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.~r,_,~..~ Rezz-~-~, 214 (I~,~) 233-252 ~.fUT LM783 233 Overlapping direct repeats stimulate deletions in specially designed derivatives of plasmid pBR325 in Escherichia cell Elias Balbinder, Cheryl Mac Vean and Robert E. Williams Department of Biochemistry, Bio2~hysics and Genetics and Colorado Cancer Center, University of Colorado Health Sciences Center, 4200 E. 9th Ave., Box B-121, Denver, CO 80262, and International Center for Cancer Research and Developmental Biology, Denver, CO (U.S.A.) (Received 19 December 1988) (Revision received 6 March 1989) (Accepted 6 April 1989) Keywords: Deletions on plasmlds; Plasmids, deletions on; DNA, single-stranded, misalignments; Chloramphenicol acetyltransferase Summary Current misalignment mutagenesis models have identified certain sequences such as direct and inverted repeats, which can stabiliT¢ transient misalignments on single-stranded DNA, as major structural parame- ters for deletions. We have constructed derivatives of the plasmid pBR325 to investigate further the relative roles of such sequences under controlled conditions. The plasmid derivatives pOCE15, pRS1 and pRS4, were obtained by eloulng fragments of the same approximate size (60-64 bp) but different sequence into the unique EcoR1 site of the chloramphenicol acetyl transferase (cat) gene of plasmid pBR325. The insert of pOCE15 is a perfectly palindromic lac operator fragment. Both pRS1 and pRS4 ean~j the same non-palindromic fragment but differ from each other in the sequence at the right (3') end of the insert. Plasmid pRS4 differs from pRS1 by a 9-bp duplication containing an additional EcoRl site at the 3' end of the insert. This arrangement yields overlapping imperfect 17-18 bp and perfect 11 bp direct repeats at the deletion termini and creates multiple opportunities for the stab'flization of misaligned intermediates. The deletion rate, measured from the reversion of chloramphenicol sensitivity (Cms) to resistance (Cm~), was always highest in pRS4, intermediate in pOCE15 and lowest in pRS1, with an approximate ratio of 100:10:1. We have also obtained evidence for the participation of RecA in the genesis of ddetions in these pBR325-derived plasmids. Deletions are an important class of genetic rearrangements and have been found to be at the basis of a number of cancers and other genetic diseases (Yunis, 1983; F.scot et al., 1986; Monaco Correspondence: Dr. ~ Balbind~r, Department of Biochem- istry, Biophy~es and Genetles, B-121, Unk.ers~ty of Colorado Health Sdene~ Center, 4200 E. 9th Ave., Denver, CO g0262 (u~.). et al., 1985). They have been extensively studied at the molecular level, mainly in prokaryotes, and most of them are explained by misalignment mutagenesis models. These propose that deletions arise from the resolution of transient misalign- ments formed by slippage of single-stranded stretches of DNA during replication and stabi- lized by sequence homologies such as terminal direct repeats, intervening inverted repeats (palin- dromes) or a combination of both (Albertinl ct al., ~027-5107/89/S03.50 © 1989 Elsevier Science PubIL~ers B.V. (Biomedical Divifion) 40000083