Copyright 8 1997 by the Genetics Society of America Genetic Analysis of the Chlamydomonas &nhadtii I-CmI Mobile Intron Homing System in Escherichia coli Lenny M. Seligman,*’tKathryn M. Stephens,*Jeremiah H. Savaget and Raymond J. Monnat, Jr.* *Department of Pathology, University of Washington, Seattle, Washington 98195-7705 and ?Department of Biology, Pomona College, Claremont, California 91 71 1 Manuscript received May 22, 1997 Accepted for publication September 17, 1997 ABSTRACT We have developed and used a genetic selection system in Eschm’chia coli to study functional require- ments for homing site recognition and cleavage by a representative eukaryotic mobileintron endonucle- ase. The homing endonuclease, I-CreI, was originally isolated from the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. I-CreI homing site mutants contained base pair substitutions or single basedeletions that altered the rate of homing site cleavage and/or product release. I-CreI endonu- clease mutants fell into six phenotypic classes that differed in in vivo activity, toxicity or genetic domi- nance. Inactivating mutations clustered in the N-terminal 60% of the I-CreI amino acid sequence, and two frameshift mutations were isolated that resulted in premature translation termination though re- tained partial activity. These mutations indicate that the N-terminal two-thirds ofthe I-CreI endonuclease is sufficient for homing site recognition and cleavage. Substitution mutations altered in four potential active site residues wereexamined DZON, Q47H or R70A substitutions inactivatedendonuclease activity, whereas S22A did not. The genetic approach we have taken complements phylogenetic and structural studies of mobile intron endonucleases and has provided new information on the mechanistic basis of I-CreI homing site recognition and cleavage. OBILE intron endonucleases are a diverse family between intron-plusand intron-minus strainsor, in the M of proteins encoded by introns that canself- case of bacteriophage mobile introns, in the context splice at the RNA level or, as protein introns (“in- of infection. Mobile intron endonuclease homing sites teins”) , at the protein level. The endonucleolytic activ- differ markedly fromthe recognition and cleavage sites ity of these proteins can promote lateral transfer (or of other site-specific endonucleases. For example, the “homing”) of their encoding introns to specific sites type I1 restriction endonucleases recognize and cleave in complex genomes.The intron homing cycle (Figure short (4-8 bp) DNA sequences that are often perfectly 1A) is initiated and targeted by a site-specific DNA dou- twofold sequence-symmetric or palindromic (ROBERTS ble strand break made by the intron-encoded endonu- and HALFORD 1993). Mobile intron homing sites, in clease in a homing site allele that lacks the intron. A contrast, are considerably longer (15-40 bp), and often copy of the intron containing the endonuclease open display little or no twofold sequence symmetry (LA” reading frame is transferred to the cleaved homing site BOWITZ and BELFORTlP93; MUELLERet al. 1993; BEL by DNA double strand break repair or gene conversion FORT and ROBERTS1997). (LAMBOWITZ and BELFORT1993; MUELLERet al. 1993; The length and specificity of cleavage of many hom- BELFORTand PERLMAN1995; BELFORTand ROBERTS ing sites,their location in physiologicallyimportant host 1997). The inserted intron is replicated with host DNA genes and the ability of many mobile introns to self- and expresses the intronencoded endonuclease, thus splice at the RNA or protein level suggest that intron retaining the potential for homing to other endonucle- homing may have arisen as a form of “smart” DNA ase-sensitive, intron-minus alleles. parasitism: once present within an essential gene, a mo- Mobile introns encoding endonucleases have been bile intron can self-splice at the RNA or protein level identified in organelles, and in a few instances the nu- to minimize deleterious effects on the host and thus clei, of many different unicellular eucaryotes. They have insure intron propagation with the potential for lateral also been identified ineubacteria, in Archea and in bac- transfer (reviewed in LAMBOWITZ and BELFORT1993; teriophage genomes. Homing of these encoding in- MUELLERet al. 1993; BELFORTet al. 1995; BELFORTand trons occurs inthe context of mating or transformation ROBERTS1997). We have developed and used a genetic system inEsch- erichia coli to studyfunctional requirements for homing Cmresponding author: Raymond J. Monnat, Jr., Department of Pa- thology, University of Washington, Box 357705, Seattle, WA 98195 site recognitionand cleavage by 1-CreI, a eucaryotic mo- 7705. E-mail: [email protected] bile intron endonuclease. The 888-bp I-CreI mobile in- Genetics 147: 1653-1664 (December, 1997) 16.34 I.. M. Seligman et 01. A B intron+ allele ho m ing site resolution site homing / Ir'l " I dsb repair FIGL'KEl."I-CreI mobile intron homing cyclc and genetic assav for I-Crel homing site recognition and cleavage in E coli. (A) Homing of theI-Crel mobile intron is initiated hy a site-specific DNA double strand break macle by I-Crd endonuclease encoded hy the mobile intron open reading frame (filled box of intron' allele, at top center). The I-Crd homing site is located in the C. rei17/tnrd/ii chloroplast 23s rRNA gene (open hox, top left). The cleaved homing site is a substrate for double strand break repair off the intron-plusallele. Repair transfers a copy of the intron to the cleaved recipient allele (dsh repair, hottom center). Resolution (resolution, top right) generates two alleles containing the mobile intron, eachflanked hv a disrupted I-CreI homing site. (€5) A genetic assay for 1-Crel endonuclease function was estahlished in E. coli by placing the I-Crel endonuclease open reading frame ona plasmid, pBE, under the control of arahinose operon regulatory sequences (the arn promoter, r, and araC regulator) and an I-Crel homing site on an FI28 F' Factor acljacent to a kanamycin resistance gene (F'S). Expression of active I-Crel endonuclease leads to the loss of the F' kanamscin marker, presumably due to homing site cleavage. Mutations that inactivate either endonuclease function or that render the homingsite resistant to cleavage generate cells that retain kanamycin resistance. Homing with intron transfer does not occur in this assay due to lack of the 888-hp mohile intron (see A). tron was originally identified in the chloroplast genome in conjunction with recent biochemical data and the of the unicellular green alga Clzlnmyiomonm rhllnrdtii X-ray crystal structure of native I-CreI protein (HEATH (ROCHAIX andMAI.NOE 1978; ROCHXIXe/ nl. 198.5; LE- ~t ol. 1997; STEPHENSet nl. 1997; K. M. STEPHENS,unpub MIEUX et nl. 1989). This intron canself-splice at theRNA lished results), would begin to provide a detailed pic- level and can home to copies of the 23s rRNA gene ture of the structural andmechanistic bases for homing that lack the mobile intron (HERRISet nl. 1990; DPRREN- site recognition and cleavage by I-Crd, and by other RERGER and ROCHAIX 1991; THOMI'SOSand HERRIS similar eucaryotic mobile intron endonucleases. 1991; DCRRENRERGERet nl. 1996). The 163-residue I-CreI endonucleaseprotein is en- MATERIALSAND METHODS coded by a 489-bp open reading framewithin the I-Crd Bacterialstrains and media: The following E. coli K-12 mobile intron. The I-Crd recognition and cleavage site strains used in this studv were kindly supplied by Dr. COLIN is a 19-24-bp sequence in the 3' end of the C. rpinhnrdtii MASOII. (Department of Genetics, University of Washington): chloroplast 23s rRNA gene. This site displays limited, CCl36, [ F12X (lac~)/A(lac+ro),am, naM, nrgE(am), thi, rpoB]; twofold sequence symmetry around four central basep CJ236, [dull zing1 /hi-l reAl/pCJl05(cnm'F')]; CSH116, [am airs that are asymmetrically cleaved to generate four lnlllL)5 zn~-502::7'n10A(g)c,/-lac)5 rpsI.]; DB6438, [lac A(lac, pro) Xlll, 1poB argE mptB m1tt7198J;MC1000, [arnD139 A(nm, base, 3'0Hextended cohesive ends. Intron insertion /~u)7697A(Inr)X74 gnKgalK /hi rpsl,].Standard growth media occurs 1 bp .5' to the cleaved centralfour basepairs were used (MILLER1992). Where required,they were supple- of the I-CrpI homing site, separating the halves of the mented with kanamycin (50 pg/ml), carhenicillin (100 pg/ homing site to generate an intron-plus allele that is mi), chloramphenicol (30 pg/ml), or arahinose (0.2%w/v). endonuclease-resistant (ROCHAIXet nl. 1985; LEMIELIX Plasmid constructions: pL-S, containing a I-Crd homing site and akanamvcin-resistance genefrom Tn5, was con- pt nl. 1989; THOMPSOSd nl. 1992; D~RRESRERGERand structed from pLrec, a 12-kh plasmid containing two E. coli ROCHAIX 1993; see Figure2). Car% genes that weregenetically inactivated by XhoI linker Our aim in initiating a genetic analysis of the I-Crd insertion or hy linker insertion and truncation (HERZING and homing system was to develop a facile genetic system MM' 1993). First, a 35-hp oligonucleotide containing a wild- to determine sequence requirements for I-CrrI intron type 24-hp I-CreI homing site sequence and an adjacent XOaI site was ligated into pLrec that had been partially digested homing siterecognition and cleavage. We reasoned with XhoI. An XI~OI-BSIBIfragment containing the Tn5 kana- that a genetic analysis of endonuclease and homingsite mycin resistance gene from plasmid pLS200 (SEI.IGMAN and requirements for homingsite recognitionand cleavage, M.ASOII. 1994) was then ligated into the BstBI and remaining I-CreI Homing1655 System Mutants &I sites of pLrecCre2 to generate pLs. The orientations of I-CreI homing site of pLs to the F' factor F128 (HOLLOWAY the oligonucleotide and restriction fragment were verifiedby and%@W 1996) of E. coli strain CC136. The resulting F'S restriction digestion and DNA sequencing. pLac+ was iso- recombinants were selected by isolating kanamycin-resistant lated after pLs transformation into E. coli, followed by screen- colonies after mating, followed by screening for carbenicillin ing for the generation of Lac+ colonies on indicator plates.