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~ University of Colorado Health Sciences Center Dr. Elias Balbln~'r Box B-121 4200 Ea.~t Ninth Avtnu¢ University Hc~.~pi~ls ..... ~..',.~: (303) 27~5518 School ~f Nursing Schc~l of D~.ntis~ru" Fax: (3f.O) 270-5~215 School of P~armzcy Graduate Sch~:~l Dr. Arthur Eisenberg Council for Tobacco Research 900 Third Avenue New York City, NY 10022 Aug~st~-3 ,'; 1994 Dear Dr. Eisenberg I am submitting a preliminary application for a research project entitled " Rearrangement mechanisms ingrowing and resting bacteria-A model system for carcinogenesis" The proposed duration of the project is three years and ~the. projected budget is $ 84,800 per year in direct costs (see page 4). Thank you very much for your consideration. Sincerely yours Elias Balbinder,PhD Research Professor Dept. of Biochemistry, Biophysics and Genetics Box B 121 Telephone # (303)270-5518 Fax # (303) 270-8215 40000080

1 PROJECT PLA~- ~earrangement meghanisms in growing and resting bacteria- A model, system for carcinogenesis. Recent reports that repetitive DNA sequences important in human genetic disease are highly unstable and mutation prone and that hereditary colorectal carcinoma cell lines are defective in mismatch repair (1,2) have shown that predictions based on decades of reseach with the bacterium E.coli were correct (3"), strength- ening the relevance of bacterial model systems for studies of mutations in cancer. It is also clear from recent studies in E.coli that important differences in mutation mechanisms exist between actively growing and stationary phase (starvation stressed) cells. Mutations in actively growing cells are random and generation dependent while those in stationary phase (non-dividing) cells seem to be adaptive, i.e. advantageous in direct response to particular environmental challenges, and are time rather than generation dependent (4). Stationary phase ~bacteria and actively growing cells differ completely in their physiology (5), and it is therefore not surprising that mutations under each of these conditions occur through different mechanisms with the participation of distinct sets of genes (4,6). It has been proposed (7) that cancer cells resemble stationary phase bacteria in that mutations in them are time dependent and adaptive, thus allowing them to divide more rapidly than normal cells. We are seeking a deeper understanding of the different mechanisms involved in the production of deletions in both actively dividing and starving E.coli. Our specific objectives are presented below. I- Deletions in actively growing cells - Involvement of SOS processing. Following our demonstration (8) that in actively growing cells SOS derepression stimulated the deletion of palindromic inserts in E.coli plasmids we want to test several predictions (below) of our hypothesis (8) that transient structures which interrupt DNA replication, such as hairpins formed at palindromic sequences, can be corrected like mutagenic adducts,i~e, by SOS processing through a bypass replication mechanism., A- If hairpins formed by slippage at palindromic sites are equivalent to mutagenic lesions they will induce the SOS-response. This should be easy to determine using strains in which ~ is under control of SOS promoters (9) so that induction, of SOS functions, after introducing a plasmid carrying an unstable palindrome, can be measured byassaying for 5-galactosidase. Such strains are already in our collection. B- The stimulation of palindrome deletions in plasmids by SOS derepression ~equired the presence of excess activated RecA (RecA* 730) and UmuC÷ (8). RecA* may be required for its direct role in SOS mutagenesis, i.e. facilitation of translesion replication (i0). since different RecA* alleles stimulated spontaneous mutation to different extents in an allele specific manner (I0), it is possible that the same allele-specificity exists for SOS-stimulated palindrome deletions. If so, we expect that the same RecA'alleles 40000081

in SOS-derepressed (lexA-) strains will stimulate spontaneous point mutations, mutagen-induced 9oint mutations and palindrome deletions in plasmids to different extents, but different recA*alleles will show different spectra of mutation frequencies for each of the different events. We have a collection of isogenic strains with different RecA" alleles obtained from Dr. E.Witkin, and some of these have already been transformed with palindrome-carrying plasmids. Once all strains are transformed, the measurement of mutation frequencies is straightforward (see ref.9) c- Trinh & Sinden (11) presented evidence supporting a replication-dependent mechanism for deletion of palindromic sequences using a specially designed set of ~lasmids with asymmetric palindromic inserts in the catgene of pBR325 in a ~ecA- strain. We intend to use the same plasmid set to measure deletion frequencies in strains derepressed for SOS (8). If SOS-stimulation of palindrome deletion occurs by a bypass replication mechanism requiring DNA polymerase III, as our model predicts, we will only see a large increase in deletion frequency when the asymmetric palindrome is eliminated from the lagging strand of replication, as Trinh and Sinden have shown (11). 2-A..model for helicase II participation.~n deletion formation in growing and starving cells. A- Based on its unique phenotype and map position dli2 is a mutation in uvrD, the gene for helicase II, and resembles other null uvrD alleles in stimulating deletion incidence in growing and non-growing cells (12). It has been suggested that the normal function for helicase II is to unwind transient deletion intermediates to restore the pre-slippage configuration (13). Using a genetic approach we want to test to test a model which proposes that in growing cells helicase II is part of a replicative pathway for resolution of deletion intermediates "which requires the SOS functions RecA and UmuCD,. but in starving cells it is part of a different pathway which does not require SOS functions.The experiments will consist of .deletion frequency measurements in multiple mutant strains carrying different combinations of SOS alleles (8, 12) with and without dli2, both in growing and non- growing cells. B- We have recently isolated a set of suppressors of ~ii2 selected for total loss of Tnl0 excision. These are extremely interesting and could represent seconday mutations in--giving a very efficient helicase II, regulatory mutations for genetic functions under starvation stress control (5), or hovel'alleles for known components of the DNA metabolism machinery. At this stage they need to be mapped and characterized genetically. 3- Is rec22~l a_novel allele, of recBC which acts only in starving cells?. In plasmid pMC874 (12) deletions of 600-800 bp join the km= promoter to a promoterless ~ to give a Lac~ phenotype. In the wild type spontaneous Lac÷deletions start appearing after two days of incubation on McCo~key's medium, when cells are not actively 2 40000082

growing, and peak at about 4-5 days.Three deletion-associated events have been identified in plasmid pMC874and there may be mo~e (12). A mutation selected for increasing Lac~ deletion frequency, rec2~51, is a ~ allele with a novel phenotype (12). In this mutant deletion frequenoy is 70-fold higher that in wild type, but Lac÷ papillae appear within the same time frame as in the wild type. Reversion of lac- frameshifts requires recA_____/+ and rec~C~ in starving cells but not in growing cells (6), so it is possible that the rec2251 RecBC, but not the wild type enzyme, can give rise to specific deletions in non-dividing cells. We want to determine whether rec2251-induced deletions can appear as adaptive mutants on minimal lactose plates over time (i0 days), and whether such deletions originate from specific events in pMC874. This will be done by restriction enzyme analysis (12) and sequencing of deletion end points. 4- Initial character~atiQn .of m~ants which alter the timing of Tn~0 excision in starving cells. - I have isolated 35 mutants which alter the timing of Tnl0 excision from three different locations in the ~.coli chromosome: lac, fuc and ~alK. In some of these mutants~excision occurs early (one day) and in others much later (4-7 days). Such mutants have not been isolated before and are extremely interesting since they may represent mutations in genes which control the overall cell response to starvation (5) or novel mutations affecting control of DNA metabolism functions in stationary phase cells, among other possibilities. I propose to begin their genetic characterization and mapping. Mutants which turn out to be particularly interesting will be singled out for intensive study. REFERENCES l-T.A.Kunkel (1993) Nature 365:207-208. 2-J.Jiricny (1994) Trends in Genetics 10:164-168. 3-M.Radman and R.Wagner (1993) Nature 36__6:722. 4-P.L.Foster (1993) Ann.Rev.Microbiol. 47:467-504. 5-R.Kolter, D.A.Siegele and A.Tormo (1993)Ann.Rev.Microbiol.47:855- 874. 6-S.M.Rosenberg, S.Longerich, P.Gee-and R.S.Harris (1994). Science 265:405-407; P.L.Foster and J.M.Trimarchi (1994) Science 265:407-409. 7-B.S.Strauss (1992) Cancer Res. 5--2:249-253. 8-E.Balbinder, B.ColI, J.Hutchinson, A.S. Bianchi, T. Groman, K.A. Wheeler and M. Meyer (1993) Mut. Res. 286: 253-265. 9-C.J. Kenyon and G.C. Walker (1980) PNAS 7_/7: 2819-2823. 10- J.B. Sweasy, E.M. Witkin, N. Sinha, and V. Roegner-Maniscalco (1990) J.Baot.172~ 3030-3036. II-T.Q. Trinh and R.R. Sinden .(1991) Nature 252:544-547. 12- E.Balbinder (1993) Mut.Res. 299:193-209 13- S.W. Matson and K.A. Kaiser-Rogers (1990) Ann.Rev. Biochem. 5--9:289-329. 40000063

ESTIMATED DURATION OF DIFFERENT PHASES OF PROJECT First year: (a) Complete IA and IB (already in progress);(b) initiate 2 (A and B), 3 and 4. Second year: (a)Complete IC and 2A;(b) continue 2B ,3 and 4. Third year: (a) Complete 2A and 3; continue 2B and 4. ESTIMATED ONE YEAR BUDGET. DIRECT COSTS A) Personnel Dr. E.Balbinder (P.I., 50%) .... $40,000 (*) Technician .................. .. $20,000 • Total salaries ................. $60,000 Fringe benefits (33% sal.) ..... $19,800 Total personnel ................. $79,800 B) Supplies and Miscellaneous Consummables (culture media, enzymes, chemicals,glassware, plasticware, etc) ................ $ 4,000 Miscellaneous (fax, telephone mail, copies) ...................... $ 1,000 Total supplies and miscellaneous...$ 5,000 Total direct costs ............... $ 84,800 (*) As a Research Professor, E.Balbinder must obtain his salary from grants. 4 40000084

BIOGRAPHICAL SKETCH NAME I POSI'I~ TII~ Elias Balbinder I Research Professor EOUCATtON (Begin m~h baccalaureate or o~her irdtia! lXOfessional edison, ~ as nursing, and lnck.,de postdocfor~t [NSTITU'T1ONANOLOCA~ON Univ. of Michigan, Ann Arbor, R! Tndiana Univ., Bloomington, ]N Cargegie Tnst., Cold Spg. Rarb., Univ. of Calif., San Diego, CA DEGREE Ph.D. Postdoc Postdoc YEAR CONFERRED 1957 1957-60 1960-63 • FIELD OF STUDY Zodlog~ Zoology (Genetics Bacterial Genetics Biochemistry RESE.~CH AND PROFESSIONAL EXPERIENCE: Colluding ~ pre~nt position, i=. in ¢ta~mo~gica! orde~ Wevious emp~oyme~, expeder, ce. and hono~ Key pemonnel i~lude the principal inve~gator and any other ind~luals ~o padidp~le b the ¢~en~ developme~ ~ exeoj1~ of the WojecL Key personnel typk:aI~ ~ l~tude ~ ind',Adua~ with doctora~ = other po~=k~'~ai degree, but ~ some ~ojects ~ll Include individu~= = the maste~ ~ baccaJaumate ~vel ~ov~ded they mntnlotde in a substance way to the scientific development or exeoJPon ~ th~ ~ect. Irtctode ~ese~ membem~p on any Federal Government puM~ ~ comm~ee. List, in ~m~logical o~de~ the ~les, a8 author& a~ ¢~nptete m~mnces to ~1 ~¢ations dudng the past three years and ~ mwesen~e ea~er pu~matio~s ~ ~ ~ ap~icatio~. O0 NOT EXCEEO TWO PAGES. 1963-67 Assistant Professor of Genetics, Dept. of Bacteriology and Botany, Syracuse University, Syracuse, NY. 1967-71 Associate Professor of Genetics, Dept. of Bacteriology and Botany, Syracuse University, Syracuse, HY. 1971-76 Professor of Genetics, Dept. of Biology, Syracuse University, Syracuse, NY. 1976-82 Head, Laboratory of Genetics and Carcinogensis, AHC Cancer Research Cente~ and Hospital, Lakewood, Colorado. 1982-93 Ad3unct Professor, Dept. of Biochem/Biophys/Genetics, University of Colorado Health Sciences Center, Denver, Colorado 1993- Research Professor, Dept. of Biochem/Biophys/Genetics, University of Colorado Hea]Eh Sciences Center, Denver, Colorado Fellowships, etc. 1960-63 Zl6S-SlSS z/70-8/70 ]977-79 6/ez-9/eI Postdoctoral Research Fellow, American Cancer Society - Laboratory of Dr. David M. Bonnet, University of California, LaOolla, California. Fulbright-Hays Award to conduct research at the Department of Biology~ Univ.of Buenos Aires, Argentina. Special Fellowship of the NaEional Institute of Health. To conduct research in the laboratory of Dr. Aizo Matsushiro, Osaka University, Osaka, ~apan. Member, National Science Foundation Panel on Genetic Biology. Recipient of Fulbright-Hays Award and Visiting Professor, Univ. of Los Andes, Bogota, Colombia. To teach course in Molecular Genetics. publications..duEin9 the past three yeaKs Balbinder, E. and Waldren, C. A review of DHA metabolism in E, coll. Cell Biology Reviews ~5:105-155 (199i). Balbinder, E.,Co11,B.,Hutchinson,O.,Bianchi,A.S. and Groman,T., Wheeler,K.A. and Meyer,M. Participation of the SOS system in producing deletions in ~ plasmids. Mutn. Res. 286:253-265 (1993). Balbinder,E. Multiple pathways for deletions in Esch~ichia coli. Mut. Res. 299:193-209(1993). Levy,M.$., Balbinder,E.and Nagel,R. Effect of mutations in SOS genes on UV- induced precise excision of TnlO in Escherichia coli. Mut. Res.293:241-~#7 (1993). PHS 338 (Rev. &~l) (Fc~rm Pa;e 6) Page FF 40000085

Principal ReDresentative ~ertinent earlier oublications 1. Callahan,R.and Balbinder,E. Tryptophan operon: Structural gene cutatton creating a "promoter" and leading to 5-methyltryptophan dependence. Science ]68:]586-1589 (lg70). 2. LaScolea, L.J., and BalbindeP, E. Restoration of phosphoribosyltransferase activity by partially deleting the ~ gene of the tryptophan operon of ~.tvDhi~uriu~. J. Bact. 112:877-885 (1972). 3. Mocrina,F.L. and 8albinder, E. Plasmid associated functions of a stable F'lac. Bact. 113:1183-1191 (1973). 4. Mocrina, F.L., Balbinder, E., and Bassel, Alix, Molecular characterization of a stable F']acplasmid. Bioche~. & Biophys. Res. Com. 54:737-743 (1973). 5. Callahan,. R. III, Dooley,H.H., and Balbinder, E. A mutation to 5-methyltryptophan dependence in the tryptophan (trp) operon of Salmonella tvDhimuriU~. II. Studies of 5-methyltryptophan-dependent mutants and their revertants. Mo]ec. Gen. Genet. ]65:129-143 (1978). 6. LaScolea, L.J., Oooley, H.H., lorget, R., and Balbtnder, E. A mutation to 5-~ethyltryptophan dependence in the rt~Jl operon of Salmonella Correlation between phenotype and the wope~ties of the second enz)Ine fop tryptophan biosynthesis in a 5-methyltryptophan dependent mutant and several 5-methyltryptophan-independent revertants. Molec; ~en. Genet. 165: 145-153 (1978). 7. Angelosanto, F.A., ~orget,R., and Balbtnder, E. A ~utation to a 5-~ethyltryptophan dependence in the ~ operon of Salmonell~ tTphi~uriu~. IV. Isolation and characterization of tJ:llpro~oter mutations. Molec. Gen. Genet. 165: 8. Dooley, M. and Balbinder, E. Differences between the Anthranilate-5-phosphoribosy] pyrophospate phosphoribosyltransferases of Salmonell~ typhimurium strains LT2 and LT7. J.Gen. ~icrobiol. 112:171-179 (1979). 9. Balbinder, E., Reich, C.I., Shugarts, D., Keogh, ~., Fibiger, R., Jones, i., and Banks, A. Relative mutagentcity of some urinary~etabolites of the antitu~or drug cyclophosphamide. Cancer Research 41:2967-2972 (1981). 10. Balbtnder, E., Kerw, D. and Reich, C.I. Deletton induction in bacteria. ~. ihe Pole of mutagens and cellular error-prone repair. Hut.Reg. 112:147-168 (1983). 11. Balbinder, E., Stick, $.H. and Sharma, O.K. Complex effects of retinol on the metabolic activation of 2-a~inofluorene. Env. Hut. ~: 665-678 (1983) 12. Balbinder, E. and Kerry O. A ne~ strain of Salmonella ty~hvmurtU~ reverted by mitomycin C and N-methyl-N-nitro-N-nitrosoguanidine; a possible universal tester for Inutagentc compounds. Hut. Reg. 130:315-320 (198~). 13. Balbtnder, E. Use of pre-designed plasmtds to study deletions: strategies for dealing with a complex problem.Chapter 40, pp.378-391-ASM, Washington, D.C. In~_N~ Replication and Motaoenesl$. Eds.R.E.Moses and W.C.Su~ners (1988). 14, Balbinder,E.,HacVean,C. and ~illiams,R.E. Overlaping direct re,eats stimulate deletions on specially designed derivatives of plasmid pBR325 ]n cF.~_~jL. ~ut.Res. 214:233-252 (1989). Page 31 40000086

CURRENTLY ACTIVE GRANTS, CONTRACTS and OTHER SOURCES of FUNDS List financial support .([direct costs, only) from all sources, inctudin~ own institution. Title of Projec~ Regulation of synthesis se- cretion of salivary proteins. (co-PI; K.N.Prasad,PI] Construction of E.coli strait useful for stabilizing un- clonable DNA sequences (con- tract) Sources (give grant numbers) NIH- ROI 9EO9589 s Life Inc. Gaithersbu Total Value of C~ant (d~t 1,278,155 (5 ys.) $12,000 :g Cun-ent Annual Amount A~ilable to You $ 18,000 (22% salaryl $12,000 Date of Termination of Grant 9-30-94 (my part. 9-30-94 Identify and describe any overlap oft~s applic~ion with the above grants: No overlap- There has been no funding for the project in this application since 1992. Indicate the total annual funds available to you this year for all research projects under your supervision. 12,000 PENDING 0K PLANNED Title of Project Construction of E.coli strains .... (see above) (contract) (pending) Sources n,umb.ers) Life Tech. InOo Total Value of Grant (direct costs) $ 50,000 Avg. A~mual Amount Available to You 50,000 Total Duration (give inclusive dates) 1 year (10-1-94 thro 9-30-95) Identify and describe any overlap &this application with the above project. He overlap- Application to NSF and NIH of a prposal to study X-ray-induced deletions planned for some time this year depending on completion of preliminary experiments. tgh 40000087