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DELETIONS ON SPECIALLY DESIGNED DERIVATIVES OF PLASMID pBR325 IN ESCHERICHIA COLI by Elias Balbinder, Cheryl Mac Vean and Robert E. Williams Dept. of Biochemistry, Biophysics and Genetics University of Colorado Health Sciences Center 4200 E. 9th Ave. - B-121 Denver, CO 80262 40000225

DNA fragments cloned into the EcoR~ site of the chloramphen- icol acetyl transferase (CAT) gene of plasmid pBR325 are deleted precisely between the two EcoRl restriction sites generated as a result of the insertion. Plasmids pOCEI5, pRSl and pRS4 carry 66- 68 bp fragments inserted at this site. The insert in pOCEI5 is a perfectly palindromic la___q operator fragment .pRSl and pRS4 both contain the same non-palindromic insert, but while pRSl has a single EcoRl site at each end pRS4 is asymmetric having one EcoRl site at the 5' end and two tandem EcoRl sites at the 3' end of the insert. Deletion frequency is measured as the reversion from Cms (chloramphenicol sensitivity) to Cmr (chloramphenicol resistance). On these plasmids, the potential to form cruciforms favored deletion between the same terminal repeats by at least a factor of i0 (pOCEI5 vs pRSl) but, surprisingly, the presence of an extra EcoRl site on the 3' side of the non-palindromic insert (pRS4) increased its frequency of deletion 5-10 fold over that of a palindrome (pOCEIS). Thus the number and arrangement oZ direct end repeats can play a major role in determining deletion prone- ness. We observed a slight but consistent increase in deletion rate in recA+ over recA- cells and a somewhat higher increase in the presence of ~gA 730, a mutant allele of recA+ which makes the SOS response constitutive and also increases homologous recombination frequency. These results show that recA+ and some of its mutant alleles can stimulate the occurrence of deletions on pBR325 derived plasmids, but do not differentiate between mechanisms involving homologous recombination or SOS processing. 40000228

Deletions on {pla~. Dr. Elias Balbind Dept. of Biochem/. B-121 University 4200 E. 9th Ave. Denver, CO 80262 enter 40000227

Deletions on &plasmids Dr. Elias Balbinder Dept. of Biochem/Biophys/Genetics B-121 University of Colorado Health Sciences Center 4200 E. 9th Ave. Denver, CO 80262 40000228

I~/TRODUCTION Interest in the study of major genetic rearrangements such as deletions, duplications, translocation, etc. has been increasing in recent years, since they have been shown to be at the basis of a number of important biological events in both prokaryotes and eukaryotes such as control of gene expression, development, differentiation and evolution (Simon & Silverman, 1983; Seidman & Leder, 1978; Riley & Anilionis, 1978). They have also been implicated in the origin of human cancer and genetic diseases (Yunis, 1983; Escot & al, 1986; Monaco & al, 1985). In spite of their importance we still know very little about the mechanisms that bring them about. Deletions are a commonly occurring rearrangement and have been extensively studied at the molecular level, mostly in prokaryotes. They can occur as the result of the movement of transposable elements (Ross, Swan and Kleckner, 1979) or from the resolution of transient secondary structures forming spontaneously on DNA (Albertini et al, 1982; Glickman-& Ripley, 1984). Most of the deletions not caused by transposon movement which have been studied to date can be explained by slipped mispairing (or misalignment mutagenesis) models. Albertini et al., (1982) proposed that, during DNA replication, single stranded regions undergo slippage followed by misaligned pairing between direct repeats on the template and nascent DNA strands and ending with the deletion of the stretch of DNA 40000229

between the repeats. This model is supported by observations in many prokaryotic (Farabaugh et ~I, 1978; McCorkle & Altman, 1982; Struhl, 1981) and eukaryotic (Fedoroff, 1979; Efstratiadis et a_~l, 1980) systems, and proposes further that inverted repeats (palindromes), bracketed by the direct repeats, play an important role as stabilizers of the mispaired structures thus favoring the occurrence of deletions. Since structures of this type could form during the excision of transposons this model is also applicable to these events (Albertini e_~t a~l, 1982). However, not all deletions occur between direct repeats. To explain many deletions and complex mutations in bacteriophage, bacteria and yeast Glickman and Ripley (1984) have assigned a major role to palindromic or quasipalindromie sequences as stabilizers of misalignments through the formation of transient hairpin struc- tures which can be resolved as deletions even in the absence of direct terminal repeats. In support of both models, palindromes are~unstable in plasmids (Sadler & Tecklenburg, 1981) and bac- teriophages (Williams & Muller, 1987), can arrest DNA pol~rmerase "in vitro" (Weaver & De Pamphilis, 1984; La Duca et al, 1983; Baumel et al, 1984) and are lethal to carrier plasmids unless deleted (Hagan & Warren, 1983). While these models have identi- fied certain DNA struotures (i.e. direct and inverted repeats) as playing major roles in deletion formation, they have left entirely open one major question: how are the transient secondary structures formed between direct and/or inverted repeats resolved as deletions? This problem was first addressed, by Franklin 5 40000230

(1967, 1971) who reported that spontaneous deletions were not caused by homologous recombination but rather by processes Of "illegitimate" recombination which do not require extended regions of base sequence homology. In general, most investi- gators asking the same question have obtained similar results (Inselburg 1967; Anderson, 1970; Coukell & Yanofs~y, 1970; Foster et a~ 1981; Collins, Volckaert and Nevers, 1982; Jones, Primrose and Ehrlich, 1982; Das Gupta, Weston-Hafer and Berg, 1987) but there are two important exceptions. Sommer, Schumacher and Sedler, (1981) and Albertini et al (1982) using different A ' systems, found that the same deletions were I0 to 100-fold more frequent in recA+ than recA- cells. Thus, recA+ function participates in the formation of some deletions. Since the recA+ protein, in addition to being essential for homologous recombina- tion (clark, 1973) plays a major role in regulating the SOS response (Walker, 1984) and is needed in SOS mutagenesis (Ennis et al, 1985) it is unclear which mechanisms (homologous recom- bination, SOS processing or other) are defined by its participa- tion. One class of very large deletions postulated by Anderson and Roth (1977) to explain the elimination of large duplicated segments (up to 20% of the Salmonella chromosome) seems to be entirely dependent on recA+. These deletions may not occur by misalignment mutagenesis, however, and may represent a special case. in any event, the role of recA in deletion formation is still unresolved. The identification of the major structural parameters of 6 40000231

deletions (direct and inverted repeats) has suggested a strategy, based on the use of specially designed plasmids, for a systematic study of these rearrangements. This strategy should not only give complete control of all the factors which play a role in causing deletions, but it should also allow us to ask precise questions and obtain clear and unambiguous answers through rapid and simple experiments. Well characterized plasmids with unique restriction site within genes giving a selectable phenotype are excellent model systems to use for this purpose. We have chosen the multicopy plasmid pBR325, which has a number of unique restriction sites within genes determining resistance to the drugs tetracycline, ampicillin and chloramphenicol (Bolivar, 1978). By the simple expedient of cloning any fragment of desired size and sequence into one of these sites we inactivate the gene, so that revertants should result exclusively from the deletion of the inserted fragment. Selecting for reversion from drug sensitivity to resistance should give us only deletions, making their frequency easy to quantitate by standard procedures. In this report we described the construction of'derivatives of pBR325 by inserting fragments of the same size but different sequence into the unique EcoRl site of this high copy number plasmid, and their use to (a) test the proposition of Albertini et a~ (1982) that palindromes stimulate deletions between direct repeats and (b) to determine whether recA plays a role in the production of these deletions. In the course of these experi- ments we have uncovered a novel situation: tandem duplication of 7 40000232

a direct repeat stimulates deletion of a non-palindrome over that of a palindrome of the same size at the same location. We have also found that deletions on our pBR325 derivatives are increased slightly" by recA+ and even more by its allele recA730 (Witkin e__t al, 1982) which produces both constitutive expression of the SOS response and an increase in recombination frequency. These findings have important implications for the eventual understand- ing of the role of recA in the genesis of deletions. 40000233

MATERIALS AND METHODS Culture Media L-broth and L-agar were prepared according to standard recipes (Silhavy, Berman and Enquist, 1984). To. detect colonies with constitutive lac operon function, a diagnostic feature for plasmids carrying la__~c operator inserts, we used X-Gal plates (Smith and Sadler, 1971), prepared as described by Betz et al (1986). Chloramphenicol and ampicillin (Sigma) and tetracycline (Aldrich) were added when necessary at final concentrations of 25 g/ml. Bacterial strains and plasmids The strains employed in this investigation are listed in table i. Plasmids were introduced by transformation. Using the procedure of Wensink et al (1974) as modified by Sadler et al (1977). All plasmids employed in this investigation were con- structed as described under Results. Preparation of plasmid DNA for restriction enzyme analysis and sequencinq The procedure employed to obtain large amounts of plasmid DNA was essentially the one described by Sadler et al (1977) with minor modifications. Three liter L- broth cultures were grown in a small fermentor at 37°C with the addition of chloramphenicol to amplify synthesis of plasmid DNA (Hershfield et al, 1974). 40000234