Associate Professor and Chair
Life Science II 139
Research Specialities: Molecular Mutagenesis and DNA Repair. Mutagenesis is a complex process that reflects the ability of a cell to correctly repair DNA damage that may result from either intrinsic or extrinsic factors. For example, errors in the base sequence may be produced during normal DNA replication, an intrinsic cellular process. Alternatively, normal metabolic processes or exposure to exogenous influences may introduce covalent modification of the DNA bases that can lead to genetic changes. To prevent the occurrence of mutations, cells contain functions that can repair misincorporated or damaged nucleotides. Generally, these functions are very accurate. However, occasional mistakes arise and these errors can have profound consequences. In humans, for example, many cancers, heritable diseases and birth defects result from mutations in the DNA that alter the behavior of cells.
PhD, 1983, Indiana University School of Medicine
Over the past number of years, our laboratory has been investigating the molecular mechanisms of mutagenesis using the bacterium Escherichia coli as a model system. Our standard mutagenesis protocol involves direct DNA sequence analysis of base substitution mutations occurring within a precise region of the E. coli genome. With this reversion assay, we can observe both transitions (G:C to A:T and A:T to G:C) as well as transversions (A:T to T:A and A:T to C:G). Using this system, we have investigated ultraviolet light-induced and alkylation-induced mutagenesis. In addition, we have recently used a plasmid-based system that allows investigation of all possible single base substitution events. Transcription of either the normally transcribed or normally non-transcribed strand of the target gene can be induced in order to study the effect of high-level transcription on mutagenesis.
Transcription-induced effects on mutagenesis: The single- or double-stranded nature of DNA can affect the frequency and occurrence of different types of DNA damage and the efficiency of DNA repair. To investigate whether the more single-stranded nature of the non-transcribed DNA strand during active transcription could affect mutagenesis, we used an alkylating chemical (methyl-methane sulfonate, MMS) known to produce different levels of certain specific base adducts in single- or double-stranded DNA along with a DNA repair mechanism (AlkB) that is known to work efficiently on single-stranded DNA. The results clearly demonstrate that MMS-induced mutations are suppressed by AlkB and that in its absence, mutations occur much more frequently during high-level transcription of the gene. Additional studies using this system are currently underway.
Ultraviolet light-induced mutagenesis: Ultraviolet light (UV) can be directly absorbed by the bases in DNA. This absorption of energy can lead to chemical changes that produce DNA photoproducts including cyclobutane pyrimidine dimers (CPDs), pyrimidine-(6-4)-pyrimidone photoproducts, and a variety of minor lesions, such as the TA* photoproduct. A recently published study (Mak and Fix, 2008) showed that DNA sequence context affects UV-induced mutagenesis. Briefly, we determined that 1) the TA* photoproduct may contribute to UV mutagenesis, 2) the potential 5'-CT photoproduct (likely a CPD) may promote T to G transversions unlike its TT counterpart, 3) differential production or processing of photoproducts located in opposite strands may affect mutational outcome and 4) the local sequence environment may affect the types of photoproducts that form and, therefore, the spectrum of mutations that result. Ongoing studies are examining each of these issues in more detail.
Alkylation-induced mutagenesis: The alkylating chemical N-ethyl-N-nitrosourea (ENU) produces a wide variety of single base adducts, several of which have been implicated in mutagenesis. In 1993, we showed that A:T to G:C transitions occurred equally in the presence or absence of the inducible "SOS response". However, transversions (A:T to T:A and A:T to C:G) required a functional umuC gene product. We suggested that transitions may result from production of the promutagenic base O4-ethylthymine by ENU to produce base substitutions by simple mispairing during DNA replication. Transversions, in contrast, may result from production of O2-ethylthymine by ENU and umuC-assisted error-prone DNA replication. These suggestions were examined by Beenken et al. 2001.. In addition, an analysis of the DNA bases neighboring those that were altered during mutagenesis revealed possible effects. Transitions occurred more often when a purine was located 5' to the affected thymine residue, while transversions appeared to prefer a flanking G:C base pair (Cai and Fix, 2002).
Articles in Professional Journals
- Fix, D., Canugovi, C. and Bhagwat, A.S. 2008. Transcription Increases Methylmethane Sulfonate-Induced Mutations in alkB Strains of Escherichia coli. DNA Repair 7:1289-1297. PubMed link
- Mak, W.B. and Fix, D.F. 2008. DNA Sequence Context Affects UV-Induced Mutagenesis in Escherichia coli. Mutation Res. 638: 154-161. PubMed link
- Yang, Y. and Fix, D.F. 2006. Genetic Analysis of the Anti-Mutagenic Effect of Genistein in Escherichia coli. Mutation Res. 600: 193-206. PubMed link
- Burger, A., Fix, D., Liu, H., Hays, J. and Bockrath, R. 2003. In vivo deamination of cytosine-containing cyclobutane pyrimidine dimers in E. coli: a feasible part of UV-mutagenesis. Mutation Res. 522:145-156. PubMed link
- Cai, Z. and Fix, D.F. 2002. Neighboring Base Identity Affects N-ethyl-N-nitrosourea-Induced Mutations in Escherichia coli. Mutation Res. 508:71-81. PubMed link
- Yang, Y. and Fix, D.F. 2001. Reduction of ENU-Induced Transversion Mutations by the Isoflavone Genistein in Escherichia coli. Mutation Res. 479:63-70. PubMed link
- Johnson, J., Ding, W., Henkhaus, J. and Fix, D.F. 2001. Identification of a mutation causing increased expression of the tas gene in Escherichia coli FX-11 Mutation Res. 479:121-130. PubMed link
- Beenken, K., Cai, Z. and Fix, D.F. 2001. Overexpression of Ogt Reduces MNU and ENU Induced Transition, but not Transversion, Mutations in E. coli. Mutation Res. DNA Repair 487:51-58. PubMed link
- Dixon, K.E. and Fix, D.F. 1993. Expression of β-Galactosidase From a Hybrid Promoter:Operator Element in E. coli. FEMS Microbiology Letters 106:135- 138. PubMed link
- Fix, D.F. 1993. N-Ethyl-N-Nitrosourea-Induced Mutagenesis in Escherichia coli: Multiple Roles for UmuC Protein. Mutation Res. 294:127-138. PubMed link
- Fix, D.F. 1993. The rex Genes of Lambda Prophage Modify Ultraviolet Light and N-Methyl-N-Nitrosourea Induced Responses to DNA Damage in Escherichia coli. Mutation Res. 303:143-150. PubMed link
- Fix, D.F., Koehler, D.R. and Glickman, B.W. 1990. Uracil-DNA Glycosylase Activity Influences the Mutagenicity of Ethyl Methanesulfonate: Evidence for an Alternate Pathway of Alkylation Mutagenesis. Mutation Res. 244:115-121. PubMed link
- Gordon, A.J., Burns, P.A., Fix, D.F., Yatagai, F., Allen, F.L., Horsfall, M.J., Halliday, J.A. Gray, J., Bernelot-Moens, C. and Glickman, B.W. 1988. Missense Mutation in the lacI gene of Escherichia coli: Inferences on the Structure of the Repressor Protein. J. Mol. Biol. 200:239-252. PubMed link
- Fix, D.F., Burns, P.A. and Glickman, B.W. 1987. DNA Sequences of Spontaneous Mutation in a PolA- Strain of Escherichia coli Indicate Sequence Specific Effects. Molec. Gen. Genet. 207:267-272. PubMed link
- Fix, D.F. and Glickman, B.W. 1987. Thermal Resistance of UV-Mutagenesis to Photoreactivation in the lacI gene of E. coli ung. Mutation Res. 179:143-149. PubMed link
- Fix, D.F. and Glickman, B.W. 1987. Asymmetric Cytosine Deamination Revealed by Spontaneous Mutational Specificity in an Ung- Strain of Escherichia coli. Molec. Gen. Genet. 209:78-82. PubMed link
- Fix, D.F. 1986. Thermal Resistance of UV-Mutagenesis to Photoreactivation in E. coli B/r uvrA ung: Estimate of Activation Energy and Further Analysis. Molec. Gen. Genet. 204:452-456. PubMed link
- Glickman, B.W., Fix, D.F., Yatagai, F., Burns, P.A. and Schaaper, R.M. 1986. Mechanisms of spontaneous mutagenesis: clues from mutational specificity. Basic Life Sci. 38:425-37. PubMed link
- Glickman, B.W., Burns, P.A. and Fix, D.F. 1986. Mechanisms of spontaneous mutagenesis: clues from altered mutational specificity in DNA repair-defective strains. Basic Life Sci. 39:259-81. PubMed link
- Fix, D.F. and Glickman, B.W. 1986. Differential Enhancement of Spontaneous Transition Mutations in the lacI Gene of an Ung- Strain of E. coli. Mutation Res. 175:41-45. PubMed link
- Fix, D.F. and Bockrath, R.C. 1983. Targeted Mutation at Cytosine-Containing Pyrimidine Dimers: Studies of E. coli B/r with Acetophenone and 313 nm Light. Proc. Natl. Acad. Sci. USA 80:4446-4449. PubMed link
- Fix, D.F. and Bockrath, R.C. 1981. Thermal Resistance to Photoreactivation of Specific Mutations Potentiated in E. coli B/r ung by Ultraviolet Light. Molec. Gen. Genet. 182:7-11. PubMed link