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JOBNAME: DNA-REP PAGE: 379 SESS: 6 OUTPUT: Frl Apt 22 17:05:48 1933 .CLS: onetimei GRP: bok JOB: dna-~ep DIV: 6702-040 t-- -XUTHOR: SEE QUERY pI~DCC~GNED PI.~M;D$ To STUnY 'DELEF[ONS explained by errors in the action of enzymes .which normally break and join DNA, such as copoisomerases, and occur on sequences that share little or no homology (13, 32, 36). While the role of sequence homologies in the formation of deletions is well estah- lished, we know nothing about the mecha- nisms which bring them about. The mlsallgn- menc models leave this question entirely open. ~osc of what has been published to date is mainly concerned with the roles of legitimate versus illegitimate recombinakion (4, t9, 20). Legitimate (homologous) recom- bination requires extensive sequence homol- ogy and the participation of the e¢¢A + pro- tein (10), while ille.gitimate recombination (4, 19, 20) refers to a number ofrecombina- tory processes which do not require large sequence homology or the participation of the recA + protein (9, 1.3, 32, 36, 49, 54). In many bacteria and bactcrlophage systems the same deletion frequencies were observed in recA+ and recA backgrounds C3, 1.l, 1.2, 1.4, 18, 26, 27). [n two cases, e¢cA + increased deletion frequency considerably but was not essential (1, 50). These deletions were large and occurred between direct repeats, and the regions deleted could form hairpin struc- tures. The participation ofrecA + is generally interpreted as indicating that homologous recombination plays a role, and indepen- dence from recA + has been explained as the result of either illegitimate recombination (4, 20) or replication errors (1, 14). It is known, however, that the recA+ protein performs a number of functions; i.e., it is essential for the initiation of homologous recombination (103, plays a major role in regulating the SOS response (53), and .is also needed in mutagenesis (16). There i's only one published report in which the possible participation of the SOS response in dele- tion production was considered (38), with negative results. The role of cecal + in pro- ducing deletions still remains unresolved, and we are still unclear about which mecha- nisms (homologous recombination, SOS processing, or other) are defined by i~s par- ticipation. As for whether recA+.indepeno dent rearrangements are caused by errors in replication or processes related to illegiti- mate recombination, we have no informa- tion at this time. • General recombination and the various specialized recombination systems appear to be mediated by separate overall processes, but may share common components of DNA metabolism such as winding/unwind- ing enzymes, ligase, polymerases, various nucleases, and DN~-binding proteins, which also participate in DN'A repair and replication (31). This sharing of majo~ func-" clans between different processes foc DNA metabolism is one component of the com- plexity of the problem. The other is the multiplicity of transient secondary structures chat can form on DNA and which are them- selves subject to modification by such factors as location on a replicon (14), degree of superhelical tension (48), etc. Clearly the identification of functions which give rise to deletions is a major priority if we hope to understand how these originate. The best way to do this is by isolating and studying mutants which affect deletion frequency (4). However, in devising strategies co obtain these, we must keep in mind the inherent complexity of the problem. We know that there are at. least two pathways for TnlO excislon-assoclated processes (33, ~4) and, by analogy, also for deletions. Thus, select- ing mutants for their effect on a given event will probably not uncover all functions af- fecting a very similar event. It is also possible that, while there are probably several path- ways for deletions, there may also be over- laps; i.e., the same deletion can be brought about by more than one mechanism. This is strongly indicated by the observations that the frequencies of the same deletions which occurred in the absence ofrecA* were sub- stantially increased by its presence (1, 50). Keeping such facts in mind, we have devised strategies for studing deletions by following the advice of Drake (15), i.e., trying to imagine "'all the ways" in which deletions 40000278

JOBNA~,fE: DNA-REP PAGE: 380 SESS: 6 OUTPUT: Fr| Apt 22 17:03:48 [988 4~LS: one¢irnei GRP: bok JOB: dna-rep DIV: l ,38O mlghc occdr and then "designing "experi- mental attacks.., powerful enough not only to discredit the inappropriate answers but also to ferret out the as yet unimagined possibilities." We have developed a two- pronged attack based on 6) control of all the srrucrural variables for deletions through predesigned p[asmids and (ii) the use of these plasmids to study the effect of host mutants on the frequency of Structurally defined classes of deletions, and as genetic screens to isolate new mutants which in- crease deletion frequency. In the following sections we ~ill show how this approach works. USE OF PtLEDESIGNED HIGH-COPY-NU~fBER PLASMIDS TO STUDY DELETIONS The slipped mispairing mutagenesis models provide a good point of departure since they identify important structural rameters for de[etions and provide a concep- tual framework within which we can make precise predictions to be tested experimen- tally. We describe here the use ofderiva¢ives of the high-copy-number plasmid pBR325 (6) to test an important prediction of the slipped-mispairing model of Albertini et al. (!), namely, that a palindrome will delete more frequently than a nonpalindrome of comparable size between the same direct repeats, at the same position on the same repllcon. This plasmid has a number of unique restriction sites within the genes de- termining resistance to the drugs, tetracy- cline, ampicillin, and chloramphenicol (6). By the simple expedient of cloning any fragment of desired size and sequence into one of these sites, we inactivate the gent and reverrants will result exclusively from the deletion of the insert. Such a system is extremely altractlve for a number of reasons. (i) It allows us to manipulate one variable at a time while, keeping the others constant, thus making interpretation of the results BALBINOI~R straightforward and unequivocal. (ii) By se- lecting for reversion from drug sensitivity to resistance we recover only deletions, making their frequency easy to quantitate. (iii) lection for drug resistance should be vet-,/ stringent for the restoration of the normal sequence of the gent into which the insert was cloned, making extensive sequencing rever~ants unnecessary. (iv) The sequence and size of any DNA to be inserted can be chosen at will. (v) Plasmids can be easily purified for biochemical analysis quencing, restriction enzyme mapping, etc.). (vi) Plasmlds are portable and can be easily introduced into strains of any desired ge- netic background. This work was carried out with deriva- tives of pB1L325 obtained by inserting two fragments of the same approximate size (66 to 68 base pairs [bp] but different sequence into the unique EcogI site of the gent coding for the enzyme chloramphenicol acetykrans- ferase, CAT, which is responsible for resis~ rance to chloramphenicol. The CAT has been completely sequenced (2) and has a unique EcoRI site located between bp 437 and 442 within an Alibi fragment of 129 bp (Fig. IA and 2). Plasmid pOCE15 was con- strutted by Betz and Sadler (5) and contains a 66-bp inverted repeat of a /ac operator fragment. This is a palindromic sequenc.e and is capable of forming a hairpin structure (47) as illustrated in Fig. 1B-I.. Plasmids p~l and pRS~ were obtained by IL Sinden (unpublished data) as independent transfor- mants in the same experiment when pB~-325, opened at the unique EcolLI site in CAT, was cloned to the 66-bp FlaelI frag- ment of pBK322 to which EcolL[ sit~s: had mic (F~. 1B-2). Digestion with Alul, fol- lowed by electrophoresis on acrylamide gels. showed that the 129-bp AluI fragment of pBK325 had been replaced by larger frag- ments in the derived plasmids: about 195 bp in pOCE15 and pR~l and 202 bp in pR~4 (Fig. 2). Although both pRSl and plLS4 contain the same nonpalindromic insert. 40000279

~C~- ] JOBNAM"E: DNA-REP PAGE: 38l SESS: 6 OUTPUT: Fri Apt 22 17:03:48 1988 CLS: onetlmel GRP: hokJOB: dn~-rep DIV: 6705-040 pKL~DFr~.D PI.,~SMIDS TO STUDY DE~L~10~$ they differ from each other in one interest- ing respect, as shown by the sequences of the insert termini (Fig. IC): while pRSI has a single Ec0Kl site at each end of the insert, pRS6 shows an asymmetry since at the 5' end it has one while at the 3' end of the insert it has two ErolLI sites in tandem con- figuration. This size difference of about 8 bp is clearly observed on 5~ and even more clearlF on 15.~o acrylamide gels (Fig. 2B and C). A de~ai~ed analysk of rcvercams of all three plasmlds, which included phenotype 40000280

jO'B'NAME: DNA-REP PAGE: 382 SESS: 6 OUTPUT: Fri Apt 22 17:05:48 1988 4~..S: onerimei GRP: hok JOB: dna-rep DIV: 6702-040 I AUTHOR: SEE QUERY A B C a b c d e a b a bc d e f 12 10 715 Ft~ului 2. Acty|smidc &el ele~trophoresis of plasmid digests. (A) Single (.,tbl) and double (#llial-Et0i~l) diltests or" p|ismMi pOCEI5 {z and h) and pBR325 (c and d). run on a ~gG IcrTIsmide ge! (iT by 22 ¢m). Lanes: (=) pOCEI~. A/it| digest: (b) pore 1.5, ~II/I-E~'~I digest; (c) pBR.:32~, .#l.#.~l digest; (d) pBR52~, .41ul.EcoR.I digest; (e) dlX | 74 F/~lelll molecular size |adder, Note char Ei'+il'Ll dlgcstioi'l ofA/ld.dlgested pOCE1 ~ |rids co the disappearance of the 195-bp replacement fragment =ncl the appear=nee of fragments of tO0 and 66 bp. The foimcr corresponds to the segment bciwecn the first tl/.l sit= and EenKl slte (Fig. IA), and the second is the ~-bp inse/t. Double digests of'pBl~325 (lane d) show the disappearance u/" the ! 29-bp/!1#| fiigmenr and its repli~ement by the |O0.-bp AI~I-E¢o9~I 't'ragmen t Ilso found with pOCE I ~, but cite 66-bp insei~t ¢•nnot be seen, (D) d/ill digests ofplismld plier (lane •) =nd plt.S4 (lane b) run alongside oath o¢lter on a S ~ aerylamld¢ gel ( I 7 by 22 cm). The inseti.contalnin~ fragment ofpR.S4 runs • behind the one t'rom p~l. The difference in size between these fragments is one Ecoi~ linker (8 bpo since or=liners were used). (C) A~ul digests ofCm" plasmids and ewe Cm~ reverrams run on • l~ acrylamide gel (I.7 by 22 tin), Lanes (a) pOCE ! ~; (b) pR~4; ~c~ pRS1.; (d) pOCE! 5 revertam peg 1." (e) pOCEI~ reve~anc pEI~fl (dimec): (~ ~X174 molecular slae ladder. This gel increases resolution, for the smaller frasmenra over the ~ gel (compare separation of the t ~8-and ! 29-hp t'~agrnencs wlth chat in pane| A). Both revtrtants ofpOCE 1.~ have lost the insect ~nd recovered the |29-bp fragment, bu~ pEBfl is a dlmer consisting of one Cm~ and one Cm~ plasmld ~ thus also sho~s the presence o~ ~he 19~-bp ~gment. (From Balbindet ¢c ~1., submkt~d). rests, restrlccion enzyme analysis, and se- quencing across the deletion site and is de- scribed in a separate publication (E. Bal- binder et al., submitted"-for:publicarionY, established that the selective procedure was specific for the restoration of the single, original EcoRl site. This permits quantitative measurements of deletion frequency by a simple reversion test. Deletion rates and frequencies ot" all three plasmlds were com- pared in recA+ and rec.'l isogenic back- grounds. The fi~llowing results (Table 1) were obtained. (i) Deledon was more fre- quent on pOCEIS, which carries a palindro- mic insert, than on p1LS1, which does not, confirming the prediction of the model of Albertini cral. (1). (it) Surprisingly, deletion on pRS4, which carries the same nonpalin- dromic insert as pRS1 but has a tandem duplication ofanEcoR.[ site ac the 3' end, was 5- to 10-leo|d higher than on pOCE15, a result totally unpredicted by slipped mispair- ing models and with some important impli- cations which will be discussed below, tilt) Although these deletions occurred indepen- dently of recA+, the deletion rates as well as 40000281

JOBNAME: DNA-REP PAGE: 383 SESS: 6 OUTPUT: Fri Apt 22 17:03:48 1988 .CLS: one¢imei GRP: bok JOB: dna-rep DIV: 6703-040 I AUTHOR: SEE QUERY T^DL~ | Cm'~Crn' K=versio~ ~ccs ~nd Fccquencics" Srraln No. of Avg. no. of Cm'.-,.~' No. of cultures wlch cells/cuhur¢ reversion rate Gc-no~ypc cuhurcs no Cm" (10") (10-t) rcvers~0n$ R~vcrs}on f~cquency Low High Ave" 78 2.6 0.5 4 15 0.6 5 6 4.7 1.7 111 tO 0 1.2 >37 25 2,116 260 79 2.4 1.2 4 40 2.4 | t 2.! I0.~ 4.7 460 33 0 2.~ >20 24 2.076 362 " TE~ dclcdon rstes were cMcula,cd ,s described hy Alberdnl ~. M. (I) from ,he ~,i~ ofc~mr~ hav;~i no ,~c~ (~0. the zero te~ ~ Poisson disrribudon) a~ rcp~cnt the frc~cn~ of Cm' rcvcrs~n ~r cell ~r g~ctat~n. ~vc~on ft~ucnclcs ,re ~p~scd ~ chc number of ~' r~c~tants ~r n~r of viable cells pl~ and were c~lu~ f, om ~he cuhu,~ cont~;nln~ Cm" r~cn~; ~ low ~uc is from the cuku~¢ show~n~ the Iowcs~ t~ ~hc hi{h v~ue is from ~he ~lmr¢ showin~ ,~ highes~ humor ~te~s~l. ~e ~vera~¢ freque~ics r~t~en¢ the sve~e fium~f of t~e~a~s ih ~ e~ire sample ~8~ to 1~ ~kutes over the a~ta~e humor of viable cells ~t cuhurc. the average deletion frequencies were txvo- to fourfold higher in recA ~" than recA cells ('Fable 1). We will return to this below. IDENTIFICATION OF FUNCTIONS WHICH .AFFECT DELETION FREQUENCY As mentioned above, the role of recA ÷ in producing deletions still remains unre- solved. We need more information about what recA+ and some of its alleles do to different deletions before we can understand its role in these processes, recA730 is an allele ofrecA * which results in constitudvlty for the SOS response at all temperatures and also increases recombination frequency (56). The plasmids described in the preceding section were introduced into three isogenic strains differing only at the recA gent: one was recA÷, another was recA, and the third had the recA730 mutation. All these strains were derived from strain SC30 (56), which carries the mutation trpE65. Reversion of this allele to prototrophy is known to be increased by recA 730 particularly in the pres- ence of adenine, thus providing us wkh a marker to monitor the mutator response of this allele (56). We found chac the deletion frequency on all plasmids was increased an average of three- to fourfold by recA730 (Table 2). This increase in deletion fre- quency can be explained by enhanced par- ticipation of the SOS response or by enhanced homologous recombination. Re- gardless of whlch mechanism is finally shown to be ac work, we can say on the basis of these results and those in "Fable I that some alleles of recA÷, and perhaps r, cA ÷ itself, increase by a small factor the frequency of a class of small (66- to 68-bp) deletions cween 6-bp direct repeats represented by the ones on these constructed plasmids. PLASI~IIDS AS GENETIC SCREENS: ISOLATION OF dli (DELETION INCREASE) MUTATIONS Plasmid p1~[C874 was constructed in the laboratory of M. Casadaban (8) and was an available genetic screen to select mutants which increased deletion frequency. This plasmid (Fig. 3A) has most of the la¢ operon, except for the promoter-operator region and the first few bases in lacZ, cloned between a BamHI and a HincII-Sall site about l to 2 kilob:,ses downstream and in the same ori- entation as the Km* gone. It is phcnotypio 40000282

~_i JOBNAME: DNA-REP PAGE: 384 SESS: 6 OUTPUT: Fri Apt 22 17:03:48 1988 -CLS: onerh~ei GP~P; hok JOB: dan-rep DIV: 6705-040 - ! AUTHOR: I i SEE I~UERY'/~ T^o~.I~ 2 Effects of Different rztat Alleles on De]edon Frequency (Cm'-,Cm')° EB496 pRS! reeA 730 3.9 (4.2) 89 (!9) 19~ (30) ED497 pRS! rec,'l + 1.2 (1.2) 15 (3.2) 25 (3.8) EB49B pE~l r~¢,'| 0.93 (1) 4..7 (|) EB499 pRS4 rtrAT30 1,212 (~) ~2 (~.2) $7 (6.~) EBb00 pIL54 rtrd + 773 (1.9) 1 t (1) 20 (2) ED~OI pRS4 re¢,'l 402 (1) 10 (L) pJ~ on L-s;~t ¢~t~nin~ 2~ ~& orchto~phen~l ~ ~; 0.t.ml s~ptes ~eoptt~e dig~t~ were pla~ on L.~gar (o o~ain a v;~lc c~nt. ~ion ~g~uc~ ~ ~p~ ~ SE.M ÷ adenine(7) g~ml) tally Lac- Kin', buc ;c gives rise to papillae ac vo~ low frequency (1.6 X 10-9) on MacConkey agar. Restriction enzyme an~ysis of~la~* ~mmids showed that the ~#+ pheno~Fpe was caused by the, deletion of a 1-k,lobme fiagmenc between ~m~ and laeZ which ellm[naces the BamHI site and fuses the lac operon to the ~* (plasmid pEB7, Fig. 3B). Sequencing of the deletion termini and inte~ening fragment is in progress. We have isolated 31 mutants for their ability to stimulate the occurrence of the 1-kilobase deletion on plasmid pMC874 bF increme in'l~* papillation (18, 28). Thir- teen of these mutants were extensiveiF tested for stimulation of ~lac+ deletion frequentF and several characteristics typical of mutants for DNA-handling functions such as sensitivity to UV and methFimetha- nesulfonate, increase in spontaneous muta- tion to nalidlxic acid (Nal¢) and rifampin (~f) resistance (murator phenotype; 34), and growth at various temperatures (Table 3). With two exceptions tstrains EB323 and EB335). the increases in ~/~c"deletion frequency were low (less than lO-fold) (Table 3). Most of the mutants, however, showed phenotypes consistent with alter- ations in genes for DNA metabolism such as mutator (strains EB325, -330, -335, -350, -359, and -360) or andmurator (EB323) ef- fects, as well as increased sensitivity to mu- ragenic agents and impaired growth. The mutants were cured of pMC874 and trans- formed with pOCE15, FRSI, and pRS4, and the frequencies ofCm'---~Cm¢ deletions were determined for each strain thus obtained. Only 4 of 13 dli mutants tested (strains EB323, -325, -335, and -360) increased Cm~ deletion frequency on pRS1 and pRS4 and actually decreased (with the exception of strain EB325) deletion frequency on pOCE15. This suggests a preference in such mutants for events raking place between direct terminal repeats in the absence of palindromes. Ouc dli mutants have very different phenotypes from the tex mutants selected by Kleckner and co-workers (33-35) for in- creasing the frequency of precise excision of TnlO. In general, dli mutants were more sensitive to UV and methFImethanesulfo- 40000283

~ JOBNA~E: DNA-I~EP PAGE: 385 SESS: 6 OUTPUT: Fri Apt 22 17:03:48 X988 • CLS: onctlrn¢i GRP: bok JOB: daa-r¢~ DIV: 670-~-0~0 • AUTHOR: i SEE QUERY PAG~ PRED~IGNED PL~SMID$ TO STUDY DELETIONS --'~ Z rep I Hinc ,= EcoR 2.52 2.62 Y .Hinc u/Hpa = o.s= I|/Hpa ! s.e/o.o ~eln. breakpoint loss of BamH ~ Pst i 6.12 Hinc n/Sai i $,82 Ftc;o~=-: 3. l~),a.grams ore p|asmid pMCS74 (A) and its derivative pEB7 (~). ~;_.~ which c=rrics a O.~kdobas¢ dclccloa joining ~omu¢er. ~e diagram o[pMC874 has bccn adapted from (8). 40000284

JOBNAME: DNA-P,-EP PAGE: 386 SESS: 6 OUTPUT: Fri Apt 22 17:03:48 • CLS: onedmei GRP: bok JOB: dan-top DIV: 6703-040 Relative deletion frequency .Relafive-muttdon'" ~.eque nc3~ ~r~¢at or MuttSen Growth at ~,~ C-~ "~;c ~ ~'~" phenowpe- .~ ~ ~nsidvi~~ cemp: (pMC874) pOCE15 p~l p~4 N~I'~Nal~ ~fr ~ ~MS 30"C 42"C EB26~(WT)~ | | I i ! I I~ I~. WT Ebb23 ~0 0.4 19.3 2.2 0.5 0,16 VS VS Slow ~low" EB325 5.~ I.S~ 12.9 3 3.5 4.7 PS VS Slow Slow EB52S 1.6 0.55 t.4 0.85 t.8 2.~ ~ S Slow Slow EB330 1.4 0.7 0.6 l.t 6.3 2 ~T V~ Slow Slow EB535 20 0.34 17 2.~ 32 24.~ ~ ~ Slow Slow EB537 8.6 0.8 t 0.7 0.2 3.2 S VS Slow SIo~ EB339 3.2 t 0.5 t.2 t t.7 ~ S ~ EBb47 t 0.98 0.7 1.01 5.2 4.2 ~ ~ Slow Slow EB348 7.2 0.8 t.04 0.8 0.6 0.9 ~T ~ S~w Slow EB)~O 1.~ ~D~ 0.6 ~ 99.2 63 ~ ~ ~ EB35~ t l 0.8 0.9 1.3 2.7 ~ ~ ~ EBBS9 ; 0.7 0.4 0.5 4.3 2.8 ~ ~ ~ EB360 3,7 0.2~ 1.9 I.~ ~.3 1.04 S S Slow i hate than rex mutants, but showed lower stimulation of deletion frequency and weaker mutator phenotypes. Also, of six dll mutants tested (Table 4), only' two stimu- lated Tnl0 excision and the remaining four showed no effect or an actual inhibition of this deletion event. Thus, on the basis of these preliminary data w¢ see no obvious similarity between dli and ux mutants. On the other hand. there are some superficial similarities between some oF our mutants and others described in the literature which affect different DNA metabolism functions. For example, EB360 (Table 4) resembles a conditional lethal p*M mutant (30); slow growers with a mutator phenotype (EB325, -328, -330, and -347, Table 3) resemble mutants affected in genes for components of the DNA polymerase Ilt replication com- plex such as dna~. and dnaE (25, 29, 46). These results support the proposition that different deletions originate through differ- ent pathways and that a genetic dissection of these pathways will require specific genetic Screens. CONCLUSIONS AND FUTURE. DIRECTIONS The results we have presented illustrate how a strategy based on the use of prede- signed plasmids for the systematic study of deletions works. With the pBR325-derived plasmids, deletion frequency can be deter- mined by a simple reversion test: selection for Cmr only yields revertants that have the original sequence at and ~diacent to the EcoRI site, making extensive sequencing of revertants unnecessary and the interpreta- tion of results dear and unambiguous. In each o£ the plasmlds we have changed one single parameter, i.e., the sequence of the insert (pP.Sl :nd p&..~ versus pOCElS) or the number and arrangement of the direct terminal repeats (pl~l and pOCEI5 versus 40000285

~¢~ I JOBNAI~E: DNA-I~EP PAGE: 387 SESS: 6 OUTPUT: Fri Apr 22 17:O3:48 1988 - CLS: onetimei GRP: bok JOB: dna-rep DIV: 6705-040 AUTHOR: SEE QUERY ~87 ER'cc~ of Several dli ~ucadons on Excblon of TnlO" EB265' t 1 1 | ! t EB323 ND* t <0.4 0.1 " <0.6 9 ED325 322" 22 61 t34 <0.16 173 I:D3~5 17 12 58 2 0.1 71 EB337 lxlD <0. t 0.4 <0.3 0.3 23 EB348 0.~4 0.4 l.l 0.5 0.54 <0.|2 EB3(ff~ ND <0.1 !.3 <0.8 <0.36 ND ot" four indtl:~ndent Actual vtluct:/Od::Tn |0. 10-9;/*N*6J::Tol0, I x 10"9;tl~r-.~#::TnlO, I x tO'l;#re-Slt:TelO. I.} X l0-s. pI~4). Thus, the differences in deletion frequency for each plasmid can .result only from the difference in the single controlled variable. In a general way, our results, agree with a major postulate of slipped-mispairing models for deletions: direct and inverted repeats promote deletion events. More sp~- cifically, our observations that deletions be- tween direct repeats are facilitated by an intervening palindrome (pOCE15) are a clear confirmation of the ~redlction ofAl- bertini et al. (1), which was based on the sequencing of large lacl-Z deletions, and agree with many reports showing that palin- dromes are highly deletion prone (11, 14, 22, 24, 43, 45, 55)- Our data also show that inverted repeats are not absolutely neces- sary, however: 66-bp inserts still delete be- tween EcolLI sires, although at a decreased frequency (p1~$1 versus pOCEIS), and, more dramatically, a tandem duplication of one EcoRl site actually increases the ddetioa frequency of a non-palindrome over that of a palindrome (pRS4 versus pOCE15). This observation has several important implica- tions. First, the stability of a given stretch of DNA is not determined exclusively by its sequence, but can be strongly influenced by surrounding sequences as well. a conclusion also reached by Das Gupta etal. (14) from the study of deletions in a different system. Second, not all deletions which can be ex- plained by slipped-mlspalring models occur of necessity by the elimination of palin- dromes or resemble the excision ofTnlO ( 1, 14, 18, 22, 43). The latter represents one class of deletions, and our data suggest that there are more deletion-prone sequences in procatToti¢ genomes that we recognize to- day. Finally, we have to consider whether structures such as the highly deletion-prone one on plasmid pRS4 can be found in con- temporary procaryotic genomes. While some palindromic sequences have been re- tained in the course of evolution (21, 40, 41), it is not dear whether other potentially unstable sm~ctures have been preserved as well or have been entirely lost. With pBR325-derK'ed plasmid systems we tan study any sequence of interest, regardless of whether it exists in nature or has been eliminated in the course of evolution. Thus, such systems can be very useful in studies of the evolution of the genome. We have also shown how the portability of plasmids provides a versatile ,approach to the problem of mechanisms, either by study- ing the effect of well-identified mutations for repalr-recombinadon functions (such as recA and re:A730) or by using them as genetic screens to isolate mutants for their effect on specific deletions. The preliminary results 40000286

(~.I JOBNAI~(E: DNA-REP PAGE: 388 SESS: 6 OUTPUT: Fri Apt 22 17:03:48 1~88 ~- . ~ ~ AUTHOR: PAG~ we have presented in this report are of some interest. Table I shows that deletion rates for all pBR325-derlved plasm[ds are about twofold higher in recA* than in recA cells. Although differences of" this magnitude are difficult to establish conclusively, the fact that they are consistent for all three plasmids is difficult to dismiss. These results suggest that possibility that recA+ plays a role in causing these deletions which, although barely detectable by the fairly insensitive method of measuring rates, may not be ms,gntfican~;As mcn,oned above, delet,ons occurring w,th the same frequen~ m recA ~d retA+ cells are interfered as being reeA ~ independen~ and re~uldng ekher from replication error~, iilegR~ate recombina- tion, or ~ome ~ott of DNA cleavage which may or may not be parr of illegitimate re- combination processes (1, 4, 14, 19, 20, 22, 43). These differen~ mechanisms are not necessarily mutuallF exclusive. In facq ~he evidence available ~uggests, m we have men- cloned earlier, that mor~ than one mecha- nism may be responsible ~or causing the same deletions (see re~erence~ 1 and 50 and above). Our ob~e~ations (Table~ I and 2) are congruent with tha~ evidence in showing [har recA + participates, together wkh other unknown functions, in producing deletions. The difference is that the deletions Albemlni ec aI. (1) and Sommer e¢ al. (50) were studying could be dr~atlcally incremed by recA+, whereas ours were only slightly hanced. Regardless of whether it does this by promoting homologous rccomblnadon, by ~lowing a certain level o~ induction of the SOS respo~e, or by a combination of =hese two mechanisms, recA + seems to f~vor cer- tain deletions over others. In most c~es its contribution could be undetectablc while in others (1, 50) it can be substamial. Certain mutant alleles of r¢cA + such as recA730 are more e~cient than recA + in causing certain deletions and thus magnit~ the e~ect recA * ~o a detectable level. What this inter- pretation implies is that recA * is one o~many intracellular ~unc¢ions participating in DNA metabolism which are normally active at low levels in "Eschcrichia coil cells and can cause, accidentally, spontaneous deletions at very low frequency. If so, we would expect that "~.o,,, n~atations selected exclusively for their abil- ~ .£c'~'/9. '1/~ it}, to increase deletion frequency may turn / out to be alleles of genetic functions which, like recA730, magnify the barely detectablet effect of the wild type on the deletion proc- ess. Mutants selected for increasing the fre- quency of excision of TnlO (rex mutants; /j/~/:~;, . . - • • • .'- ..... ":-; ..... ".---/'P'~¢$~ ~" ~'T ~'~ - 33-35) fit th~s'pred~ct~on.'~TnlO excts~on ~s normally independent of the E. coil recABC te" -~ "",,. ..... ".'-.~" homologous recombination pathway, yet ~...~ _ several rex mutations are alleles of recB and recC. In these mutants, TnIO excision ap- pears to depend on an altered form of the recBC nuclease ~[~.,~'; and one of them actu- all), makes TnlO excision dependent on recA+ function (35). We are in the process of characterizing the dli mutants, and if tl~e prediction above is correct, we expect that some of them will be alleles of already identified recombination-repalr-replication genes which thus far have had no demonstra- ble participation in producing deletions. Although a final understanding of the dli mutants we have isolated awaits their complete genetic characterization and the sequencing of the endpoints of the 1-kb deletion on plasmid pMC874, both of which are in progress, the preliminary data we have presented (Tables 3 and 4) contain some interesting findings which deserve some comment. Firsr, most of the d/i mutants had no effect on deletions on the pBK325-de- rived plasmlds or on excision ofTnl 0. Thus, selecting for mutants which increase the frequency of one class of deletions will not uncover functions involved in other, dif- ferent deletions. Lundblad and Kleckner (33, 34) reported a similar finding for tex mutants. These were selected for stimulating the precise excision of TnlO but will not affect the excision of nearly precise excision remnants of the same transposon. Second, the four mutants which affected deletions on pBK325-derived plasmids stimulated prefer- 40000287