarity at symmetrically arranged nucleotide sequences (located at ends of genes or operons). This is followed by local denaturation of the region, promoted by a single-strand-specific DNA binding protein (i.e., an unwinding protein) (16-18). Hydrogen-bonding between homologous DNA molecules can then be initiated, and this allows for the subsequent propagation of hybrid DNA in the pathway to formation of the synapton structure. The mechanism presented here differs in detail from the one I proposed earlier to initiate genetic recombination; however, all other features of my genetic recombination model remain unchanged. The role of a restriction-like endonuclease and a DNA unwinding protein in initiating eukaryotic meiotic recombination: The activation of branch migration and synapsis between homologous DNA molecules at ends of genes or operons

Basic ideas are presented in Figs. 1, 2, and 3. One can envision three types of sequences (each-possessing varying degrees of symmetry) capable of initiating branch migration and synapsis with homologous DNA molecules. The first (shown in Fig. lA) possesses exact 2-fold symmetry. A sequence such as this can be envisioned to extend thousands of base-pairs in either direction and may have evolved in eukaryotes as regions containing silent information that are expressly reserved for synapsis (2) (one must postulate, however, an additional presynaptic alignment step to avoid the pairing ambiguity introduced by a sequence with exact symmetry). This sequence contains within it a symmetric site (shown in the stippled boxes) that can be recognized by a restriction-like endonuclease to nick either DNA chain (shown by the arrows). Once a nick is introduced into the double helix, an instability in the base-paired region exists and this can be amplified by binding of a single-strand-specific DNA binding protein to produce further denaturation in the region (Fig. 1B). A protein such as this can promote both the denaturation and the renaturation of DNA (16). Since the sequence possesses exact 2-fold symmetry, a partially opened hairpin structure can form and this can then elongate through the process of branch migration (Fig. 1 C and D). A similar mechanism can be envisioned for a sequence that contains blocks of exact symmetry, separated by a central region with less exact symmetry [a sequence such as this could correspond, for example, to control regions of DNA

280

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Proc. Nat. Acad. Sci. USA 72 (1975)

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FIG. 1. A schematic illustration of a mechanism to initiate branch migration between homologous DNA molecules in genetic recombination. (A) A restriction-like endonucleolytic enzyme first nicks DNA (to produce single-strand breaks) on strands of opposite polarity at symmetrically arranged nucleotide sequences (shown in the stippled boxes). (B) This results in an instability in the base-paired region that can be amplified by binding a single-strand-specific DNA binding protein (i.e., an unwinding protein) to produce further denaturation in the region. (C) Sequences possessing exact 2-fold symmetry can then "snap back" to form partially open hairpin structures. (D). These structures can elongate by branch migration. Simultaneously, denatured regions can initiate hydrogen-bonding between homologous DNA molecules. (E) Further branch migration leads to the Broker-Lehman type central structural intermediate; this structure can then undergo closure by polynucleotide ligase to form one of two types of synapton structures. See text for further discussion.

found in prokaryotes and viruses; the symmetry in its sequence may reflect the general utilization of symmetry in protein-nucleic acid interaction (refs. 19 and 20; for general reference, see ref. 2) ]. Refer to Fig. 2. DNA is first nicked and then destabilized by the unwinding protein (Fig. 2A and B). Denaturation proceeds until symmetric regions "snap back" into a partially opened looped structure (Fig. 2C). The stems on this structure can then elongate by branch migration (Fig. 2D). Finally, in Fig. 3, we consider a sequence that has isolated patches of symmetric sequences in addition to the sequence required for endonucleolytic attack. Here, a similar mechanism can be envisioned that leads to substantial denaturation without hairpin or loop formation.

Each of these mechanisms leads to regions of denatured DNA capable of initiating hydrogen-bonding between homologous DNA molecules. These hybrid DNA structures can then undergo further branch migration to form the central structural intermediate in the synapsis pathway (shown in Figs. 1E, 2E, and 3D). In the presence of polynucleotide ligase, this structural intermediate can then be converted (through closure) into one of two types of synapton structures. Depending on the type of synapton structures formed, a gene conversion event may or may not be associated with the exchange flanking markers (3). Polarity in gene conversion can reflect the formation of synapton structures at the ends of genes or ope-rons that arise by any of the three mechanisms described.

Initiation of Genetic Recombination

Proc. Nat. Acad. Sci. USA 72 (1976)

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FIG. 2. The mechanism illustrated is similar to that in Fig. 1, except that the sequence contains blocks of exact 2-fold symmetry separated by a central region with minimal symmetry. This forms a partially opened looped structure that can elongate by branch migration. See text for discussion. A role for x-rays and a DNA unwinding protein in stimulating eukaryotic mitotic recombination: The enhancement of single-strand DNA breakage randomly in the genome and the initiation of branch migration between homologous DNA molecules from within the gene?

It has been known for some time that mitotic recombination (both spontaneous and x-ray induced) and meiotic recombination share several similar features. One of these is the occurrence of gene conversion events that span large segments of the gene to give rise to coincident conversion of adjacent alleles in multifactor crosses (ref. 21, S. Fogel, personal communication). Another feature concerns the correlation of crossing-over of flanking markers with meiotic and mitotic gene conversion events; meiotic gene conversion events have been found to be accompanied by crossing-over about 50% of of the time (4), while mitotic gene conversion events are accompanied about 30% of the time by crossing-over (ref. 22, S. Fogel, personal communication). There are data, however,

that have suggested a fundamental difference between mitotic and meiotic recombination. Polarity in gene conversion has been a feature that has been constantly found in the meiotic unselected tetrad studies (see, for example, ref. 4), whereas polarity for many of these same alleles has been found to be absent or significantly reduced in spontaneous and x-ray induced mitotic recombination studies (S. Fogel, personal communication). A possible explanation is as follows. Spontaneous and x-ray induced mitotic recombination may both be processes that begin with radiation-induced single-strand breaks in DNA. In contrast to the situation in meiosis, however, these breaks occur randomly in the genome and (in the presence of x-rays) with much higher frequency. When nicks in homologous DNA molecules occur on opposite strands in neighboring regions (i.e., perhaps 5 to 50 base-pairs apart), denaturation, followed by hydrogen-bonding and branch migration between DNA molecules, can take place (akin to that shown in Fig. 3).

282

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Proc. Nat. Acad. Sci. USA 72 (1975)

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As in meiosis, two types of synaptons can form (these can give rise to either the parental or recombinant configuration of flanking markers); however, these structures now arise randomly throughout the genome. Depending on their origin with respect to the particular alleles examined, two types of gene conversion events can occur: those that show "right to left" polarity (these would be due to synaptons originating to the right of the heteroalleles), and those that show "left to right" polarity (these would be due to synaptons originating to the left of the heteroalleles). Since both types of events would be expected to arise at roughly equal frequency, one would expect to find a net lack of polarity (as detected by two-point allelic crosses) in gene conversion. This work has been supported in part by grants from the National Institutes of Health, the American Cancer Society, and the Atomic Energy Commission. This paper has been assigned Report no. UR 3490-668 at the Atomic Energy Project, the University of Rochester. 1. Sobell, H. M. (1972) "Molecular mechanism for genetic recombination," Proc. Nat. Acad. Sci. USA 69, 2483-2487.

2. Sobell, H. M. (1973) "Symmetry, in protein-nucleic acid interaction and its genetic implications," Advances in Genetics, ed. Caspari, E. (Academic Press, Inc., New York), pp. 411-490. 3. Sobell, H. M. (1974) "Concerning the stereochemistry of strand equivalence in genetic recombination," in Mechanisms in Genetic Recombination, Oak Ridge Symposium held at Gatlinburg, Tennessee, April 1-4, 1974 (Plenum Publishing Co., New York), pp. 433-438. 4. Fogel, S., Hurst, D. D. & Mortimer, R. K. (1971) "Gene conversion in unselected tetrads from multipoint crosses," Stadler Genet. Symp. 1st and 2nd, pp. 89-110. 5. Fogel, S. & Mortimer, R. K. (1974) "Gene conversion and mismatched base repair in hybrid DNA," Genetics 77, Suppl. 1974, no. 1/part 2, s22. 6. Boon, T. & Zinder, N. D. (1971) "Genotypes produced by individual recombination events involving bacteriophage f1," J. Mol. Biol. 58, 133-151. 7. Hartman, N. & Zinder, N. D. (1974) "The effect of B specific restriction and modification of DNA on linkage relationships in fi bacteriophage. II. Evidence for a heteroduplex intermediate in ft recombination," J. Mol. Biol. 85, 357369.

Proc. Nat. Acad. Sci. USA 72 (1975) 8. Broker, T. R. & Lehman, I. R. (197i) "Branched DNA molecules: Intermediates in T4 recombination," J. Mol. Biol. 60, 131-149. 9. Benbow, R. M. (1974) "Exchange of parental DNA during genetic recombination in bacteriophage 4X174," in Mechanisms in Genetic Recombination, Oak Ridge Symposium held at Gatlinburg, Tennessee, April 1-4, 1974 (Plenum Publishing Co., New York), pp. 3-18. 10. Doniger, J., Warner, R. C. & Tessman; I. (1973) "Role of circular dimer DNA in the primary recombination mechanism of bacteriophage S13," Nature New Biol. 242, 9-12. 11. Meselson, M. (1967) "Reciprocal recombination in prophage lambda," J. Cell. Physiol. 70, Suppl. 1, 113-118. 12. Herman, R. K. (1965) "Reciprocal recombination of chromosome and F-merogenote in Escherichia coli," J. Bacteriol. 90, 1664-1668. 13. Holliday, R. (1964) "A mechanism for gene conversion in fungi," Genet. Res. 5, 282-304. 14. Holliday, R. (1968) "Genetic recombination in fungi," in Replication and Recombination of Genetic Material, eds. Peacock, E. J. & Brock, R. D. (Australian Academy of Sciences, Canberra), pp. 157-174.

Initiation of Genetic Recombination 15. Radding, C. M. 16.

17. 18.

19. 20. 21.

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(1973) "Molecular mechanisms in genetic recombination," Annu. Rev. Genet. 7, 87-111. Alberts, B. M. & Fry, L. (1971) "T4 bacteriophage gene 32: A structural protein in the replication and recombination of DNA," Nature 227, 1313-1318. Hotta, Y. & Stern, H. (1971) "Meiotic protein in spermatocytes of mammals," Nature New Biol. 234, 83-86. Sigal, N., Delius, H., Kornberg, T., Gefter, M. L. & Alberts, B. M. (1972) "A DNA-unwinding protein isolated from Escherichia coli: Its interaction with DNA and with DNA polymerases," Proc. Nat. Acad. Sci. USA 69, 35373541. Gilbert, W. & Maxam, A. (1973) "The nucleotide sequence of -the lac operator," Proc. Nat. Acad. Sci. USA 70, 35813584. Maniatis, T., Ptashne, M., Barrel, B. G. & Donelson, J. (1974) "Sequence of a repressor-binding site in the DNA of bacteriophage X," Nature 250, 394-397. Fogel, S. & Mortimer, R. K. (1971) "Recombination in yeast," Annu. Rev. Genet. 5, 219-236. Wildenberg, J. (1969) "Gene conversion in synchronous yeast cell pedigrees," Ph.D. Thesis, Department of Biology, The City University of New York.

A mechanism to activate branch migration between homologous DNA molecules in genetic recombination.

A mechanism to activate branch migration between homologous DNA molecules is described that leads to synapsis in genetic recombination. The model invo...
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