Mutations by UVSW Mutations

Genes uvsW, uvsX and uvsY are dispensable for T 4 growth but are implicated in recombination and in the repair of damaged DNA. We found that large-plaque mutants arose efficiently from smallplaque uvsX and uvsY mutants at 42" and were pseudorevertants containing a new mutation in uusW. Using reconstructed double mutants, we confirmed that a mutation in uvsW partially increases the burst size and UV resistance of uvsX and uvsY mutants. At 41 the uvsW mutation completely restores the arrest in DNA synthesis caused by mutations in genes uvsX, uvsY and 46 , but at 30" it only partially restores DNA synthesis in a gene 46 mutant and does not restore DNA synthesis in uvsX and uvsY mutants. Restored DNA synthesis at 41 O was paralleled by the overproduction of single-stranded DNA and gene 32 protein. Based on these findings, we propose that the uvsW gene regulates the production of single-stranded DNA and we discuss the phenotype of uvsW mutants and their suppression of some uvsX and uvsY phenotypes. Infection of restrictive cells with am uvsW mutants revealed a defect in the synthesis of a protein of molecular weight 53,000 daltons, suggesting that this protein is the uvsW gene product. ACTERIOPHAGE T 4 genes uvsW, uvsX and uvsY are implicated in the processes of genetic recombination and in the repair of DNA damages (HAMLETT and BERGER 1975; CUNNINGHAM and BERGER 1977). From pathway analysis of DNA repair, all of these genes are associated with a set of genes involved in recombinational repair (HAMLETT and BERGER 1975) and their mutations commonly reduce ssDNA of longer than unit length whose production depends on DNA recombination (HAMLETT and BERGER 1975; CUNNINGHAM and BERGER 1977). Mutations in genes uvsX and uvsY are phenotypically similar: genetic recombination and resistance to UV irradiation decrease to similar extents (HAMLETT and BERGER 1975; DEWEY and FRANKEL 1975; CUNNINGHAM and BERGER 1977), DNA synthesis is arrested late in the infection (DEWEY and FRANKEL 1975; CUNNINGHAM and BERGER 1977) and the lethality of gene 49 (endonuclease) mutants is suppressed (DEWEY and FRANKEL 1975; CUNNINGHAM and BERGER 1977; CONKLING and DRAKE 1984a; YONESAKI, MIYAZAKI and MINAGAWA 1984). Wu, YEH and EBISUZAKI (1 984) showed by genetic crosses that dar mutations, which were discovered as suppressors of gene 59 mutations (WU and YEH 1975), and uvsW mutations, which were found as UV-sensitive, minute-plaque variants (HAMLETT and BERGER 1975), are in the same gene. The phenotype of uvsW mutants differs from that of uvsX and uvsY mutants. UV resistance and genetic recombination are less reduced by uvsW mutations than by uvsX and uvsY B Genetics 115: 219-227 (February, 1987) mutations (HAMLETT and BERGER 1975; CUNNINGHAM and BERGER 1977; see also Table 2). Only the uvsW mutants are sensitive to hydroxyurea (HU) (HAMLETT and BERGER 1975; Wu and YEH 1975), they synthesize DNA continuously (HAMLETT and BERGER 1975; Wu and YEH 1975), and they overproduce gp32 (KRISCH and VAN HOUWE 1976; WU and YEH 1978a). They suppress the arrested DNA synthesis caused by mutations in genes 46 and 47 (which determine an exonuclease activity) (CUNNINGHAM and BERGER 1977) and in gene 59 (Wu and YEH 1975; CUNNINGHAM and BERGER 1977), but have been reported not to suppress arrested DNA synthesis caused by mutations in uvsX and uvsY (CUNNINGHAM and BERGER 1977). The lethality of gene 49 mutations is not suppressed by uvsW mutations (CONKLING and DRAKE 1984a). In this paper, we show that mutations in uvsW are intergenic suppressors of mutations in uvsX and uvsY and discuss the mechanism of suppression and the phenotype of uvsW mutants. MATERIALS AND METHODS Bacteria and phages: E. coli B40sul (sui+) was the permissive host and BB (sa-) and BE (su-) were nonpermissive hosts for am mutants. ED8689 (suhsdRtrp-) was used as a carrier of plasmids. Mutant phages used were a m H l 1 (gene 23), amN134 (gene 3 3 ) , amN88 (gene 44) , amNI30 (gene 46) , amBL292 (gene 55), amEl120 (gene 62) and m22 (gene uvsw). amxb (gene uvsx) and amyd (gene uusr) were described previously (YONESAKI, MIYAZAKI and MINACAWA 1984). The additional uvsW mutations amwa3, amwcl, amwd2 and tswl were isolated in this study. 220 T. Yonesaki and T. Minagawa Plasmids: pHTL8 is a chimeric plasmid, carrying a 1 1.6kb T 4 DNA insertion in pBR322 at a Sal1 site, on which nine known T 4 genes (including uvsY and uvsW) are located (TAKAHASHI and SAITO 1982). T h e insert has several sites for PstI and Hind111 (Figure 1). With either restriction enzyme, we subcloned T 4 DNA fragments (pBSK1 and pBSK6) or deleted DNA fragments (pHTL8-H1, pHTL8H3, pHTL8-P2 and pHTL8-P3) in pBR322 (Figure 1). Assay for HU sensitivity: Addition of 0.2 ml of 100 mg/ ml HU and a few drops of a log-phase culture of indicator bacteria was made to 2.5 ml of melted PG (MINAGAWA and RYO 1978) soft agar and poured on 20 to 25 ml PG hard agar in a 9-cm dish. T h e phage (ca. 108/ml) to be assayed was transferred to the plate by a platinum loop or printed by a sterile nailhead as described in the next section and the plate was incubated at 37". Our bacterial strains could not grow at 30" on the HU-containing plate but did grow well at 37" and 42". Assays for UV sensitivity: Phage suspensions were irradiated under a Toshiba 15 W germicidal lamp at a dose rate of 0.8 J m-' sec-'. As a routine assay for isolating UV-sensitive clones, a plaque was sampled with a toothpick, suspended in one drop of M9 in the hole of a microtitration tray, and transferred to appropriate plates before and after UV irradiation for 2.5 min. To confirm the UV sensitivity, w e used the semiquantitative method of MINAGAWA, YONESAKI and FUJISAWA (1 983). Two drops of each phage suspension (ca. 1 O"/ml) were put in seven adjacent holes of a microtitration tray, which was then covered with another tray lapped by aluminum foil. Every 40 sec, the top tray was moved to uncover an additional hole, so that suspensions received 240, 200, 160, 120, 80, 40 and 0 sec of UV irradiation. Irradiated suspensions were transferred onto agar plates, which had been seeded with appropriate host cells, using nailheads which had been set into a plastic plate so as to fit the holes on the tray. After incubation, the wild type produced a lysed area even after 240 sec of irradiation, while uvsWdid not produce lysis after more than 200 sec and uvsXand uvsYdid not produce lysis after mpre than 120 sec. To get survival curves, a 5-ml phage suspension (ca. lo8/ ml) in a Petri plate was irradiated for appropriate periods. Marker-rescue assay: Mutated genes were identified by tests scoring UV sensitivity which was measured semiquantitatively by printing phage suspensions on plates which contained BB cells and UV-killed amxb, amyd, m22 or amwcl (2 X 1 0* particles/plate). UV-killed phages were prepared by irradiating 5 ml of phage suspensions (ca. 2 X 109/ml) in a Petri plate for 2.5 min. We also screened for the uvsW gene by scoring the HU sensitivity of the mutants. Phage suspensions (ca. 108/ml) were transferred to HU plates with a platinum loop; the plates had been seeded with ED8689 cells transformed by a plasmid carrying the whole or an appropriate fragment of the uvsW gene. Measuring DNA synthesis: BB cells were grown to a density of 5 X 10s/ml in M9A (MINAGAWA and RYO 1978) at 37". They were spun down and resuspended in the same volume of fresh M9A. After incubation for 10 min at the desired temperature, they were infected with phage at a multiplicity of infection (m.0.i.) of five. Five min after infection, 0.1 volume of a mixture containing deoxyadenosine (2 mg/ml), thymidine (50 pg/ml) and ['Hlthymidine (40 pCi/ ml) was added. At times, 20-pl aliquots were removed and blotted on filter paper discs (Toyo No. 2). T h e discs were rinsed in ice-cold 10% trichloroacetic acid (TCA), then in acetone, and dried. Their radioactivities were counted by a Packard liquid scintillation counter. Measuring ssDNA with S1 nuclease: T o avoid the complexity of encapsidation of T 4 DNA, the mutation amHl I in gene 23 (major head protein) was included in the genetic background. Infected BB cells were labeled as above except that the final ['Hlthymidine was at 10 ECi/ml. At 40 min after infection, 100 yl of culture were removed and quickly cooled in ice water. T h e infected cells were spun down, resuspended in 20 pI of a mixture of 20 mM Tris-HCI (pH 7.4), 2 mM EDTA and 200 pg/ml egg white lysozyme, and incubated for 30 sec at 37 O . Two pl of 10% SDS were added to the suspension which was further incubated for 30 min to completely lyse the cells. T h e lysate was diluted 25-fold with distilled water and two 135-pl aliquots were prepared. Both received 15 pI of S1 buffer (2 M NaCl, 0.5 M sodium acetate at pH 4.4 and 0.5 mM ZnS04) and one received 1 pl of S1 nuclease ( lo6 units/ml). After incubating at 30" for 30 min, 140-pl samples were applied to filter paper discs, which were washed with 10% TCA, then with acetone, and dried. Radioactivities were measured and S 1 -sensitive fractions were expressed as the fraction (difference of radioactivity between mock incubated and S1 -treated)/(radioactivity of mock incubated). Electrophoresis of T4 proteins and measurement of gp32: BB cells were grown to a density of 5 X 108/ml in M9A, collected and resuspended in 0.5 volume of M9. After 20 min incubation at the desired temperature, the cells were infected at a m.0.i. of five. T 4 proteins were labeled with L[3H]leucine (5 pCi/ml) for appropriate periods, electrophoresed through SDS-10% polyacrylamide gel and detected by fluorography as described previously (YONESAKI et al. 1985). To estimate gp32 synthesis, infected cells were labeled at two infection periods; the first was when the total synthetic rate of the wild-type was maximum, and the second was when this rate was reduced. Gp32 bands on X-ray films were quantitated by tracing with a Toyo digital densitorol (model DMU-33C) at 620 nm, and relative intensities were expressed by normalizing the value to that of the wild type at the first period at the same temperature. Chemicals: Chemicals were purchased from the following sources: ~[~H]leuc ine (specific activity, 64 Ci/mmol) and ['Hlthymidine (specific activity, 4 1 Ci/mmol) from Amersham; hydroxyurea from Nakarai Kagaku; deoxyadenine and thymidine from Sigma; S1 nuclease from Riken; restriction enzymes PstI and Hind111 from Takara Shuzo.