Environmental & Molecular Toxicology

John B. Hays

Professor
Dept: EMT (1087B Ag Life Sciences Bldg)
Ph.D., University of California -San Diego
Phone: 541-737-1777
Fax: 541-737-0497
haysj@science.oregonstate.edu

Genomic-Stability Functions. Cells defend the integrity of their genomes - three billion DNA base-pairs in human beings - with astonishing success. Critical to low DNA replication error rates (10^-10 - 10^-9 mistakes per generation per bp) are evolutionarily conserved mismatch-repair (MMR) systems. Environmental mutagens such as solar UV-B radiation, oxyradicals, and carcinogenic chemicals cause DNA damage that threaten genomic stability, but these are countered by cellular DNA-repair and other damage-toleration systems.

Biochemistry of Mismatch Repair in Human Nuclear Extracts. Mismatch-repair in human cells begins with recognition of mismatches and certain DNA lesions by heterodimeric MSH6•MSH2 (hMutSα) proteins. To analyze local DNA-sequence contexts affect recognition, we measure binding of hMutsα to base-mismatches and DNA lesions in different contexts. After mismatch recognition, MMR proteins must specifically identify the new DNA strand and initiate its excision.

Excision is efficiently provoked in human nuclear extracts by mismatches/lesions in plasmid substrates. Preexisting nicks in the substrates mimic in vivo excision-initiation signals (probably growing DNA ends). Special techniques allow us to insert virtually any mismatch or DNA lesion into any sequence context in MMR substrates [WANG and HAYS, 2002a] and to sensitively and quantitatively analyze MMR excision kinetics [WANG and HAYS, 2002b]. Mismatch-recognition complexes might activate excision-initiation sites by (I) sliding to them along the DNA or (II) contacting them through space by DNA bending (Figure 1). We used special techniques to insert DNA-hairpin or protein (streptavidin) blockades in mismatch-nick paths. The absence of any effect on MMR excision initiation supported model II [WANG and HAYS, 2003; WANG and HAYS, 2004]

Current work focuses on understanding how DNA-sequence-contexts affect MMR recognition specificity, determining which proteins mediate MMR excision and how they do it, and elucidating mechanisms of MMR-dependent signaling from DNA lesions to cell-cycle-checkpoint and apoptosis pathways. Insights gained will increase understanding of how MMR deficiencies increase susceptibility to cancer.

Genomic Stability and Somatic Mutation in Plants. Since plants lack reserved germ lines, they must minimize spontaneous and environmental mutagenesis in the dividing meristematic cells that eventually give rise to flowers and seeds. We proved that mismatch repair is essential for plant genomic stability, by demonstrating rapid accumulation of mutations in MSH2-defective Arabidopsis: (Figure 2): after five generations, 34 of 36 independent lines showed defects [LEONARD et al, 2003; HOFFMAN et al 2004] Plants employ an extra mismatch-recognition protein MSH7; Arabidopsis MSH7•MSH2 and MSH6•MSH2 are specialized for certain subsets of base-mismatches {Wu et al 2003]. Studies of MMR roles in long-term plant genomic stability and in antagonism of inter-species crossing continue. Plants express homologs of the specialized DNA translesion synthesis polymerases (TLS polymerases) that in other organisms facilitate more-or-less accurate DNA synthesis past hard-to-copy DNA lesions. We are biochemically characterizing Arabidopsis translesion polymerases in vitro, and studying in vivo effects of TLS inactivation.

Plant Bioindicators for Environmental Genotoxins. Because of their fixed locations, plants can detect sites where genotoxic pollutants in soil, groundwater or air pose hazards to sustained human occupancy. Its short growth cycle and genetic pliability make Arabidopsis ideal for detection of mutagenic and cytotoxic pollutants. To increase sensitivity and specificity, we are incorporating into Arabidopsis a series of transgenes that report specific mutation events, at rates as low as 10^-8 per cell division, and are selectively inactivating Arabidopsis DNA repair pathways and TLS polymerases. The aim is to construct sets of biomonitor plants with specificity for UV radiation, reactive oxygen radicals (or their elicitors), nuclear radiation, and heavy metals.

Figure 1:

Figure 2:

Mismatch repair in nuclear extracts. (A) Recognition complex forms at T:G mismatch and pre-excision complex forms at preexisting nick separated by streptadin (square) bound to biotinylated nucleotide (oval). Recognition complexes activate the pre-excision complexes by (B1) sliding to it or (B2) contacting it through space. Our work [WANG and HAYS, 2003; WANG and HAYS, 2004] favors model B2. (c) Excision proceeds towards the mismatch but cannot (here) pass the blockade.

Mutation accumulation in fifth generation of mismatch-repair-defective Arabidopsis. (A) wt plants, (B-F) selected morphological mutants, (G) wt flower, (H) mutant flower.

 

Selected References:

• HAYS, J.B., HOFFMAN, P.D., and WANG, H. (2005). Discrimination and versatility in mismatch repair. DNA Repair. 4(12):1463-74.
• HOFFMAN, P.D., WANG, H., LAWRENCE, C.W., IWAI, S., HANAOKA, F., and HAYS, J.B. (2005). Binding of MutS mismatch repair protein to DNA containing UV photoproducts, "mismatched" opposite Watson--Crick and novel nucleotides, in different DNA sequence contexts. DNA Repair. 4(9):983-93.
• PANDELOVA, I., HEWITT, S.R., and HAYS, J.B. (2005) Fluoroimaging-based immunoassay of DNA photoproducts in ultraviolet-B-irradiated tadpoles. Methods Mol Biol. 291:29-38.
  
• HOFFMAN, P.D., LEONARD, J.M., LINDBERG, G.E., BOLLMAN, S.R. and HAYS, J.B. (2004). Rapid accumulation of mutations during seed-to-seed propagation of mismatch-repair-defective Arabidopsis. Genes Dev. 18:2676-2685.
• GARCIA-ORTIZ, M.V., ARIZA, R.R., HOFFMAN, P.D., HAYS, J.B., and ROLDAN-ARJONA, T. (2004). Arabidopsis thaliana AtPOLK encodes a DinB-like DNA polymerase that extends mispaired primer termini and is highly expressed in a variety of tissues. Plant J. 39(1):84-97.
• WANG, H. and HAYS, J.B. (2004). Signaling from DNA mispairs to mismatch-repair excision sites despite intervening blockades. EMBO J 23:2126-2133.
  
• WANG, H. and HAYS, J.B. (2003). Mismatch repair in human nuclear extracts: Effects of internal DNA-hairpin between mismatches and excision-initiation nicks on mismatch correction and mismatch-provoked excision. J. Biol. Chem. 278:28686-28693.
• WU, S.-Y., CULLIGAN, K. LAMERS, M. and HAYS, J, (2003). Dissimilar mispair-recognition spectra of Arabidopsis DNA-mismatch-repair proteins MSH2*MSH6 (MutSalpha) and MSH2*MSH7 (MutSgamma) . Nucleic Acids Res. 31:6027-6034.
• LEONARD, J.M., BOLLMAN, S.R. and HAYS, J.B. (2003). Reduction of stability of Arabidopsis genomic and transgenic DNA-repent sequences (microsatellites) by inactivation of AtmsH2 mismatch-repair function. Plant Physiol. 133: 328-338.
• YOUNG, L.C., HAYS, J.B., TRON, V.A., and ANDREW, S.E. (2003). DNA mismatch repair proteins: potential guardians against genomic instability and tumorigenesis induced by ultraviolet photoproducts. J Invest Dermatol. 121(3):435-40.
• HAYS, J.B. (2002). Arabidopsis thaliana , a versatile model system for study of eukaryotic genome-maintenance functions. DNA Repair, 64:1-22.
• WANG, H. and HAYS, J.B. (2002a). Mismatch repair in human nuclear extracts: Quantitative kinetic analyses of excision of nicked circular mismatched DNA substrates, constructed by a new technique employing synthetic oligonucleotides. J. Biol. Chem., 277:26136-26142.
• WANG, H. and HAYS, J.B. (2002b). Mismatch repair in human nuclear extracts: Time courses and ATP requirements for kinetically distinguishable steps leading to tightly controlled 5´to 3´ and aphidicolin-sensitive 3´ to 5´ mispair-provoked excision. J. Biol. Chem. 277:26143-26148.

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