DNA. However, the specific molecular properties that give a critical evolutionary advantage to DNA are still unknown.

Homologous recombination, which occurs between homologous chromosomes or sister chromatids, is another general attribute of a DNA genome. Homologous recombination is a type of genetic rearrangement that occurs through the breakage and rejoining of DNA molecules within a stretch (some hundreds to thousands of base pairs) of identical or very similar (i.e., homologous) sequences. Homologous recombination maintains the integrity of the genome through the accurate repair of various types of DNA damage, especially double-stranded breaks. For the repair of double-stranded breaks, an intramolecular intact strand is not available as a template. At the same time, homologous recombination is a mechanism that can confer genetic diversity on the genome of a species by the rearrangement of alleles, variations of which were acquired by mutations. This is believed to contribute to evolution.

Sexual reproduction occurs in a variety of organisms, especially eukaryotes. It has a tight connection with homologous recombination, in that, at an early stage of meiosis, cells induce homologous recombination by which each chromosome recombines with its homologue. Mutations causing deficiencies in meiotic recombination generally result in the nondisjunction (or random sorting) of homologous chromosomes during meiosis (see refs. 5–7 for review). Thus, sexual reproduction is regarded as a system that ensures that all chromosomes undergo recombination in each sexual generation.

Homologous recombination occurs through a general intermediate, the Holliday intermediate, in which a pair of parental DNA molecules are connected by heteroduplex joints. These are duplexes of complementary strands each of which is derived from each parental DNA molecule (8–10). The heteroduplex has been shown to extend to a size large enough to cover an entire gene, as revealed by the size of co-conversion tracts. The heteroduplex joint ensures the exact alignments of multiple genes at a homologous sequence, allowing them to be recombined without disturbing their coding frames.

Various genes involved in homologous recombination are well conserved from viruses and bacteria to higher eukaryotes, including human beings. In particular, heteroduplex joints generally are formed by a reaction between homologous double-stranded DNA and single-stranded DNA, mediated in vivo as well as in vitro by the RecA/Rad51-family of proteins (11–15). Proteins in this family include UvsX of a bacterial virus (Escherichia coli phage T4: ref. 16), RecA of various bacteria, RadA of archaea, and the Rad51-family proteins found in various eukaryotes from yeast to human beings (refs. 17 and 18, and see ref. 19). Some of the RecA/Rad51-family proteins were demonstrated by electron microscopy to form conserved, right-handed, spiral filamentous structures either by themselves or around DNA that is either single-stranded or double-stranded (20, 21), and to some extent to have conserved amino acid sequences within the core region. It is remarkable that UvsX is only slightly conserved in amino acid sequence (22), but is well conserved in its biochemical functions and three-dimensional structure (16, 23, 24). The three-dimensional structure of DNA-free RecA, as solved by x-ray crystallography, revealed a right-handed helical filament (25), which is consistent with that obtained by electron microscopy as described above.

Single-stranded DNA tails are created by the processing of double-stranded breaks for the initiation of homologous recombination during meiosis in vivo (26, 27). It has been demonstrated in vitro that heteroduplex joints are formed by a reaction between homologous double-stranded DNA and single-stranded DNA mediated by the RecA/Rad51-family of proteins (11–16). E. coli RecA is the prototypical protein that promotes heteroduplex joint formation (11, 12). Various combinations of DNA molecules have been shown to be substrates for heteroduplex joint formation by RecA. The formation is especially efficient when one of a pair of DNA molecules has a single-stranded region within a sequence homologous to its partner double-stranded DNA (28–30). RecA forms a Holliday intermediate in vitro when the substrates are a pair of linear double-stranded DNA molecules, one of which has at least one single-stranded tail (31). Natural closed-circle double-stranded DNA is negatively supercoiled, which stimulates the RecA-mediated heteroduplex joint formation. When double-stranded DNA is negatively supercoiled, RecA by itself can pair more than 50% of substrate double-stranded DNA molecules (ca. 10 kbp) with homologous single-stranded DNA molecules to form heteroduplex joints (D loops) in vitro within 2–3 min at 37°C in Mg²⁺—and ATP-dependent reactions (12, 30). Heteroduplex joint formation by Rad51 from yeast or human is much slower and less efficient compared with RecA, and efficient heteroduplex joint formation by Rad51 requires coactivator proteins such as a single-stranded binding protein (RPA) and Rad54 (13–15, 32).

The molecular mechanisms of heteroduplex joint formation have been extensively studied by using RecA-mediated reactions as a model system. The heteroduplex joint formation promoted by RecA has been experimentally separated into two phases: homologous pairing and strand exchange (33, 34). Homologous pairing is the formation of the core of the heteroduplex, which then is stabilized by strand exchange. Strand exchange is associated with the unidirectional extension of the joint (unidirectional branch migration) by several kilobase pairs (34, 35) and can integrate mismatches (36) and heterologous sequences (37) into a heteroduplex. RecA has DNA-dependent ATPase activity (38), and ATP is required for heteroduplex joint formation (11, 12, 35). ATP hydrolysis is not required for homologous pairing but is required for unidirectional branch migration to bypass heterologous sequences (35, 39, 40), and ATP hydrolysis stimulates the detachment of the protein from the DNA for the recycling of RecA (41–43).

The reaction steps have been identified in detail in the case of homologous pairing and strand exchange by RecA. In homologous pairing, the RecA polypeptide first polymerizes on single-stranded DNA (29, 44–46) to form a spiral filamentous structure around the DNA (47–49). The secondary structure of the single-stranded DNA is removed during this step (48), which is stimulated by SSB (single-strand binding protein; refs. 50 and 51). Then double-stranded DNA binds to the RecA-single-stranded DNA filament without any need for sequence homology (44). The search for sequence homology between the single-stranded and double-stranded DNA occurs within this complex, and upon the recognition of homology, a core heteroduplex is formed (44, 52–54). For the stabilization of the core heteroduplex, strand exchange follows as described above.

In the formation of double-stranded DNA from complementary single-stranded DNA molecules (annealing), once the secondary structure of the DNA has been unfolded, all of the bases of the single-stranded DNA are available to form a stretch of Watson-Crick base pairs. The mechanism of annealing thus can be explained by simple collisions of DNA molecules. On the other hand, in homologous pairing, all of the bases in the double-stranded DNA are already involved in Watson-Crick base pairs and do not appear to be available to form base pairs with the single-stranded DNA. Thus, how sequence homology (identity or complementarity) is recognized between double-stranded and single-stranded DNA, and how the base pairs of the parental