Ancient ideas about chromosomal recombination and crossing-over in meiosis. Pre-existing chromosomes (DNA duplexes) are shown as either solid or open single lines; newly synthesized chromosomes are shown as broken single lines. Explanations are in the text.

The influence of various combinations of cutting and repression on phage replication and recombinant yield. (A) Cutting at the unique restriction sites precludes formation of the wild-type recombinant. As in Fig. 2A, spi6 and nin5 regions are shown as open rectangles when they are present in the phages, whereas the short vertical lines mark the corresponding deletions. Chromosomes with double-strand breaks at the unique XhoI sites are marked cut, whereas unbroken chromosomes aremarked uncut. Exchanges (left) are indicated by diagonal lines leading to particular outcomes (right). (Top cross) Neither parent is cut, so both the wild-type (WT) and the double-deletion recombinants are possible. (Second from top cross) Both parents are cut, so only the double-deletion recombinant is possible; (two bottom crosses) one of the parents is cut, so two recombinants are formed, the double-deletion one and a recombinant which is not different from one of the parents. (B) The two parental phages were injected into either Rec⁺ or ΔrecA host cells containing various combinations of cutting and repression functions. Total DNA was extracted at the indicated postinjection times, cut with NcoI, and analyzed by blot hybridization with probe 1. (Top) Crosses in Rec⁺ cells; (bottom) crosses in ΔrecA mutant cells. Rec⁺ strain is WA800, ΔrecA mutant is AK24. Phages are MMS2660 (Δnin5) and MMS2663 (Δspi6). Plasmids supplying the XhoI cutting and cI repression functions are pPaoRM3.8 (XhoI), pK116 (cI), and pK107 (XhoI + cI); when no function is supplied, cells are plasmid-free.

Monoparental infections do not yield recombinant bands. The four parental phages were injected alone or in two cut-by-uncut combinations (controls) into Rec⁺ cells harboring pK107 (cutting + repression). The total DNA was extracted at the indicated postinjection times, cut with NcoI, and analyzed by blot hybridization with probe 1. The host strain is WA800; phages are MMS2660 (Δnin5 cuttable), MMS2661 (Δnin5 uncuttable), MMS2662 (Δspi6 uncuttable) and MMS2663 (Δspi6 cuttable).

Genetic requirements of the recombinant formation. Two parental phages in a single cut-by-uncut combination were injected into various host strains harboring pK107 (cutting + repression). The total DNA was extracted at the indicated postinjection times, cut with NcoI, and analyzed by blot hybridization with probe 1. recD strain is WA800, wild-type strain is AB1157, recBC mutant is JC5519, recD recF mutant is AK61, recD dnaE mutant is AK64. Phages are MMS2660 (Δnin5 cut) and MMS2662 (Δspi6 uncut). Incubation was at 42°C to inactivate DnaEts protein.

The two principal deletion substrates used in this study and the two complementary recombinant products. (A) A schematic diagram (not to scale) of the two principal substrate DNAs, the two complementary recombinants, and the probes used in this study (for probe specifications, see Materials and Methods). The spi6 and nin5 regions are boxed in the DNAs that retain them; boxes are absent in the DNAs having the corresponding deletions. Also shown are the unique XhoI restriction sites for in vivo cutting and the NcoI restriction sites used in the subsequent in vitro analysis. The sizes of the fragments generated as a result of in vivo and subsequent in vitro cutting are indicated. (B) A blot with two freely replicating parental λs, cut with NcoI and rehybridized to three region-specific probes. Nonspecific probe (1) hybridizes to the region between deletions detecting both Δspi6 and Δnin5 phages. nin5⁺-specific probe identifies only Δspi6 phages and the wild-type recombinant; spi6⁺-specific probe identifies only Δnin5 phages and the wild-type recombinant. A weak cross-hybridization in lanes j, n, and o is due to overloading, which causes a small fraction of Δspi6 DNA to migrate with the Δnin5 band and vice versa. Explanations are shown only for the window at left. (M1) MW markers for the parental phages; (M2) MW markers for the recombinant phages.

RecA-catalyzed double-strand end repair primes DNA synthesis. (A) The four parental phages in four pairwise combinations were injected into either Rec⁺ or ΔrecA host cells harboring pK107 (cutting + repression). The total DNA was extracted at the indicated postinjection times, cut with NcoI, and analyzed by blot hybridization with probe 1. (Top) Crosses in Rec⁺ cells; (Bottom) crosses in ΔrecA mutant cells. Rec⁺ strain is AK3, ΔrecA mutant is AK24. Phages are MMS2660 (Δnin5 cut), MMS2661 (Δnin5 uncut), MMS2662 (Δspi6 uncut) and MMS2663 (Δspi6 cut). Judging by 10-min time points, in vivo cutting at XhoI sites seems to be inefficient, as if the result of a rapid methylation of the injected DNA. However, at least 90% of the cuttable, yet uncut, parental DNA is likely to come from the adsorbed phages that failed to inject, as revealed by cutting the 10-min samples with XhoI in vitro. (B) The scheme of XhoI-stimulated formation of double-deletion recombinant with the simultaneous replication of the uncut parent. The phage duplex chromosomes are shown as single lines; boxes designate the presence of spi6 and nin5 DNA regions; short vertical lines mark the position of the corresponding deletions; cos sites are shown as chevrons. (step 1) Two parental phages aligned by the regions between the two deletions; (step 2) Δspi6 phage opened at the unique XhoI site; (step 3) RecA-catalyzed end invasion created a replication fork between the cut Δspi6 phage and the uncut Δnin5 phage; (step 4) replication fork copying the uncut Δnin5 phage. Note the formation of double-deletion DNA at the site of recombination.

Double-strand end repair far from the cI-containing interval primes DNA synthesis. (A) A schematic diagram (not to scale) of the two alternative substrate DNAs, the two complementary recombinants, and the probe (2) used in this alternative setup. The b519 and spi6 regions are boxed in the DNAs that retain them; boxes are absent in the DNAs having the corresponding deletions; the nin5 region, present throughout, serves as the position reference. Also shown are the unique XhoI restriction sites for in vivo cutting and the EagI and BsiWI restriction sites used in the subsequent in vitro analysis. The sizes of the fragments generated as a result of in vivo and subsequent in vitro cutting are indicated. (B) The three parental phages in two pairwise combinations were injected into either Rec⁺ or ΔrecA host cells harboring pK107 (cutting + repression). The total DNA was extracted at the indicated postinjection times, cut with BsiWI + EagI and analyzed by blot hybridization with probe 2. Lanes a-f are from crosses in Rec⁺ cells; lanes j-o are from crosses in ΔrecA mutant cells. Rec⁺ strain is AK3, ΔrecA mutant is AK24. Phages are IS4 (Δb519 cut), λAK1 (Δspi6 cut) and MMS2662 (Δspi6 uncut).

Exchange (break–join) versus repair (break–copy) in contemporary schemes of recombination. A–D represents the break–join, exchange-oriented pathway (Clark and Sandler 1994; Kowalczykowski et al. 1994; Taylor and Smith 1995; Eggleston and West 1996), whereas A, B, E, and F (Asai et al. 1993; Kuzminov 1995a; Cox 1998) or A–C, G, and H (Kuzminov 1996b) are the break–copy, repair-oriented pathways. DNA duplexes are depicted as double lines; the strands to be cut are indicated by small arrows. For simplicity, only one of the two possible orientations of Holliday junction resolution is shown. (A) Two homologous DNA duplexes, the black one with the double-strand end, the white one intact; (B) the double-strand end, with the help of homologous pairing activity of RecA protein, invades the intact duplex; (C) the intact duplex is nicked in the invasion loop, and one of the invading strands is linked with the nicked strand; (D) the Holliday junction is resolved, yielding two recombinant DNA molecules; (E) the invading end primes DNA synthesis; (F) the Holliday junction is resolved, completing the replication fork; (G) the Holliday junction is removed by sliding towards the double-strand end, which creates a replication fork framework; (H) DNA synthesis begins at the replication fork framework.