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~_~_.._~ University of Colorado Health Sciences Center £,-121 270-5518 Fax: (303) 270~2-15 420~) Ea~! Nir~th A%enue Dcmcr. Cch3rado ~0262 University Hosl~itals Solace! of Medi~ne School of Nursing Seh~)l of DemistD, School of Pharm.a~- GraSuate Schozl Dr. Arthur Eisenberg Council for Tobacco Research 900 Third Avenue New York City, NY 10022 Dear Dr. Eisenberg I am submitting a preliminary application for a research project entitled " Rearrangement mechanisms in growing 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 77~t" gbriversitv ,,f C, dr2r.,ib~ He~;lth Scier, ees Ce*uer is crm:u*~itted tc* eq:~ttl t.Tpornmitv a~zd a.~Tn?:ative actii;~, 40000074

~ University Colorado Health Sciences Center of Dr. FA-:z~ Ez!b':n-~:r B~x P,-121 FI:,cr.~: 003) 270-5518 270-8215 Der~cr. Ct~h~rado ,~0262 UrAver~ity Sch~l (~f Medicine Sch~:~l of Nursing S-c.hotfi of Dentist~" Gmd~at~ School Dr. Arthur Eisenberg Council for Tobacco Research 900 Third Avenue New York City, NY 10022 August--3,~ 1994 Dear Dr. Eisenberg I am submitting a preliminary application for a research project entitled " Rearrangement mechanisms in growing 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 40000075

1 PROJECT PL~- Rearrangement mechanisms 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 hair~ins formed at palindromic seq~.ences, can be corrected like mutagenlc adducts,i.e, by SOS processlngthrough 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 lacZ is under control of SOS promoters (9) so that induction of SOS functions, after introducing a plasmid carrying an unstable palindrome, can be measured by assaying for B-galactosidase. Such strains are already in our collection. B- The stimulation of palindrome deletions in plasmids by SOS derepression required 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 (10). 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 1 40000076

in SOS-derepressed (lexA-) strains will stimulate spontaneous point mutations, mutagen-induced point 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 (ll) presented evidence supporting a replication-dependent mechanism for deletion of palindromic sequences using a specially designed set of plasmids with asymmetric palindromic inserts in the cat gene of pBR325 in arecA- 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 (ii). 2-A model for helicase II..participation in 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 ingrowing 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 dli2 selected for total loss of Tnl0 excision. These are extremely interesting and could represent seconday mutations in uvr~ giving a very efficient helicase II, regulatory mutations for genetic functions under starvation stress control (5), or novel alleles for known components of the DNA metabolism machinery. At this stage they need to be mapped and characterized genetically. 3- Is rec225~ a novel allele of recBC which acts only in starvinq 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 McConkey's medium, when cells are not actively 2 4OOOOO77

growing, and peak at about 4-5 days.Three deletion-associated events have been identified in plasmid pMC874 and there may be more (12). A mutation selected for increasing Lac÷ deletion frequency, rec2251, is a recBC allele with a novel phenotype (12). In this mutant deletion frequency 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 recBC÷ in starving cells but not ingrowing 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-induceddeletions can appear as adaptive mutants on minimal lactose plates over time (I0 days), and whether such deletions originate from spec±fic events in pMC874. This will be done byrestriction enzyme analysis (12) and sequencing of deletion end points. 4- Initial characterization of mutants which alter the timing of Tnl0 excision in starving cells. - I have isolated 35 mutants. which alter the timing of Tnl0 excision from three different locations in the E.coli chromosome: lac, fu__cand galK. 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.Ktlnkel (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. 4_/7:467-504. 5-R.Kolter, D.A.Siegele and A.Tormo (1993)Ann.Rev.Microbiol.4__7:855- 874. 6-S.M.Rosenberg, S.Longerich, P.Gee and R.S.Harris (1994) Science 26__~5:405-407; P.L.Foster and J.M.Trimarchi (1994) Science 26__~5:407-409. 7-B.S.Strauss (1992) Cancer Res. 5_~2:249-253. 8-E.Balbinder, B.Coll,~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 77: 2819-2823. i0- J.B. Sweasy, E.M. Witkin, N. sinha, and V. Roegner-Maniscalco (1990) J.Bact.172: 3030-3036. II-T.Q. Trinh and R.R. Sinden (1991) Nature 35__/2:544-547. 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. 3 40000078

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. 40000079