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M~ Rese=rch, 28~ (1~;~3) 2>'3-265 ~ 1~93 Els~der Se~e PuSl~h-~rs B.V. ALl r~,~s re~':=d C~.)27-ff107/93/$O&c.~ 253 MUT 05226 Participation of the SOS system in producing deletions in E. coli plasmids Elias Balbinder, Bryan Cell, Jeff Hutchinson, Anthony S. Bianchi *, Tobi Groman *, Kevin A. Wheeler * and Matt Meyer * Department of Biochemistry, B~ophysics and Genetics, and Colorado Cancer Center, University of Colorado Health Sciences Center, 4200 E. Ninth Auenue, P.O. Box B-122, Denver, CO 80262, USA (Received 7 January 1992) (Revision received 22 September 1992) (Accepted 25 September 1992) Keywords: SOS ~stem, participation; Eseheriehia coil plasmids; Plasmids, E. coli Summary The participation of the SOS response in the deletion of palindromic and non-palindromic inserts of about 66 and 100 bp cloned within the EcoRl site of the ehloramphenicol acetyl transferase (cat) gene of plasmid pBR325 was tested after introducing the derived plasmids into strains containing different combinations of let.A, recA and urauC alleles and the auxotrophie mutation trpE65. This allowed for a coml~arison of deletion frequency in the plasmids, measured as the reversion of ehloramphenicol sensitivity to resistance (CmS~ Cmr), to l~oint-mutation frequency measured from the reversion of trpE65 to ttyptophan independence (Trp-~ Trp +). We found that the spontaneous deletion frequency of palindromic inserts was increased by the overproduction of activated ReeA * and UmuC÷ in lexA (Def) backgrounds but the deletion of the non-palindromic inserts was unaltered. Overproduction of ReeA+ had no significant effect on deletion incidence but it did increase Trp-~ Trp÷ reversions. The SOS stimulation of palindrome deletions paralelled the SOS mutator effect of certain recA and urauC alleles on Trp---> Trp÷ reversions, suggesting that some form of SOS processing was responsible for the observed increases. The results further suggest that the SOS effect on deletions depends on the distinction between palindromy vs. non-palindromy, rather than on the sizes or sequences of the inserts or those of the terminal homologies bracketing them. Spontaneous deletions in prokaryotie replicons can occur through a variety of mechanisms and include the resolution of unstable transient inter- * Summer Sludent Fellows of the Colorado Cancer Center. Correspondence: Dr. Elias Balbinder, Department 0£ Bio- chemistry, B|t~physics and Oe~n,~t.[cs and Colorado Cancer Center, Uni','ersi~ of Colorado Health Sciences Center, 4209 E. Ninth Avenue, P.O. Box B-121, Denver, CO 80262, USA. mediates formed in the normal course of DNA metabolism (Albertini et al., 1982; Ripley and Glickman, 1983), intermoleeular recombination events (Dianov et al., 1991; Mazin et al., 1991; Singer and Westlye, 1988) or mistakes in the movement of transposable elements (Kleckner et al., 1979). A large number of deletions occur between direct terminal repeats, which often flank inverted repeats (palindromes). According to mis- alignement mutagenesis models, these sequences can transiently stabilize unstable intermediates such as hairpins or erucfforms which may form on 40000124

254 single-stranded stretches of DNA as the result of slippage during replication (Albertini et al, 19~2; Balbinder et al., 1989; DasGupta et al., 19S7; Glidanan and Ripley, 1984; Jones et al., 1982; Meulien et al., 1981; Ripley and GlicI~man, 1983; Singer and Westlye, 1988; Weston-Hafer and Berg, 1989, 1991). Cruciform structures in plas- raids have actually been shown to be present, in vivo (Zheng et al., 1991). Transient-deletion in- termediates could be removed by either a recom- binational event, misreplieafion across the base of a misaligned secondar~ structure, or nuclease ex- cision of protruding hairpins or unp:aired loops (refs. above). It is possible that all 3 mechanisms may participate to different extents in the forma- tion of the same deletions. The possibility that the SOS response could participate in giving rise to deletions and other rearrangements has been suggested (Eehols, 1981; Little and Mount, 1982) and supported by the demonstration that certain duplications in the E. coli chromosome were ineremsed by derepression of the SOS regulon and the participation of the reef recombination pathway (Dimpfl and Eehols, 1989). One important unanswered question con- cerns the role of recA in deletion formation. Most deletions reported in E. coli occur with the same frequency in recA+ and tee_d- cells (Franklin, 1967, 1971; Inselburg, 1967; Jones et al., 1982; Das Gupta et al., 1987) but some are stimulated by recA + either dramatically (Sommer et al., 1981; Albertini et al., 1982; Goldberg and Mekalanos, 1986) or by low but detectable factors (Williams and Muller, 1987; Balbinder et al., 1989). Aibertini et at (1982) found that certain deletions in lacl were increased 25-fold in rec,,l÷ as compared to recA-baekgrounds, yet Miller and Low (1984) found that induction of the SOS response in a tif-1 (recA44D strain at 42°C in- creased the frequency of base substitutions but not frameshifts or deletions in lacL rec.,'1441 codes for a genetically activated ReeA* which leads to derepression of SOS functions at 42"C in tlxe presence of excess adenine (Walker, 1984; Miller and Low, 1984). For hlstofieal reasons the partici- pation of rer.A* in deletion formation has been considered mainly in terms of its role in homolo- gons recombination (Clark and Margulies, 1965) and some deletions may be produced through such mechanism (Whoriskey et al., 1991). How- ever, RccA is a multifunctional protein and plays essential parts in DNA repair and SOS mutagen- esis in addition to homologous recombination (for review see Walker, 1984). In mutagenesis alone it plays three major roles: (1) facilitates the cleaving of LexA, the repressor of the SOS regulon thus regulating the expression of the SOS genes, (2) facilitates the proteolyfic activation of UmuD to UmuD' which is essential for mutagenesis (Burkhardt et al., 1988; Nohmi et al., 1988; Shina- gawa et al., 1988) and (3) plays a direct role, presumably in translesion replication (Dutreix et al., 1989; Sweasy et al., 1990). In this report we explore the possibility that certain deletions in derivatives of plasmid pBR325 occur through the participation of SOS functions and that the in- volvement of recA in these events is related to its roles in mutagenesis rather than recombination. Materials and methods Culture media L-broth and L-agar were prepared according to standard recipes (Silhavy, Berman and En- quist, 1984). Chloramphenicol and ampieillin (Sigma) and tetracycline (Aldrich) were added when neeessat3, at final concentrations of 25 p.g/ml. Selection o1~ Trp + revertants was on mini- real medium E (Vogel and Bonnet, 1956) supple- mented with 2.5% (v/v) nutrient broth (SEM, Witkin et al., 1982). Bacterial strains The strains employed in this investigation are listed and their modes of origin indicated in table 1A. Strains Nos. 3, 7, 8 and 9 are from the collection of E. Witkin. All the others shown in the table were obtained by us either through by the loss of TnlO, detected by the procedure of Maloy and Nunn (1981), or the replacement of selected markers by bacteriophage P1 mediated transdttction, as we will describe next. Strains EB706 (No. 11), EB714 (No. 14) and EB799 (No. 17) were derived from SC30SP (EB699, No. 8) by replacing respectively, the following markers: recA730 by recA + in EB706 and ArecA306 in EBT14, and umuC ÷ by umuC122 :: Tn5 in EB799. Strains EB709 (No. 12) and EB710 (No. 15) were 40000125

255 TABLE 1 BACTERIAL STRAINS USED IN THIS INVESTIOATION (A) ,.,:tm/r~ rwz can3~g p!asrr, Ma No. Strain Relevant Original Origin or reference ree.A /era UmuC Other designation 1 EB218 A306 + + (X re_zA*) 2 EB6g8 + + + 3 EB222 + + + 4 EB788 + 71 ::Tn5 122::Tn5 5 EBB15 730 71::Tn5 122::Tn5 6 EBSI3 7:10 + 122 :: To5 7 EB221 730 + + 8 EB699 730 71 :: Tn5 + 9 • EB701 7:70 + 36 10 EB30I + + + 11 EBT06 + 71 :: Tn5 + 12 EB709 + + 36 13 EB305 A306 + + 14 EB714 A306 71 :: Tn5 + 15 EB710 A306 + 36 16 EB797 730 + 122 :: Tn5 17 EB799 730 71 :: Tn 5 I22 :: Tn5 18 EBB01 + + 122 :: Tn5 19 EBB19 A306 71 ::Tn5 122 :: Tn5 20 EB~I7 A306 + 122 :: Tn 5 21 EB821 + 7/:: Tn5 122 :: Tn5 sdC300 ::Tnl0 trpE65 srlC300 :: TnlO trpE65 srlC300 :: Tnl.O fadR261 :: TnlO trpE65 trpg65 trpE65 trpE65 trpE65 trpE65 trpE65 trpE65 trpE65 trpg65 trpE65 trpE65 fadR261 :: TnlO trpE65 fadR261 :: TnlO trpE65 fadR261 :: Tnl 0 trpE65 fadR261 :: TnlO trpE65 fadR261 :: TnlO trpE65 fadR261 ::Tnl0 ESIx)45 SC30RP-uvrA SC30RP EST'2590 SC30 SC30SP SC~0 umuC36 Tess'man and Pete~soa 0985) E. Witkin Witkin et aL (1982) P.K. Peterson This work 'Tet~ revertant of EB799 (No. 17, below) Tets rev. of EB797 (No. 16, below) Wilkin and Kogoma (1984) Witldn and I~goma (1984) E. Witkin This work, Derived from EB222 by loss of TnlO This work, Pt, EB698 ×EB699 This work, PI, EB698 xEBT01 This work" PI, EB218 ×EB301 This work, Ft, EB218 ×EB699 This work. Pz, EB218 x EB701 This work, PI, EB788 × EB221 This work, P. EB788 ×EB699 This work, Pz, EB788 XEB301 ~ work, P1, EB218 XEB815 Tlais work" Pz, EB218 XEBS13 Th~ work" Pz, EB222 ×EBB15 40000128

TABLE 1 ~B) StraL'~s dedced from (.A) ~oce carr~n~ pfasmid pOCEI5 No. Strains Plasmid in Strains 22 EB389 pOCE15 EB301 23 EB716 pOCEI5 EB709 24 EBB07 pOCE15 EB801 25 EB306 pOCE15 EB221 26 EB312 pOCE15 EB305 27 EB724 pOCE15 EBT01 28 EB728 pOCE15 EB710 29 EBSZ5 pOCEI5 EBB17 30 EBB04 pOCE15 EB797 31 EB726 pOCE15 EB706 32 EB829 pOCE15 EB821 33 EB722 pOCE15 EB699 34 EB730 pOCE15 EB714 35 EB823 pOCE15 EB799 36 EB827 pOCE15 EB8|9 (A) Origin of strains with various combinations of recA, ler.A and umuC alleles. (B) Strains in A transformed with plasmid pOCE15. For details of strain construction see Materials and methods. derived from SC30 umuC36 (EB701, No. 9) by replacing recA730 with recA+ (EB709) and ArecA306 (EBT10). EB301 (No. 10) is Witldn's SC30RP (EB222, No. 3) minus TnlO and was used as the recipient in transduction crosses for the construction of strain EB305 (No. 13) by replacing recA + with ArecA306, and EBB01 (No. 18) by replacing umuC + with umuC122 :: Tn5. Strain EB797 (No. 16) was obtained by replacing the umuC-~ allele of EB221 (No. 7) with umuC122 :: Tn5. As donors in Pl-mediated transduetion, strains EB698 (No. 2) and EB222 (No. 3) were used as sources of recA + and EB218 (No. 1) was the source of ArecA306. All 3 strains carried the closely linked marker srlC300 ::Tnl0 which al- lowed for selection of tetracycline resistant (Tetr) transduetants. EST2590 (EB788, No. 4) from the collection of Tessman and Peterson was used as donor of umuC122 :: Tn5 t "aking advantage of its linkage to fadR261 :: TnlO which also allowed for the selection of Tetr transduetants. These were tested for simultaneous transfer of Tn5 by resis- tance to kanamycin (Kin0 and a concomitant drop in spontaneous Trp--> Trp÷ reversion fre- quency (Tessman and Peterson, 1985; Sweasy et al., 1990), as well as for their genoWpe as de- sen'bed below. Strain EBB15 (No. 5) is EB799 minus TnlO and was the recipient for the con- struction of EBB19 (No. 19) and EBB21 (No. 21) by replacing recA730 with ArecA306 in EBB19 and recA + in EBB21 respectively. Strain EBB13 (No. 6) is EB797 minus TnlO and was used to construct EB817 (No. 20) through the replace- ment of recA730 with ArecA306. Transduetants were tested for genotype by us- ing them as donors or recipients in transduction crosses. Linkage of umuC122 :: Tn5 to fadR261 :: TnlO was confirmed by using lysates grown in EB797 (No. 16), EB799 (No. 17) and EB801 (No. 18) to transduce EB221 (No. 7) and testing the Tet' transductants recovered in these crosses for Kmr and a simultaneous drop in Trp- -~Trp+ reversion frequency from about 200 revs/plate to about 10-30 (compare Nos. 24, 30 and 35 to No. 25 in Table 2). The genotypes of EB817 (No. 20) and EB819 (No. 19), which car- ried srlC300 ::Tnl0 arecA306 from EB218 and EB821 (No. 21) where the recA730 of EB815 was replaced by srlC300 :: TnlO recA+ of EB222, were confirmed by using lysates grown on these strains to transduce EB699 (No. 8) or EB221 (No. 7) and testing Tetr Srl- transduetants from these crosses for a 10-30-fold drop in Trp-~ Trp + reversion frequency (compare Nos. 29, 32 and 36 to Nos. 25 and 33 in Table 2, and Nos. 19 and 21 to Nos. 7 and 8 in Table 3). The genotypes of EBB13 (No. 6) and EB815 (No. 5) were confirmed by replac- ing umuC122 :: Tn5 with umuC ÷ fadR621 :: TnlO and testing Kms transduetants for an increase in Trp-~ Trp ÷ reversion frequency to the level of EB221 (~ 200 rev./plate) or EB699 (~ 500-600 revs./plate) (compare Nos. 30 and 36 to Nos. 25 and 33 in Table 2). Plasmids The construction of the plasmids used in these investigations has been descn'oed (Betz and Sadler, 1981; Balbinder et al., 1989; Sinden et al., 1991). The relevant characteristics of these plas- raids are shown in Fig. 1 and will be described under Results. The plasmids were introduced into the desired strains by transformation as deser~ed previously (Balbinder et al., 1989). 40000127

TABLE 2 FREQUENCIES AND RATES OF SPONTANEOUS Cms ~ Cmr DELETIONS IN PLASMID pOCE15, AND Trp- ~ Trp+ REVERSIONS IN THE ISOGENIC STRAINS CARRYING DIFFERENT COMBINATIONS OF recA,/exA AND umuC ALLELES LISTED IN TABLE 1B No. Strain Relevant genotype Revs. Trp- .-* Trp+ recA lexA umuC Rates Frequencies Number of colonies/ Rel. plate x10-9 Rel. A B X10-~ Rel. xlO-g Rel. 22 EB309 + + + 1.5 4- 3 1 44 4-14 t 474- 15 1 13.84- 2.7 1 23 EBTt6 + + 36 2.54- 1.5 0.17 9 + 5 0.2 134- 6 0,3 21.34- 4.1 1.5 24 EBB07 + + 122::Tn5 4 0.3 16.5+ 5 0.4 35_+ 13 0.7 8.4+ 1.9 0.6 25 EB306 730 + + 17 + 1 1.1 84 4-13 1.9 108+ 27 2.3 1824- 3.5 13.2 26 EB3t2 A306 + + 7.5+ 0.7 0.5 17 + 4 0.4 24+ 8 0.5 6.44- 2.2 0.46 27 EB724 730 + 36 7.54- 0.9 0.5 36 + 5 0.8 33+ 8 0.7 25.3+ 5.6 1.8 28 EB728 A306 + 36 6 4. 1.4 0.4 28 ±11 0.64 34_+ 13 0.72 10.2± 4.5 0.74 29 EB825 A306 + 122::Tn5 7 ± 1.4 0.5 29.5+ 5 0.7 32_+ 16 0.7 19.84- 3.5 1.4 30 EBB04 730 + 122::Tn5 13.5+ 9 0.9 44 1 39+ 15 0.$ 26.74- 4.5 1.9 31 EB726 + 71::Tn5 + 25 4..18 1.7 54 4. 3 1.2 72+ 20 1.5 94.2_+10.8 5.8 32 EB829 + 71::Tn5 122::Tn5 20 1.3 100 4-20 2.3 1614- 32 3.4 37.2_+ 6.1 2.7 33 EB722 730 7]::Tn5 + 116 4.22 7.7 723 4.32 16.4 6674-200 14.2 631 ±87 45.7 34 EB730 A306 71::Tn5 + 30 2 152 4.48 3.4 2224- 70 4.7 21.1± 9.5 1.5 35 EB823 750 71::Tn5 122::Tn5 42 4. 9 2.8 267 4-50 6.1 197:i: 75 4.2 36 _+ 6.6 2.6 36 EB827 A306 71::Tn5 122::Tn5 25 4.17 1.7 180 4.1 142+ 39 3 20.34- 6.1 1.5 Deletion rates were calculated by the method of Luria and Delbriick (1943) as modified by Lea and Coulson (1949). The values represent the rate of deletion per cell per generation and were obtained from the expression R = m/N, where m is the mean number of mutations per sample and Ar is the average viable cell count per culture. The value of m was determined from the median of the distribution of the number of Cmr revertants per culture (r0), and ra was calculated from the expression ro/m using the values in Table 3 of Lea and Coulson (1949). N was always obtained from direct viable counts. The results are from two independent experiments of 90 cultures each. The deletion frequencies are expressed as the number of Cmr revertants per 109 cells plated on selective agar. The frequencies in column A were calculated ~om data obtained in the experiments performed to determine deletion rates, by taking the average number of Cmr for the ninety cultures in each experiment and dividing by the average final cell count per culture. The numbers represent the average of two independent experiments. The frequencies under colunm B were from platings taken from overnight cultures of each strain and represent the averages of at l~ast 6 independent measurements. The Trp+ reversion frequencies were obtained as de~crlbed under Materiats and methods and are the averages of at least 6 independent experiments. Columns labelled Rel give the relative rates of deletion and reversion with wild-type taken as unity.

Deletion rates and frequencies Deletions were measured by the reversion of chloramphenicoI sensitivity to resistance (Cms ~ Cmr). Deletion rates and frequencies were calcu- lated as described in the legend to Table 2 (Bal- binder et al., 1989). Trp- to Trp + reversions To assay for spontaneous Trp ~ revertants, cells were grown in L-broth overnight on a shaker at 37°C, washed twice and resuspended in the same volume of saline solution, after which 0.1-ml aliquots were plated on SEM and incubated at 30°C for 3 days (Witkin et al., 1982), when the number of colonies in each plate was counted. To exclude jackpots of large numbers of Trp+ rever- tants, simultaneous platings were done on E agar and incubated for 2 days at 37°C. The values obtained are shown in Tables 2 and 3 as numbers of Trp+ revertants per plate, but are equivalent to mutation frequencies since the final number of cells on SEM plates is always constant (Demeree and Calm, 1953). R~gult~ (A) Strains and plasrnids To determine whether deletions could origi- nate through the intervention of the SOS system we introduced plasmids derived from pBR325 (Fig. 1) into strains carrying various combinations of lexA, recA and umuC alleles constructed as described under Materials and methods and listed in Table 1A. We used the following mutants. (1) recA730 produces a genetically activated ReeA* and shows constitutive expression of SOS functions, as well as an increase in homologous recombination and spontaneous mutation fre- quency (mutator phenotype, Witkin et al., 1982; Sweasy et al., 1990). (2) ArecA306 is a deletion, and thus a null allele of recA, and decreases both spontaneous TABLE 3 FREQUENCIES OF SPONTANEOUS Cms ~ Cm" DELETIONS IN 5 DERIVATIVES OF PLASMID pBR325 (FIG. 1) AND Trp---,Trp+ REVERSIONS IN ISOGENIC STRAINS CARRYING DIFFERENT COMBINATIONS OF re¢4, lex.4 AND umuC ALLELES No. Relevant genotype Non-palindromes Palindromes Trp- --~ Trp÷ recA le.r.A umuC pRS1 pRS4 pOCE15 F14C F14S revs. t0 + + + 1.5+0.6 985+189 475:15 241+ 90 25__. 9 165:5 (I) (I) (1) (1) (I) (i) 7 730 + + 2.1+1.2 857+331 108+ 27 269+ 84 34+14 2105:65 (1.4) (0.9) (2.3) (1.1) (1.4) (13) t3 ..1306 + + 1.55:0.5 5355:215 244- 8 375 305:5 55:3 (1) (0.54) (0.5) (1.5) (1.2) (0.3) 11 + 71::Tn5 + 2.7+_0.6 801+188 72+ 20 653+-100 18+- 8 625:" 17 (t.8) (0.8) (1.5) (2.7) (0.7) (4) 8 730 71::Tn5 + Z2+- 0.5 11375:308 667-1-200 1501:[:493 73+15 5105:167 (1.5) (1.15) (14.2) (6.2) (3) (32) 21 + 71::Tn5 122::Tn5 2.8_+1.1 1047_+308 1615:32 587+160 335:12 275:7 (1.9) (1.Off) 0.4) (2.4) (1.3) (1.7) 19 A306 71::Tn5 122::Tn5 1.6+0.2 881+335 1425:39 729_+149 37_+ 9 55: 3 (1.1) (0.9) (3) (3) (1.5) (0.3) The plasmids were introduced into some of the strains listed in Table 1A and identitied here by the numbers used in that table. The result~ for pOCE15 are from Table 2 and are included here for ease in tmmparing results. Cms ~ Cmr deletion frequencie~ were calculated as for B in Table 2. Reversion from Trp- --* Trp÷ is as per legend to Table 2. The numbers represent the averages of at least 6 independent measurements. Deletion frequencies are expressed as the number of Cmr revertants per 109 cells plated on selective agar, and Trp- --* Trp+ rever~oa as the number of Trp÷ revertants l~r plate. 40000129

and induced mutagenicity (Tessman and Peter- son, 19S5; Sweasy et al., 1990). (3) h~r.A71 :: Tn5 is a null, or/era (Def), allele of l~c~l and results in full derepression of the SOS system (Krueger et al., 1983; Tessman and Peterson, 1985). (4) umuC36 lowers the mutagenic response after induction by mutagenie agents, but it is leaky and has a small to non-detectable effect on spontaneous mutations (Kato and Shinoura, 1977; Sargentini and Smith, 1984). (5) umuC122 :: Tn5 is a null allele of umuC and results in decreased mutagenicity (Tessman and Peterson, 1985). Although the plasmids we utilized in this study have been described in detail before (Betz and 259 Sadler, 1981; Balbinder et al., 1989; Sinden et al., 1991), their relevant characteristics (Fig. 1) will be briefly reviewed here to help the reader. In the first two plasmids, pRS1 and pRS4, the insert is a non-palindromic 64-bp Haelll fragment of plasmid pBR322 with EcoR1 linkers added. Be- cause of the orientation of the insert in both plasmids, the terminal homologies include adja- cent sequences to the left of each EcoRl linker which generate terminal 17-bp repeats (18 bp in pRS4) with a 15/17-bp (or 15/18-bp) homology. They differ from each other in one respect: in pRS1 the insert is flanked on each side by the same 8-bp sequence which includes an EcoR1 site, while in pRS4 there is an additional copy of the 8-bp repeat on the 3' side as a consequence pRS1 pRS4 pOCE15 F14C F14S Gl~c~ TT6AT~,~TIIG TAXAACTA~:A. TTA~CA~;~.g; G~¢CI:GAT#,T TTAATTTATA (:{~G6CCU:TC TGCTAA.TGTA GTTTTA~CA¢ TATCAATTCG~ Fig. 1. Relevant sequences of the pBR325-derived plasmids employed in this investigation. (PO Sequence of the 129-bp Alul fragment of pBR325 eontainlng the unique EcoR1 site (boxed). Alul sites are underlined. (B) Sequences of fragments cloned into the EcoR1 site of pBR325 generating at least one such site (boxed) at each end. "[he perfect hairpin structure potentially formed in pOCE1.5 is shown. The extended direct retreats in pRS1 and the overlapping direct repeats of pRS4 are underlined. Mismatches in the repeats are indicated by asterisks. For more details see text and also Balbinder et al. (1989) and Sinden et al. (1991). 40000130

of a 9-bp tandem duplication. This duplication creates a second perfect ll-bp direct repeat in pRS4 which overlaps with the imperfect 18-bp one. The presence of these overlapping direct repeats increased the deletion frequency of the insert more than 500-fold in pRS4, presumably because of the possible formation of multiple deletion intermediates between the terminal di- rect repeats (Balbinder et at., 1989; see aLso Table 3). The other three plasmids, pOCE15, F14C and F14S carry palindromic inserts differing in size and sequence between flanking EcoR1 sites. pOCE15 contains a 60-bp perfect palindrome consisting of an inverted repeat of a/ac operator fragment (Betz and Sadler, 1981; Balbinder et al., 1989). Plasmids F14C and F14S (Sinden et al., 1991) both carry the same 100-bp palindrome, the difference between them being in the center 14-bp sequences which are non-palindromic in F14S (Fig. i). Deletion of the cloned inserts was measured by the reversion from Cms --, Cmr and point mu- tations in the chromosome by reversion of trpE65 from Trp-~Trp+. Thus we could compare the effects of the SOS alleles on both deletions and point mutations in the same strains. (B) Deletions in pOCE15 The transformed strains carrying the plasmid pOCE15 are listed in Table lB. Table 2 shows in detail the results of a series of experiments in which Cm~o Cm~ deletions in that plasmid as well as Trp-~ Trp÷ mutation frequencies in the chromosome were measured in these strains. To insure that the results were reproducible the fre- quency of deletion incidence was measured both as deletion rates, that is the frequency of deletion per cell per generation, and as deletion frequen- cies expressed as the number of Cmr revertants per viable cells plated. As indicated in the legend to Table 2, the latter were measured in two different ways. As the table shows the results were highly reproducible, and are summarized below. (A) Full derepresslon of the SOS regulon in a /erA(Def) strain making activated ReeA* and UmuC+ (EB722, No. 33) increased the frequency of Cm~ -~ Cmr reversion, i.e. deletion of the palindromic insert in pOCE15, between 8-16-fold and point mutations 46-fold over wild-type (com- pare Nos. 33 and 22). (B) Replacement of urnuC + by urnuC122 :: Tn5 (EB823, No. 35) in the above strain resulted in a 3-fold lowering of deletion and a 20-fold lowering of point mutation frequencies. (C) In contrast to ReeA*, overproduction of ReeA+ did not result in a major increase in deletion incidence but did yield a 7-fold increase in point-mutation frequency (EB726, No. 31). (D) In order to observe large increases in point mutation and deletion incidence, both ReeA*730 as well as UmuC+ had to be overproduced since increases of the same magnitude were not ob- served in lexA + backgrounds (compare EB722, No. 33 to EB306, No. 25). However, even in lexA+ backgrounds recA730 brought about a 13- fold increase in point mutation frequency (EB306, No. 25) which dropped 7-fold upon replacement of umuC ÷ by umuC122 :: Tn5 (EB804, No. 30) or in the presence of umuC36 (EB724, No. 27). These results show that enhancement of the deletion of the palindromic insert in pOCE15 paralelled the stimulation of Trp-~ Trp ~: rever- sion, suggesting that the former can be brought about by a form of SOS processing which re- quires ReeA*, rather than RecA+, and UmuC+. We also observed that in /exA(Def) strains, replacement of recA730 by either recA+ or ztrecA306 resulted in a reproducible lowering of deletion incidence, but not always to wild-type levels (compare No. 33 to Nos. 31, 32, 34, 35 and 36). The significance of these observations is not dear at this time, but they could indicate the presence of some SOS-regulated functions, in addition to recA and umuC, which are also con- tributing to overall deletion frequency. " (C) Deletions in other plasmicls Since, as Table 2 shows, SOS functions essen- tial for mutagenicity can stimulate the deletion of the 60-bp perfect palindrome in plasmid pOCE15, it was important to determine whether this would also be the ease with other inserts cloned into the EcoR1 site of pBR325, palindromes as well as non-palindromes. In the experiments summarized in Table 3 we compare the deletion frequencies of these inserts in various genetic backgrounds. The results show the following. 40000131

CA) Full SOS dereprcssion and overproduction of RceA.* and UmuC+ (No. 8) increased the deletion frequency of the F14C and F14S palin- dromes to different extents but had no effect on the deletion of the same non-palindromic insert between different terminal repeats in pRS1 and pRS4. (B) Deletion enhancement by SOS derepres- sion seems to favor perfect palindromes (F14C vs. F14S). The difference between pOCE15 and FI4C suggests that it may also favor smaller palin- dromes as well. (C) pRS1 and pRS4 differ only in their termi- nal repeats and deletion in the latter is more than 500-fold higher than the former (No. 10, see also Balbinder et al., 1989) yet they are both unaf- fected by SOS derepression. These results show that SOS processing stimu- lates the deletion of palindromic but not that of non-palindromic inserts in pBR325. The stimula- tion of palindrome deletion seems to be influ- enced by palindrome size (pOCE15 vs. F14C) or the extent of palindromy, i.e. whether the palin- drome is perfect or not (F14C vs. F14S). Both non-palindrornie inserts were unaffected and they differ from each other only in the nature of their terminal repeats. These results are consistent with the interpretation that the SOS effect depends on palindromy vs. non-palindromy, i.e. the configu- ration of the transient intermediate, rather than on the sequences of the palindromic inserts or the direct terminal repeats. (D) Spontaneous mutation frequency Spontaneous mutation frequency (Trp--> Trp+ reversions) increased when SOS was geneti- cally derepressed (compare Nos. 10 and 11 and Nos. 7 and 8, Table 3; and Nee. 33 and 25 and 31 and 22, Table 2), and this increase was eliminated when umuC + was replaced by urnuC122 :: Tn5 (compare blos. 11 and 21, Table 3; and Nos. 25 and 30, Nee. 31 and 32, and Nos. 33 and 35 in Table 2). Both in the presence of lex,4+ or /exA(Def), reversion frequency was highest in the presence of recA730, lower in the presence of recA + and lowest in strains carrying the null mutation ArecA306 (compare Nos. 10, 7 and 13, and Nos. 11 and 8 in Table 3; and also Nee. 22, 25 and 26, and Nos. 31, 33 and 34 in Table 2). These results are in full agreement with those reported by other workers (Tessman and Peter- son, 1985; Sweasy et al., 1990). (E) Increase in Cmr frequency is not due to a selective advantage of Cmr revertants To eliminate the possibility that the 8-16-fold increase in the frequency of Cms ~ Cmr deletions obsezved in strain EB722 (No. 33, Table 2) was due to a selective advantage of Cmr over Cms cells in cultures of this strain, we performed reconstruction experiments in which Cm" EB722 cells were mixed with those of a Cmr revertant derived from it in a ratio of approximately I0:1, and the mixture cultured in L broth for about 25 cell generations. This was at least 4-5 times as many generations as the cells were allowed under the conditions used in Cm~ Cmr reversion measurements. At intervals of about 5-6 genera- tions, aliquots from the cultures were plated on LB agar plus chloramphenicol to determine whether Cm~ cells showed any signs of having a selective advantage. We saw no evidence of this, on the contrary the ratio of Cm~/Cm~ colonies decreased gradually with increasing time of cul- ture, if anything. In contrast, when the 10:1 mixture of Cm~-Cmr cells was cultured in L broth plus ehloramphenicol, Cmr cells overtook the Cm~ ones completely within less than 10 cell genera- tions. These experiments were repeated 3 times and the resuRs were completely reproducible. We can conclude, then, that the large increases in Cmr revertants observed in strain EB722 were not due to a selective advantage of drug-resistant cells under non-selective conditions. (F) Increase in. Cm" reversion is not the result of higher plasmid copy number It is known that upon derepression of SOS, chromosomal DNA replication can begin at sev- eral sites other than the normal replication origin oriC (Witkin and Kogoma, 1984). This may be related to repression of the gene for RNaseH, rnh, in mutants expressing the SOS regulon con- stitutively (Ouifiones et al., 1987). RNaseH is essential for the synthesis of an RNA primer needed for the normal initiation of replication at the colE1 orig~m by DNA polymerase I. Absence of RNaseH allows replication of pBR322 in E. 40000132

262 coli mutants lacking DNA pol~anerase I by an alternative mode requiring neither the poly- merase nor RNaseH (Kogoma, 1984, 1986). Thus, the poss~ility that the increases in Cmr rever- tants we observed in lexA(Def) strains were the result of increased plasmid copy number rather than actual deletion of the palindromic inserts had to be taken into account. This possibi/ity can be rejected, however, from the following consid- erations. (1) Significant increases in deletion frequency were seen only in EB722 (No. 33, Table 2) which is recA730 lerA (De0, but not in str'aJa't EB726 (No. 31, Table 2) which is recA + lexA (De0. The only way these differences in deletion frequencies could be explained as being the result of differ- ences in plasmid copy number would be by a differential effect of these two rec/1 alleles on plasmid replication. Plasmid pBR325 was derived from pBR322 by cloning a fragment carrying the cat gene into the latter (Bolivar, 1978) and both plasmids have the same colE1 origin of replica- tion. Sweasy et aL (1990) showed that there were no differences in the copy number of plasmid pBR322 derivatives in/exd (Def) strains carrying rec/1730 or recA +. (2) The increase in palindrome deletion fre- quency is umuC+-dependent and there is no evidence that UmuC plays a role in either chro- mosome or plasmid replication. (3) If the increase in deletion frequency for palindromic inserts in a recA730 lex.,d(Def) back- ground was a reflection of increased plasmid copy number, we should expect similar increases for all plasmids including pRS1 and pRS4, but this was not observed (Table 3, strain No. 8). (4) In estimates of relative plasmid copy ntun- ber for all the strains in Table 2 by measuring the ampicillin concentration necessary to decrease plating efficiency by a factor of 10 (Uhlin and Nordstrom, 1977; Cesareni, 1981) no differences were found. Discussion The results xve have presented show that over- production of genetically activated ReeA* 730 and UmuC+ in lexA(Def) strains enhanced the dele- tion of 60- and 100-bp palindromic inserts in the EcoR1 site of plasrnid pBR325, but did not affect the deletion of a 64-bp non-palindromic fragment be~,een different direct repeats cloned into the same site of this plasmid. No major increase in the deletion incidence of palindromes was ob- served when ReeA+ instead of ReeA*730 was overproduced (Table 2, No. 31; Table 3, No. 11) or in a recA730 lexA+ background where RecA*730 and/or UmuC+ were probably not being overproduced to the same extent (Table 2, No. 25; Table 3, No. 7). DNA sequence analysis of spontaneous Cm~ revertants has shown that in all cases examined the revertant plasmids con- tained a restored, intact cat gene: the inserts and one copy of the EcoR1 sequence had been pre- cisely deleted (Balbinder et al., 1989; Sinden et al., 1991). Spontaneous mutation frequency (Trp-~Trp÷ reversions) also increased when SOS was genetically derepressed and was highest under the same conditions stimulating deletion frequency, namely in lexA(De0 backgrounds overproducing RecA730 and UmuC+. As with deletions, spontaneous mutation frequency de- creased when umuC+ was replaced by umu- C122 :: Tn5. Both in lexA (Def) as well as lexd+ backgrounds spontaneous reversion frequency was always highest in the presence of recA730, lower in the presence of tee,4+ and lowest in strains car~qcing the null mutation ArecA306. These re- suits are identical to those reported by Sweasy et al. (1990) in their demonstration of a UmuCD- dependent direct role for RecA in mutagenesis and show that, in a general way, enhancement of palindrome deletions in the plasmid system we employed in these experiments paralclled closely the stimulation of Trp-~Trp+ reversion in a mechanism which requires high levels of UmuC+ and RecA*. Thus the deletion of palindromic inserts and the incidence of point mutations look like different manifestations of the SOS mutator effect. In strains expressing the SOS response constitutively, spontaneous mutation rates can be elevated by as much as 50-fold (Walker, 1984; Sweasy et at., 1990), and this mutator activity requires the amplification (or overproduction) of UmuDC as well as RecA, with genetically acti- vated RecA* having a stronger mutagenic effect than RecA+ (Dutriex et al., 1989; Sweasy et at., 1990). Thus it appears that the role of ReeA* in 4OOO0133