Role of DNA Ligase in the Illegitimate Recombination That Generates bio-Transducing Phages in Escherichia coli

Corresponding author: Hideo Ikeda, Microbial Chemistry, Center for Basic Research, Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan., ikeda-h{at}kitasato.or.jp (E-mail)

Communicating editor: G. R. SMITH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We studied the role of DNA ligase in illegitimate recombination in Escherichia coli. A temperature-sensitive mutation in the lig gene reduced the frequency with which bio-transducing phages were generated to 10–14% of that of wild type under UV irradiation. Reintroduction of the lig gene into this mutant restored the frequency of recombinant phage generation to that of wild type. Furthermore, overexpression of DNA ligase enhanced illegitimate recombination by 10-fold with or without UV irradiation. In addition, when DNA ligase was present in only limited amounts, UV-induced or spontaneous illegitimate recombination occurred exclusively at hotspot sites that have relatively long sequences of homology (9 or 13 bp). However, when DNA ligase was overexpressed, most of the illegitimate recombination took place at non-hotspot sites having only short sequences of homology (<4 bp). Thus, the level of ligase activity affects the frequency of illegitimate recombination, the length of sequence homology at the recombination sites, and the preference for recombination at hotspots, at least after UV irradiation. These observations support our hypothesis that the illegitimate recombination that generates bio-transducing phages is mediated by the DNA break-and-join mechanism.

ILLEGITIMATE recombination takes place at low frequencies but occurs ubiquitously in both prokaryotes and eukaryotes. It is characterized by recombination between nonhomologous sequences or short homologous sequences at two different DNA sites and it induces genomic rearrangements. Examples of illegitimate recombination are the chromosomal aberrations in Drosophila occurring after X-ray irradiation (MULLER 1927 ) and the development of the deletion mutant in the rII gene when bacteriophage T4 is treated with nitrous acid (TESSMAN 1962 ). Illegitimate recombination is also seen in Xenopus (LEHMAN et al. 1994 ) and mammals (ROTH and WILSON 1988 ). In addition, specialized transducing phages are formed by illegitimate recombination (CAMPBELL 1962 ; FRANKLIN 1967 ; KUMAGAI and IKEDA 1991 ). For example, while the prophage is normally precisely excised at both ends (attR and attL) in the Escherichia coli genome, occasionally a recombination event occurs between substantially nonhomologous DNA segments that results in the production of the specialized transducing phages containing E. coli DNA (bio or gal).

There are two types of illegitimate recombination that lead to the formation of bio-transducing phages, namely, short-homology-dependent and short-homology-independent illegitimate recombination (SHDIR and SHIIR, respectively; SHIMIZU et al. 1997 ). UV-light-induced illegitimate recombination is an example of SHDIR, as sequence analysis of the recombination junction of bio-transducing phages produced by UV light irradiation reveals 4–10 bp of homologous DNA (YAMAGUCHI et al. 1995 ). In contrast, the illegitimate recombination mediated by DNA gyrase is classified as SHIIR because it gives rise to bio-transducing phages lacking any homology at recombination junctions (SHIMIZU et al. 1997 ). The DNA gyrase subunit exchange model postulates a likely mechanism by which illegitimate recombination can result from DNA gyrase (IKEDA et al. 1982 ), while the mechanism of SHDIR remains controversial.

FRANKLIN 1967 has proposed two models by which the illegitimate recombination that generates specialized transducing phages could occur. Both models have been also used to explain SHDIR in general (for reviews, see EHRLICH et al. 1993 ; MICHEL 1999 ). One is the "slipped mispairing model." In this model, a replication fork slips down a region of the DNA strand during DNA replication, thus resulting in a deletion mutation. The other is the "DNA break-and-join model," also known as the "nonhomologous end-joining (NHEJ)" or "double-strand break and end-joining" model, and it occurs in yeast and higher organisms (TSUKAMOTO and IKEDA 1998 ; LIEBER 1999 ). This model consists of three steps, namely, DNA double-strand break, DNA end processing, and end joining.

We previously suggested that the DNA break-and-join model is more likely to describe the mechanism leading to the illegitimate recombination resulting in bio-transducing phages than the slipped mispairing model (UKITA and IKEDA 1996 ). If this mechanism indeed pertains to the SHDIR-generating phages, it would be expected that DNA double-strand breaks would occur at two sites in the E. coli genome flanking the prophage, causing the DNA fragments including phage DNA to be excised. Next, the DNA ends would be processed by exonucleases or other enzymes preparatory for end joining, and, finally, the DNA ends would be ligated, probably by DNA ligase, thus producing the specialized transducing phages. It is well known that E. coli DNA ligase catalyzes the covalent joining of DNA strands (ZIMMERMAN et al. 1967 ). This enzyme requires that the ends being ligated are cohesive, while in contrast, T4 DNA ligase can ligate both blunt and cohesive DNA ends (SGARAMELLA 1972 ). DNA ligases participate in nonhomologous end joining in E. coli, yeast, and mammalian cells (CONLEY and SAUNDERS 1984 ; CRITCHLOW et al. 1997 ; SCHAR et al. 1997 ; TEO and JACKSON 1997 ; WILSON et al. 1997 ).

Here we investigate the role of E. coli DNA ligase in the SHDIR that generates bio-transducing phages and show that it is indeed involved at least after UV irradiation. An E. coli mutant carrying a temperature-sensitive mutation in the lig gene has a lower frequency of bio phage formation than the wild type while overexpression of DNA ligase enhances the frequency. The lig mutation also affects the length of sequence homology at the recombination sites, and the preference for recombination at hotspots. This involvement of E. coli DNA ligase strongly supports the mechanism postulated by the DNA break-and-join model.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Bacterial strains and plasmids:
The bacterial strains used in this study are indicated in Table 1. Ymel was used to titrate the total number of phages. An E. coli P2 lysogen, WL95, was used to titrate Spi^- phages. E. coli N1624 and N2668 were generously supplied by Dr. H. Ogawa. Mini-F plasmids pMF3, pLG914, and the runaway plasmids pSY343 and pLP312 were generously supplied by Dr. Y. Ishino (ISHINO et al. 1986 ). E. coli lysogens HI1699, HI1669, HI2688, HI2714, HI2689, HI2715, HI2678, HI2679, HI2680, and HI2680 were used to analyze the formation of Spi^- phages. The P1kc phage that was grown in E. coli N1126 polAts recQ101::cat was kindly provided by Dr. J. Kato.

Media:
Media used in this study have been described previously (YAMAGUCHI et al. 1995 ). YP broth was used to grow bacteria or to prepare Spi^- phage from single plaques. trypticase agar was used to titrate Spi^- phages. agar was used to titrate total phages.

Spi^- assay:
Measurement of the frequency of Spi^- phage was as described (UKITA and IKEDA 1996 ). The lysates were plated on either E. coli Ymel or an E. coli P2 lysogen to determine the total phage and Spi^- phage numbers, respectively. The frequency at which Spi^- phages arose was determined as the ratio of the Spi^- phage titer to the total phage titer in the phage lysate. The numbers of Spi^- phages thus generated were used as estimates of the bio phage numbers.

Independent isolation of Spi^- phages:
An E. coli lysogen was grown to 2 x 10⁸ cells/ml in YP broth at 30° and the culture was divided into 48 tubes. Phages were induced as described above (although in this case with 50 J/m² of UV light). The phage lysates thus obtained were plated on a lawn of E. coli WL95 on a trypticase agar plate. Only one Spi^- phage plaque was picked from each lysate, diluted, and replated on a agar plate with E. coli Ymel for the isolation of single phage clones. The phages thus obtained were amplified by the standard method and the amplified phages were used for the analysis of the recombination sites.

Localization of recombination junctions by PCR and sequence analysis:
Localization of recombination junctions in bio-transducing phages was determined by the procedure described by ONDA et al. 1999 . Serial PCRs and restriction-enzyme digestion with EaeI or PvuII were then used to examine the recombination sites. ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) was used to sequence the recombination junctions.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Effect of a temperature-sensitive mutation in E. coli DNA ligase on the frequency of Spi^- phage generation:
To examine the role of E. coli DNA ligase in the illegitimate recombination that generates bio-transducing phages, the wild-type strain (HI1699) and a strain with a heat-sensitive mutation in the lig gene (the ligts7 mutant, HI1669) were lysogenized by infection of cI857 and subjected to the Spi^- assay. This assay utilizes the Spi^- phenotype as a marker for bio-transducing phages (IKEDA et al. 1995 ). Specialized transducing phages generated from the prophage by illegitimate recombination usually contain the E. coli genes gal or bio that are adjacent to the phage genome. Most of the bio-transducing phages are thus defective in the red-gam region of phage DNA and can form plaques on an E. coli P2 lysogen lawn (Spi^- phenotype), whereas normal phages cannot. Thus, it is possible to select Spi^- phages from the phage pool. The number of Spi^- phages is assumed to be the same as that of bio-transducing phages since previous experiments have shown that most Spi^- phages are bio phages (IKEDA et al. 1995 ; YAMAGUCHI et al. 1995 ).

In the Spi^- assay, the lysogens are exposed at 42° for the heat induction of the prophage, and in these conditions, the ligase activity in the ligts7 mutant is considerably reduced (GOTTESMAN et al. 1973 ). Table 2 shows that the frequency of Spi^- phages generated from the E. coli ligts7 mutant after UV irradiation was between 10 and 14% of that of the wild-type strain. Thus, E. coli DNA ligase is involved in the illegitimate recombination generating bio-transducing phages. This was confirmed by introducing the E. coli lig⁺ gene into the E. coli ligts7 mutant by transforming it with pLG914. This plasmid contains a functional E. coli lig gene (ISHINO et al. 1986 ) and was constructed with mini-F plasmid pMF3 as a vector. As a control, the wild-type strain was transformed with pMF3. The Spi^- assay results with these lysogens are shown in Table 2 and indicate that the introduction of the lig gene into the E. coli ligts7 mutant (resulting in the lysogen denoted as HI2715) caused the Spi^- phage frequency to rise to the value of the wild-type lysogen.

View this table: [in this window] [in a new window]   Table 2. Effect of the ligts7 mutation on illegitimate recombination during formation of Spi- phage

Effect of overexpression of DNA ligase on the frequency of Spi^- phage generation:
Four lysogens (HI2678, HI2679, HI2680, and HI2681) were prepared by transformation of the wild-type strain (HI1699) or ligts7 mutant strain (HI1669) with the high-copy-number plasmids pSY343 or pLP312 (see Table 1). The pLP312 plasmid was constructed by cloning the E. coli lig gene into pSY343, causing overproduction of E. coli DNA ligase. Each lysogen was then subjected to the Spi^- assay to examine the effect of DNA ligase overexpression.

Surprisingly, the frequency of Spi^- phage formation in the lysogens overexpressing E. coli ligase was 10 times higher than that in the pSY343-transformed lysogens, both with and without UV-light irradiation (Table 3). This indicates that the frequency of Spi^- phage formation depends on the concentration of DNA ligase in the cell, probably because increased DNA ligase activity enhances the rejoining of broken DNA ends. That the frequency of Spi^- phage formation is increased even in the absence of UV-light irradiation suggests that the DNA breakage occurs spontaneously at a certain frequency and that such spontaneously broken DNA is mostly not rejoined under the normal DNA ligase activity.

Distribution of recombination sites in bio-transducing phages:
The sites of recombination were examined in bio-transducing phages independently isolated from the wild-type strain (HI1699), the lysogen overexpressing the lig gene (HI2679), the E. coli ligts7 mutant (HI1669), and the E. coli ligts7 mutant reconstituted with the lig gene (HI2715) after irradiation by UV light followed by heat induction. The recombination junctions of these phages were assessed by serial PCRs using several primers, because recombination junctions were derived from the parental E. coli and phage genomes; thus, the recombination junctions in turn indicated the parental recombination sites.

Fig 1 depicts the structure of prophage DNA and its recombination site. The recombinant bio phages were generated by the illegitimate recombination of DNA and E. coli DNA, as shown in the figure, as are the sites of recombination that produced the bio phages in the various lysogens studied here. Some recombination occurred at hotspots I or II, as the PCR products were of the same size as those from a previously isolated bio-transducing phage known to be recombined at hotspot I or II. This was confirmed by sequence analysis. Recombination at hotspots I and II has been previously observed in both UV-light-induced and spontaneous illegitimate recombination (YAMAGUCHI et al. 1995 ; HANADA et al. 1997 ).

View larger version (82K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Distribution of recombination sites on bio-transducing phages. (A) Prophage map and site of illegitimate recombination. A thick line indicates DNA and a thin line indicates E. coli DNA. The numbers indicate the map coordinate on the genome (SANGER et al. 1982 ) or the E. coli genome numbered from the attR site (OTSUKA et al. 1988 ). The recombinant bio phage, WL1 (see B), was generated by the illegitimate recombination at the sites as indicated. B–G show the distributions seen in the indicated strains with or without UV irradiation. The dose of UV irradiation was 50 J/m2. The boxes marked as hotspot I or II indicate a group of bio-transducing phages that are produced by recombination at hotspot I or II.

In bio-transducing phages from the UV-light-irradiated wild-type cells, 69 and 19% of all bio phages were generated by recombination at hotspots I and II, respectively. Thus, bio-transducing phages in the wild type are frequently produced by illegitimate recombination at hotspot I or II. In contrast, bio phages from the cells overproducing E. coli DNA ligase and irradiated with UV light had frequently recombined at nonhotspot sites. bio phages from the ligts7 mutant with UV irradiation, however, were all generated by recombination at the hotspots, with the number of phages recombined at hotspot II (58% of all bio phages) now exceeding those recombined at hotspot I (42%). When this mutant lysogen was reconstituted by the introduction of one copy of the lig gene, however, the sites of recombination were similar to that occurring in the wild-type strain.

In bio-transducing phages from the wild-type cells without UV irradiation, 42 and 23% of all bio phages were generated by recombination at hotspots I and II, respectively. bio-transducing phages from the ligts7 cells without UV irradiation were exclusively generated by recombination at hotspot II (96% of all bio phages), as was observed in bio phage from the ligts7 cells with UV irradiation.

Analysis of recombination junctions of bio phages isolated from lysogens:
Recombination junctions are derived from E. coli and phage genomes and show the site where the recombination took place. To more precisely examine the recombination event, the nucleotide sequences of the recombination junctions in the bio phages were determined.

Fig 2 shows examples of sequences of the recombination junctions. Fig 2A or Fig 2B denotes recombination at hotspots I or II, indicated by the boxes of 9 or 13 overlapping base pairs, respectively. The phage WL22 is an example derived from the wild-type strain, HI1699, with UV-light irradiation, and it shows 7 overlapping base pairs in its recombination junction (Fig 2C). The phages HL7 and HL48 are from the lysogen overexpressing DNA ligase, HI2679, with UV-light irradiation and are examples of phages containing only one or two overlapping nucleotide(s) in their recombination junctions (Fig 2D and Fig E). The phage FL21 is an example from the E. coli ligts7 mutant reconstituted with the lig gene, HI2715, with UV-light irradiation (Fig 2F). The phages WS2, WS4, and WS10 are from the wild type without UV-light irradiation (Fig 2G and Fig H).

View larger version (55K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Nucleotide sequences of junctions derived from bio-transducing phages. The sequences of hotspot I (A) and hotspot II (B) were detected in bio phages obtained from UV-irradiated cells (50 J/m2) of HI1699, HI1669, HI2715, and HI2679 and from unirradiated cells of HI1699 and HI1669. The map coordinates for phage and bacterial sequences are indicated. Phage numbers WL22, HL7, HL48, FL21, WS2, WS4, and WS10 (C–H) correspond to those indicated in Fig 1. The boxed sequences represent homology at recombination sites between the parental recombination sites.

Fig 3 indicates the distribution of lengths of overlapping sequences in the bio phage junctions. In the bio phages isolated from the UV-light-irradiated wild-type cells (HI1699), there are 4 or more overlapping base pairs (Fig 3A). The average length of overlapping nucleotides in all of the sequenced bio phages isolated from the wild-type strain was 9.5 bp. On the contrary, many of the bio phages from the UV-light-irradiated cells overproducing DNA ligase (HI2679) showed a shorter length of overlapping nucleotide(s) than that of the wild-type strain (Fig 3B). Nineteen bio phages including HL48 overlapped by only a single base pair, while 7 phages including HL7 overlapped by 2 bp. Six phages including HL11 overlapped by 3 bp. Thus, phages with overlaps of <4 bp constitute 67% of all the bio phages isolated from the strain overexpressing ligase, with the average overlap length being 3.9 bp. Thus, overexpression of E. coli DNA ligase markedly increases the frequency of phages with shorter overlapping sequences. In contrast, recombination junctions of all the bio phages from the UV-light-irradiated ligts7 cells (HI1669) overlapped by 9 or 13 bp, with overlaps of 13 bp being more frequent than overlaps of 9 bp (Fig 3C). The average length of overlapping sequence in these phages was 11.3 bp. It seems as though relatively long stretches of homologous sequence between the E. coli and phage DNAs are required for the formation of bio phages when DNA ligase is present only in low concentrations. When the lig⁺ gene is introduced into the ligts7 mutant, the bio phages derived from this isolate bear on average 8.5 bp of overlapping sequence at their recombination junctions (Fig 3D), and thus mostly revert to what is seen in phages derived from the wild-type strain.

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Distributions of lengths of overlapped nucleotide(s) at recombination sites. The dose of UV irradiation was 50 J/m2.

We have also examined the lengths of overlapping sequences in the bio phage junctions formed spontaneously at nonhotspot sites. In bio phages isolated from the unirradiated wild-type cells (HI1699), overlapping nucleotides were 1 or 2 bp in some cases and 7 or 8 bp in other cases (Fig 3E). The average length of overlapping nucleotides in all of the sequenced bio phages was 8.1 bp. On the contrary, most of the recombination junctions of all the bio phages from the unirradiated ligts7 mutant (HI1669) occur at hotspot II sites, which are overlapped by 13 bp (Fig 3F). The average length of overlapping sequences in these phages was 12.7 bp, confirming that longer stretches of homologous sequence are required for the formation of bio phages when the ligase activity is limited.

It should be noted that we were not able to detect any bio phages without at least one overlapping nucleotide in all of the sequenced recombination junctions. We therefore concluded that the short homology plays an important role in this type of illegitimate recombination.

Effect of the ligts mutation on the frequency of spontaneous Spi^- phage generation:
The experiment in Table 1 implied that the frequency of formation of spontaneous bio-transducing phages is not affected when the ligase activity is reduced. Since the frequency of spontaneous illegitimate recombination is increased in the recQ mutation background (HANADA et al. 1997 ), the effect of ligts mutation on the frequencies of spontaneous illegitimate recombination can be more accurately examined under this condition. The frequency of spontaneous bio-transducing phage formation in the ligts7 mutant was increased by the recQ mutation, as was observed by HANADA et al. 1997 , and this level is comparable to that in the recQ lig⁺ strain (Table 4). Therefore the frequency of spontaneous bio-transducing phage formation is not significantly affected by the ligts mutation in the recQ mutation background.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Here we report our studies on the role of E. coli DNA ligase in the illegitimate recombination that generates bio-transducing phages. The ts7 mutation limiting the expression of DNA ligase reduced the frequency of bio-transducing phages formed after UV irradiation to 10–14% of that of the wild-type strain. Ligase activity in a strain carrying the ligts7 mutation has been studied in vivo by measuring formation of covalently closed circular phage DNA molecules after infection by phage of homoimmune lysogens at 42°. The ligase activity of the ligts7 strain is reduced to 15% of the wild type (GOTTESMAN et al. 1973 ). The reduced ligase activity is consistent with the reduced frequency of illegitimate recombination in the ligts7 mutant. Introduction of the E. coli lig⁺ gene into the ligts7 mutant caused a return to wild-type frequencies. These observations indicate that DNA ligase is important in the generation of bio phages at least after UV irradiation, and this is further supported by the fact that overexpression of DNA ligase enhances by 10-fold the frequency of illegitimate recombination. In addition, when only a limited amount of DNA ligase is present, illegitimate recombination takes place between relatively long homologous sequences (9–13 bp), while, when the ligase is present in higher concentrations, recombination, at least after UV irradiation, takes place between relatively short homologous sequences (3 bp or less). These observations thus show that reduction or augmentation of ligase activity can alter the frequency of illegitimate recombination, and that the length of homology at recombination sites depends on the degree of DNA ligase activity (Table 5).

Phages from the ligts7 mutant, which has low DNA ligase activity under the conditions used, had on average the longest homologous sequences of all four strains examined (11.3–12.7 bp vs. 3.9–9.5 bp in a lig⁺ host). This suggests that when DNA ligase is present in only low activity, longer sequences of homology between E. coli and phage DNA are required to hold together the broken DNA ends during ligation. That overexpression of DNA ligase increases the frequency of bio phages with short homologous sequences of 3 bp or less (as well as increases the overall frequency of bio phage formation) is probably because very short cohesive ends of broken DNA, which cannot be ligated under normal levels of DNA ligase activity, can be ligated when high levels of DNA ligase activity are present.

All of the bio-transducing phages examined showed overlapping nucleotide(s) in their recombination junctions (see Fig 3). This is probably due to E. coli DNA ligase requiring cohesive DNA ends for ligation.

Our group has previously proposed that the mechanism of illegitimate recombination proposed by the double-strand break-and-join model may be applicable in the case of bio-transducing phages (YAMAGUCHI et al. 1995 ; UKITA and IKEDA 1996 ). That the reduction or augmentation of ligase activity affects the frequency of illegitimate recombination as well as the length of homologous sequences at the recombination junction strongly supports our hypothesis. As an alternative model, the "microhomology priming" model has been proposed (LEHMAN et al. 1994 ), in which DNA ends carrying a single-stranded overhang bind to each other with short homologous sequences. A transient joint is stabilized initially by primed DNA synthesis and then ligated. However, according to this model, the reduced or overproduced ligase activity might not affect the formation of recombinant DNA, because the joint molecule is already stabilized by microhomology priming before ligation. In another alternative model devised to explain illegitimate recombination, namely, the "slipped mispairing model," DNA ligase is not thought to be the primary enzyme in the reaction, although it may be involved in replication by joining Okazaki fragments and/or by mismatch repair. While this model could explain the low frequency of bio phage in the ligts7 mutant as being due to low DNA ligase activity, it cannot explain why overexpression of DNA ligase greatly enhances the frequency of bio phages and facilitates ligation with relatively short cohesive ends. We thus conclude that the break-and-join model is more likely than the microhomology priming model or the slipped mispairing model to explain the mechanism of illegitimate recombination that generates bio-transducing phages.

E. coli DNA ligase is known to recircularize linearized plasmids (CONLEY and SAUNDERS 1984 ) and thus it is quite likely that this ligase is responsible for joining the broken DNA ends in illegitimate recombination. It has been reported that the RecJ exonuclease can also stimulate the illegitimate recombination that generates bio-transducing phages (UKITA and IKEDA 1996 ). RecQ protein has also been reported to affect the frequency of the illegitimate recombination. As applied to the DNA break-and-join model, it is believed that this enzyme, which has 3'-5' helicase activity, processes DNA ends by disrupting the intermediate product formed by the annealing of DNA ends before the ligation step (HANADA et al. 1997 ). Exonucleases affect illegitimate recombination not only of the E. coli chromosome but also of plasmids (KEIM and LARK 1990 ; ALLGOOD and SIHAVY 1991 ; YAMAGUCHI et al. 2000 ). It thus appears that many kinds of illegitimate recombination are mediated by the DNA break-and-join mechanism.

This study also provides an explanation of why illegitimate recombination frequently takes place at hotspots. We found that when DNA ligase was limited, most, if not all, of the illegitimate recombination took place at either hotspot I or II, where the homology between parental sequences is relatively long (9 or 13 bp, respectively). However, when concentrations of DNA ligase were high, most of the illegitimate recombination, at least after UV irradiation, took place at nonhotspot sites where parental sequences have relatively short sequences of homology (3 bp or less). These observations suggest that preferential recombination occurs at hotspots because of the longer sequences of homology at these sites. However, it should be noted that some nonhotspot sites normally not favored for recombination also have long (10–12 bp) sequences of homology (SHIMIZU et al. 1997 ; this article). This suggests that in addition to the dependence on DNA ligase activity, other factors might also influence the preferential recombination occurring at the hotspots. One of the factors is RecJ exonuclease, although the role of RecJ in the preferential recombination is not known yet (UKITA and IKEDA 1996 ).

The frequency of spontaneous bio-transducing phage formation (i.e., without UV irradiation) was not significantly affected when the ligase activity was reduced by the ts7 mutation. This observation was confirmed by the experiments in the recQ mutation background. The above results and the nucleotide sequence analyses of the junctions showed that the ligts7 mutation does not affect the frequency of spontaneous illegitimate recombination, but affects the length of sequence homology at the recombination sites (12.7 vs. 8.1 bp in a lig⁺ host) as well as the preference for recombination at hotspots in spontaneous illegitimate recombination (Table 5). It is possible that impaired DNA replication and/or DNA repair in the ligts7 mutant triggers double-strand breaks in DNA, resulting in promotion of illegitimate recombination and thus compensating for the reduction of spontaneous bio-transducing phage formation mediated by the defect of DNA ligase. At any rate, the role of DNA ligase in spontaneous illegitimate recombination may be essentially the same as that in UV-light-induced illegitimate recombination.

	FOOTNOTES

¹ Present address: Funakoshi Co. Ltd., 9-7-2 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

	ACKNOWLEDGMENTS

We thank Drs. H. Ogawa, Y. Ishino, and J. Kato for providing us with bacterial strains and plasmids. We appreciate encouragement by Drs. R. Funakoshi, Y. Yogo, and S. Ohmura throughout this work. The work was supported by Grants-in-Aid for Scientific Research (B) and Scientific Research on Priority Areas (B) to H.I. from the Ministry of Education, Science, Sports, and Culture of Japan.

Manuscript received May 10, 2000; Accepted for publication February 13, 2001.

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