Escherichia coli MutM Suppresses Illegitimate Recombination Induced by Oxidative Stress

Corresponding author: Hideo Ikeda, Department of Molecular Biology, Institute of Medical Science, University of Tokyo, P.O. Takanawa, Tokyo 108-8639, Japan., ike{at}ims.u-tokyo.ac.jp (E-mail)

Communicating editor: R. MAURER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DNA damage by oxidative stress is one of the causes of mutagenesis. However, whether or not DNA damage induces illegitimate recombination has not been determined. To study the effect of oxidative stress on illegitimate recombination, we examined the frequency of bio transducing phage in the presence of hydrogen peroxide and found that this reagent enhances illegitimate recombination. To clarify the types of illegitimate recombination, we examined the effect of mutations in mutM and related genes on the process. The frequency of bio transducing phage was 5- to 12-fold higher in the mutM mutant than in the wild type, while the frequency in the mutY and mutT mutants was comparable to that of the wild type. Because 7,8-dihydro-8-oxoguanine (8-oxoG) and formamido pyrimidine (Fapy) lesions can be removed from DNA by MutM protein, these lesions are thought to induce illegitimate recombination. Analysis of recombination junctions showed that the recombination at Hotspot I accounts for 22 or 4% of total bio transducing phages in the wild type or in the mutM mutant, respectively. The preferential increase of recombination at nonhotspot sites with hydrogen peroxide in the mutM mutant was discussed on the basis of a new model, in which 8-oxoG and/or Fapy residues may introduce double-strand breaks into DNA.

OXIDATIVE stress is generated by external sources, such as ionizing radiation, oxidants, and peroxides, and also by endogenous metabolism. Oxidative stress is known to induce various types of DNA damage that have been implicated in mutagenesis and carcinogenesis, as well as in other degenerative diseases (AMES 1989 ; BREIMER 1990 ; FARR and KOGOMA 1991 ). In addition, oxidative stress generated by normal cellular metabolism has also been shown to injure DNA and other components and is believed to be a cause of aging. It has been estimated that an enormous number of oxidative hits take place in the DNA of normal cells (AMES et al. 1993 ).

Reactive oxygen species, such as superoxide radical, hydrogen peroxide, and hydroxyl radical, all damage bases and sugar residues and also cause strand breaks (IMLAY and LINN 1988 ). Hydrogen peroxide is generated in all aerobic cells as the result of normal cellular metabolism and produces hydroxyl radicals and related oxidants in the presence of trace amounts of metals (HALLIWELL and GUTTERRIDGE 1986 ; HENLE and LINN 1997 ). Cytosine glycol, thymine glycol, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 4,6-diamino-5-formamidopyrimidine, 7,8-dihydro-8-oxoguanine (8-oxoG), etc. are produced by the reaction of hydrogen peroxide with DNA (HALLIWELL and ARUOMA 1991 ).

Among the various kinds of DNA damage caused by oxygen radicals, 8-oxoG, which results from the damage of deoxyguanine at the C-8 position, has been studied extensively. Damage to the 8-oxoG residue induces a G:C to T:A transversion both in vivo and in vitro because adenine is misincorporated to the opposite site of 8-oxoG residue during DNA replication (MORIYA et al. 1991 ; SHIBUTANI et al. 1991 ). The transversion-specific mutator genes, mutM and mutY, have been shown to participate in the repair of this damage (MICHAELS et al. 1992 ). In a mutM mutant, the frequency of G:C to T:A transversion is specifically increased 10-fold over the wild type (MICHAELS et al. 1991 ). MutM protein has been found to be a formamidopyridine-DNA glycosylase and is able to remove 8-oxoG residues from DNA (MICHAELS et al. 1991 ; TCHOU et al. 1991 ). The mutY mutant shows the same unidirectional mutator phenotype as the mutM mutant (NGHIEM et al. 1988 ). MutY protein is also a DNA glycosylase and is able to remove an adenine base from the A:8-oxoG pair (AU et al. 1988 , AU et al. 1989 ; MICHAELS et al. 1990 ). Furthermore, another mechanism for suppressing 8-oxoG-induced mutagenesis has been previously reported (MAKI and SEKIGUCHI 1992 ). A mutT mutation specifically increases the A:T to C:G transversion 1000 times over the wild type (YANOFSKY et al. 1966 ). The MutT protein preferentially degrades 8-oxo-7,8-dihydrodeoxyguanosine triphosphate to the monophosphate, thus preventing 8-oxo-G:A mispairing in DNA.

There are a few reports showing that recombination is affected by oxidative stress. It is known that irradiation of Drosophila melanogaster with X rays induces chromosome aberrations (MULLER 1927 ). Hydrogen peroxide or other oxidative stress has been also reported to generate sister-chromatid exchanges in mammalian cells (OYA et al. 1986 ; LARRAMENDY et al. 1987 ) and illegitimate recombination in bacteria (OUCHANE et al. 1997 ). The molecular mechanism of recombination by oxidative stress, however, is not fully understood.

Illegitimate recombination is a class of recombination that takes place between sequences of little or no homology, and results in gene rearrangements such as deletions, translocations, or insertions of the chromosomes. Illegitimate recombination can be classified into two classes, short-homology-dependent illegitimate recombination (SHDIR) and short-homology-independent illegitimate recombination (SHIIR; SHIMIZU et al. 1997 ). The former class of recombination takes place between short regions of homology within plasmid or chromosomal DNA. These regions usually contain 4–10 bp of homologous DNA (YAMAGUCHI et al. 1995 ; UKITA and IKEDA 1996 ; SHANADO et al. 1997 ). The latter class of recombination occurs between sequences with virtually no homology and is mediated by DNA gyrase (SHIMIZU et al. 1995 , SHIMIZU et al. 1997 ). Here we show that hydrogen peroxide increases the frequency of SHDIR and that MutM protein suppresses this increased illegitimate recombination, especially illegitimate recombination at nonhotspot sites. These results suggest that the specific DNA lesions that are repaired by the MutM protein, 8-oxoG and/or formamidopyrimidine (Fapy), induce SHDIR.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Bacterial strains:
The bacterial strains used in this study are indicated in Table 1. Escherichia coli MK601, MK602, MK603, and MK604 were generously supplied by Dr. Y. Nakabeppu. E. coli HI2382, HI2383, HI2384, and HI2385 were used for the analysis of the formation of Spi^- phages. Total -phage was titrated on E. coli Ymel, and the number of Spi^- phages was counted by titration on an E. coli P2 lysogen, WL95.

Media:
Media used in this study were 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.

Exposure of E. coli lysogens with hydrogen peroxide:
An E. coli lysogen, HI2382, 2383, 2384, or 2385, was grown to 2 x 10⁸ cells/ml in YP broth at 30°. Various concentrations of hydrogen peroxide aqueous solutions were added to the culture and the prophage was induced at 42° for 15 min with aeration. The bacteria were centrifuged at 3000 rpm for 15 min, resuspended in fresh YP broth after removal of the supernatant, and incubated at 37° for 2 hr. After addition of two drops of chloroform, the lysate was centrifuged at 3000 rpm for 15 min to obtain a supernatant.

Measurements of the frequency of Spi^- phage:
The frequency of Spi^- phage was determined by calculating their relative titer on an E. coli WL95, representing the number of Spi^- phages in the lysate, to the titer on E. coli Ymel, representing the number of normal -phages in the lysate. For titration of Spi^- phages, 2 x 10⁷ phages were spread on a lawn of WL95 on a trypticase agar plate.

Independent isolation of Spi^- phages induced by hydrogen peroxide:
E. coli lysogen was grown to 2 x 10⁸ cells/ml in YP broth at 30°. Hydrogen peroxide solution was added to the culture to give a final concentration of 2.0 mM and the prophage was induced at 42° for 15 min with aeration. The bacteria were collected by centrifugation at 3000 rpm for 15 min, resuspended in 100 ml of fresh YP broth, and divided into 50 small tubes. Each tube containing 0.5 ml of the culture was incubated at 37° for 2 hr. Phage lysates thus obtained were diluted and plated on a lawn of E. coli WL95 on a trypticase agar plate. A plaque was picked from each plate, diluted, and replated on a agar plate with E. coli Ymel for isolation of single clone of phage. A single plaque from each agar plate was isolated. The phage from the plaque was amplified by the standard method and the amplified phage was used in an experiment for the analysis of the recombination site.

Identification of bio transducing phages and localization of recombination junctions by PCR:
Analysis of bio transducing phages by PCR was performed as described by SHIMIZU et al. 1995 . Identification of bio transducing phages was performed by using three sets of primer oligonucleotides: #184 (bp 27411–27430 of DNA), #185 (bp 28068–28049 of DNA) and bio-in (bp 210–191 of the attB site). A PCR with the primer set of #184 and #185 amplifies the DNA fragment of 657 bp as an indicator for attP site that results from the normal excision of -prophage. The set of #184 and bio-in amplifies the DNA fragment of 513 bp derived from the attR site, which is carried by bio transducing phages generated by an abnormal excision. Localization of recombination junctions in bio transducing phages was determined by serial PCRs with the following primers: #388 (bp 35091–35072 of DNA), #402 (bp 34521–34502 of DNA), #401 (bp 34002–33983 of DNA), #387 (bp 33559-33540 of DNA), #408 (bp 33122–33103 of DNA), #389 (bp 1012–1031 of E. coli bio), #390 (bp 1896–1915 of E. coli bio), #391(bp 3026–3045 of E. coli bio), #392 (bp 4185–4204 of E. coli bio), #404 (bp 4761–4780 of E. coli bio), #393 (bp 4925–4708 of E. coli bio). On these PCRs, occurrence of amplification shows that the DNA of bio transducing phage contains the sequences of two primers, i.e., it shows that the recombination took place within the region between two primers. Restriction enzymes, EaeI and PvuII, were also used to examine the recombination sites. PCR products amplified by #391 and a primer located at DNA (#388, #402, #401, #387, or #408) were incubated with EaeI or PvuII, the site of which is located at bp 3328 or at bp 3706 of the E. coli bio gene, respectively. The occurrence of digestion shows that the recombination took place between the restriction-enzyme site of the E. coli bio gene and the DNA site indicated by the primer used.

Sequencing of recombination junctions:
A recombination junction in a recombinant bio transducing phage was amplified by PCR. The PCR products were sequenced using an ABI PRISM 310 genetic analyzer (PE Applied Systems, Foster City, CA).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Frequency of Spi^- phage is enhanced by hydrogen peroxide:
Excision of -prophage mediated by illegitimate recombination occurs at a low frequency, resulting in the formation of specialized transducing phages (FRANKLIN 1971 ). The phage DNA usually comprises the bacterial genes, gal or bio, which are adjacent to the phage genome integrated in the bacterial genome of a lysogen, and most of them have defects in the red-gam region of -phage DNA. The Spi^- assay can detect the frequency of the specialized transducing phages quantitatively (IKEDA et al. 1995 ). The assay makes use of the characteristic that bio transducing phages carry defects in red and gam genes. These phages can form plaques on a lawn of an E. coli P2 lysogen (Spi^- phenotype), while normal -phages cannot. The Spi^- phage is thus easily selected out of a large number of phages, and the number of the Spi^- phages was assumed to be the same as that of bio transducing phages.

To examine the effect of hydrogen peroxide on the formation of the bio transducing phage, E. coli lysogens grown to mid-log phase were exposed to hydrogen peroxide and incubated at 42° for 15 min to induce growth of -phage. The cells were then centrifuged to remove hydrogen peroxide, allowed to lyse by incubation at 37°, and phage lysates were prepared to measure the frequency of Spi^- phages. As shown in Figure 1, the frequency of Spi^- phage was increased in a dose-dependent manner. In an E. coli lysogen, HI2382, the frequency of Spi^- phage formation by exposure to 2 mM of hydrogen peroxide was about 260-fold higher than the frequency without hydrogen peroxide.

View larger version (20K): In this window In a new window Download PPT slide   Figure 1. The effect of mutations in the mutM or its related genes on Spi- phage formation in the increasing amount of hydrogen peroxide. E. coli lysogens were exposed to hydrogen peroxide, and phage lysates were prepared as described in the text. The frequency of Spi- phages was defined as the ratio of the number of Spi- phages to the number of total phages. The frequencies in a wild-type strain (HI2382), mutM strain (HI2384), mutT strain (HI2383), and mutY strain (HI2385) are represented as open circles, open squares, solid squares, and open triangles, respectively. Four independent experiments were performed and standard errors are indicated on the values of wild-type strain (HI2382) and mutM strain (HI2384).

The numbers of total phages per cell, burst sizes, were 54, 17, 15, and 7.0 in the increasing order of hydrogen peroxide concentration, i.e., 0, 0.5. 1.0, and 2.0 mM, in the wild-type strain. The stimulation of the frequency of Spi^- phage formation by hydrogen peroxide is not due to the decrease in the total number of phages, because the titer of total phages after exposure to 2 mM of hydrogen peroxide was not <14% of that without hydrogen peroxide. However, the frequency of the Spi^- phage was increased 190-fold or >190-fold, depending on the strains used here.

Effect of the mutM mutation on frequency of Spi^- phage:
The E. coli lysogen HI2382 wild type and its mutM, mutT, and mutY derivatives were subjected to the Spi^- assay. The frequency of Spi^- phage in the HI2384 mutM lysogen was 5- to 12-fold higher than that of the wild type (Figure 1). In contrast, the frequency of Spi^- phage in the HI2383 mutY or HI2385 mutT strain was comparable to that of the wild type. The mutM, mutY, and mutT genes are known to be transversion-specific mutators and are involved in the repair of 8-oxoG residues. Among these genes, only the mutM gene was shown to be effective in suppressing illegitimate recombination. The differential effects of these mutator genes on illegitimate recombination are discussed below.

Distribution of recombination junctions in bio transducing phages:
Spi^- phages are reported to have recombination sites ranging from int to git in the genome and from bioA to uvrB in the bacterial genome (YAMAGUCHI et al. 1995 ). To examine the properties of Spi^- phages induced by hydrogen peroxide, independent Spi^- phages were isolated from two lysogens, HI2382 wild type and HI2384 mutM, after exposure to hydrogen peroxide. The locations of recombination sites were deduced by serial PCRs using several primers as described in MATERIALS AND METHODS. All Spi^- phages isolated were bio transducing phages because they had the attR site. The bio transducing phages, HW3, HW10, HW12, HW14, HW28, HW30, HW32, HW38, HW42, HW43, HM18, and HM50, produced the same-sized PCR products with the set of primers, #404 and #408. From the sequence analysis, these PCR products were found to have a unique recombination junction, which is formed by recombination at Hotspot I, seen in UV-induced or spontaneous illegitimate recombination (YAMAGUCHI et al. 1995 ). Figure 2 shows the distribution of recombination sites. HW8, HW16, and HW29 produced the same-sized PCR products and were found by sequence analysis to have the same recombination site. HW22 and HW45 also produced the same-sized PCR products and had the same recombination site. The recombination site of W22 and W45 was the same site as that of U4 and U31 bio transducing phages induced by UV from int lysogen, HI1631 (SHANADO et al. 1997 ). These two sites could be hotspots for illegitimate recombination (called Subhotspot I or II in Figure 2). From the results of PCR analysis, the bio transducing phages, which recombined at hotspot sites, were not observed in the mutM strain, except for HM18 and HM50. The latter two were shown to be formed by recombination at Hotspot I. The percentage of phages that have recombination sites at Hotspot I were 22 and 4% of the total bio transducing phages, in the wild-type and mutM lysogens exposed to hydrogen peroxide, respectively. Both ratios, particularly that of the mutM strain, are lower than that of UV-irradiated and spontaneous bio transducing phage formation. The ratio was 57% for UV-irradiated bacteria and 77% for unirradiated bacteria (YAMAGUCHI et al. 1995 ). These results show that a larger number of bio transducing phages were formed by recombination at nonhotspot sites in the presence of hydrogen peroxide than at hotspot sites, i.e., Hotspot I and Subhotspots I and II.

View larger version (26K): In this window In a new window Download PPT slide   Figure 2. Prophage structure and distribution of recombination sites. (A) Prophage structure. Gray bar indicates E. coli genome region and the rectangle between gray bars indicates genome. The nucleotide numbers described under genome correspond to the length from cos site on genome map. The numbers described under E. coli genome show the nucleotide number from attR site. (B and C) Distribution of recombination sites on the formation of bio transducing phages in a wild-type strain (B) and the mutM strain (C) by exposure to hydrogen peroxide. Vertical lines indicate the map coordinates of genome and horizontal lines indicate the map coordinates of E. coli bio and uvrB genes. These nucleotide numbers correspond to the numbers described in A. The boxes marked Hotspot I, Subhotspot I, and Subhotspot II indicate a group of bio transducing phages that are produced by recombination at hotspots.

Nucleotide sequences of recombination junctions of bio phages from lysogen exposed to hydrogen peroxide:
In addition to bio transducing phages that are recombined at Hotspot I or subhotspots, sixteen other phages were picked randomly from the wild-type or the mutM strain and subjected to sequencing. Recombination junctions were amplified by PCR with proper primers, the PCR products were isolated, and the sequences of recombination junctions, which were derived from parental E. coli and -phage genomes, were determined. Figure 3 shows the sequences of the recombination junction at Hotspot I (A), Subhotspot I (B), Subhotspot II (C), and some sequences at nonhotspot sites out of each of the sixteen phages. In HW5, HW7 (Figure 3I), HW31, HM23 (Figure 3N), and HM38, there is an overlap of 1 bp at the junction. Similarly, an overlap of 2 bp in HW11 (Figure 3H), HW13, HW17, HW27, HW44, HW48, HM1, HM2, HM10, HM11, HM12, HM13, HM32, and HM41 (Figure 3M); an overlap of 3 bp in HW2, HW15, HW37 (Figure 3G), HM17, HM34, and HM37 (Figure 3L); an overlap of 4 bp in HW36 (Figure 3F), HM7 (Figure 3K), and HM31; and an overlap of 5 bp in HW9 (Figure 3E) and HW39 are found. There are overlaps of 7 and 8 bp in HW21 (Figure 3D) and HM20 (Figure 3J), respectively. Figure 4 shows the distribution of length of overlapped nucleotide(s) in the junction of bio transducing phages sequenced. All the sequences of recombination junctions that we examined had overlapped nucleotide(s) within their recombination sites.

View larger version (56K): In this window In a new window Download PPT slide   Figure 3. Nucleotide sequences of junctions derived from bio transducing phages in a wild-type strain (HI2382) and the mutM strain (HI2384) by exposure to hydrogen peroxide. The recombination junction sequences of phages recombined at Hotspot I, Subhotspot I, Subhotspot II, and nonhotspot sites are described. The junction sequences at nonhotspot sites had one or some overlapping nucleotide(s) and we list examples from both strains. The boxed sequences represent homology at recombination sites or around the recombination sites between the parental recombination sites. The map coordinates for phage and bacterial sequences are indicated.

View larger version (20K): In this window In a new window Download PPT slide   Figure 4. Distributions of lengths of overlapped nucleotide(s) at recombination sites in wild-type strain HI2382 (A) and in mutM strain HI2384 (B). The total numbers of bio transducing phages of which recombination junctions were sequenced are 32 and 18 in the wild-type strain and mutM strain, respectively. The y axes in A and B indicate the ratios of bio transducing phages examined and x axes indicate numbers of nucleotide(s) overlapped between DNA and E. coli genome. All bio transducing phages examined showed overlapped nucleotide(s) at their recombination sites.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Illegitimate recombination is an extraordinary recombination event that was first identified by FRANKLIN 1967 . Normally a prophage present in an E. coli genome is excised precisely at both ends, attR and attL. Very infrequently, precise excision does not occur but the E. coli DNA fragment adjacent to the prophage genome is integrated to generate transducing phage DNA. Illegitimate recombination has been observed in prokaryotes as well as eukaryotes and takes place between regions of little or short homology at two different sites of DNA(s) and results in genome rearrangement (MULLER 1927 ; TESSMAN 1962 ). In this experiment, we showed that oxidative stress generated by hydrogen peroxide increases the frequency of illegitimate recombination up to 260-fold during the formation of bio transducing phage. To our knowledge, this is the first quantitative experiment indicating that illegitimate recombination is affected by oxidative stress. Furthermore, the formation of bio transducing phage is 5- to 12-fold more frequent in the mutM strain than in the wild-type strain upon exposure to hydrogen peroxide, although the frequencies in the mutT and the mutY strains were similar to those of the wild type at each dose of hydrogen peroxide. These results indicate that MutM, but not MutT or MutY, can repair hydrogen peroxide-generated DNA damage, which induces illegitimate recombination.

As described above, the mutM, mutT, and mutY genes are involved in 8-oxoG-induced mutation. It is known that 8-oxoG is generated in bacterial DNA by exposure to hydrogen peroxide (KASAI et al. 1986 ). When 8-oxoG is formed in a DNA strand, MutM protein removes the 8-oxoG residue and the subsequent repair system reverses the mutation to an original G:C base pair. MutY protein does not remove the 8-oxoG residue but removes the misincorporated adenine residue on the opposite side of the 8-oxoG residue. It is therefore thought that the persistence of 8-oxoG in DNA may induce illegitimate recombination in the mutM mutant but not in the mutY mutant. Illegitimate recombination is thought to occur shortly after exposure to hydrogen peroxide. There may not be enough time for the generated 8-oxo-7,8-dihydrodeoxyguanosine triphosphate to be incorporated into DNA strands and therefore MutT activity is not involved in the occurrence of 8-oxoG residues.

It has been reported previously that Fapy residues are formed by the reaction between DNA and hydrogen peroxide (DIZDAROGLU 1992 ). The MutM protein excises Fapy residues as well as 8-oxoG residues (TCHOU and GROLLMAN 1993 ). Hence the Fapy lesion may also be involved in the hydrogen peroxide-induced illegitimate recombination of the mutM strain. Even if this were possible, MutY and MutT proteins would not be involved in the suppression of the illegitimate recombination.

Besides the lesions that can be repaired by MutM protein, it is known that hydrogen peroxide generates a variety of types of DNA damage, for example, thymine glycol, cytosin glycol (HALLIWELL and ARUOMA 1991 ), apurinic/apyrimidinic sites (POVIRK and STEIGHNER 1989 ), and strand breaks (MASSIE et al. 1972 ). It is also possible that there are multiple types of lesions by hydrogen peroxide that trigger illegitimate recombination.

Illegitimate recombination is classified into two classes, SHIIR and SHDIR (SHIMIZU et al. 1997 ). It is presumed that the illegitimate recombination induced by hydrogen peroxide belongs to SHDIR, because all bio transducing phages examined were recombined with overlapping nucleotide(s) between -phage DNA and E. coli genome. In the SHDIR induced by UV, none of the generated bio transducing phages recombined without overlapping nucleotide(s) have been found (YAMAGUCHI et al. 1995 ; UKITA and IKEDA 1996 ; SHANADO et al. 1997 ), while the SHIIR, which is mediated by DNA gyrase, does not require any short region of homology (SHIMIZU et al. 1997 ). It is likely that an annealing step between broken DNA ends is necessary for SHDIR and therefore it is thought that overlapped sequences near broken DNA ends play an important role in this event. Illegitimate recombination induced by hydrogen peroxide, however, has different characteristics compared to UV-induced illegitimate recombination. In the illegitimate recombination induced by hydrogen peroxide, the recombination at nonhotspot sites is more remarkable than that at hotspot sites.

Hotspot I and subhotspots were observed as one of the recombination sites of the wild-type strain, although hotspots were not predominant in the recombination sites of the mutM strain. Because the frequency of the Spi^- phage in the mutM strain is 5-fold higher than in the wild type, these results indicated that the recombination at nonhotspot sites takes place with a higher frequency in the mutM strain compared to the wild type and that the frequency of illegitimate recombination at Hotspot I may be at a similar level in both strains. The increased frequency of the recombination at nonhotspot sites in the mutM strain is probably due to 8-oxoG and/or Fapy formed in DNA.

The question remains as to how illegitimate recombination is induced by oxidative stress and how it is suppressed by MutM function. As for the formation of bio transducing phage, double-strand breaks of the bacterial chromosome are thought to be required for the excision of the prophage as was discussed by UKITA and IKEDA 1996 . We assume that the 8-oxoG or Fapy species of DNA damage would somehow induce double-strand breaks during DNA replication and that the breaks would result in the formation of bio transducing phage through the joining of DNA ends. YAMAGUCHI et al. 1995 suggested that thymine dimers produced by UV-irradiation induce double-strand breaks at a specific region, which contains direct repeat sequences. In contrast, hydrogen peroxide produces other types of lesions, 8-oxoG and/or Fapy, which may induce double-strand breaks at other sites of DNA. To test this possibility, localization of break sites induced by UV-irradiation and oxidative stress must be determined at a nucleotide sequence level.

	FOOTNOTES

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

	ACKNOWLEDGMENTS

We thank Drs. Y. Nakabeppu, H. Maki, M. Sekiguchi, and T. Nohmi for providing us with bacterial strains. This work was supported by Grants-in-Aid for Scientific Research Category B and Scientific Research on Priority Areas to H.I. from the Ministry of Education, Science, Sports, and Culture of Japan and by the Uehara Memorial Foundation.

Manuscript received August 31, 1998; Accepted for publication November 5, 1998.

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