The long-term objectives are to explain biochemically how human mismatch-repair (MMR) systems act in vivo to suppress induction of mutation by DNA lesions, yet promote lesion-triggered cell-cycle arrest and apoptosis. The focus here is on two representative classes of environmentally-induced mutagenic and carcinogenic lesions, UV photoproducts and polycyclicaromatic-hydrocarbon (PAH) adducts.
The aim of the specific components of this exploratory work is to provide a well-defined basis for future studies-both expanded biochemical analyses and work with cultured cells and transgenic mice.
Aim 1 is to analyze (ATP-sensitive) binding, by the (purified) human MMR-recognition proteins hMutS1 and hMutS2, of DNA containing defined cyclobutane-pyrimidine-dimer (CPD) photoproducts or PAH-purine adducts-opposite """"""""matching"""""""" or """"""""mismatching"""""""" nucleotides. The MMR targets here are, respectively, T[CPD]T/AG vs. T[CPD]T/AA in various contexts, and B[a]P-N2G:C vs. B[a]P-N2G:T and B[c]Ph-N6A:T vs. B[c]Ph-N6A:C. Targets bound twice as well as homoduplex DNA in electrophoretic-mobility-shift arrays will be used for further experiments.
Aim 2 is to determine, using complete cell-free extracts, which Aim 1 lesion targets activate incision, by the essential MMR protein hMutL1, of model circular substrates (containing preexisting defined nicks in non-lesion strands) and concomitantly provoke 3'-5'excision along the shorter paths from substrate nicks to targets. Ability to activate MutL1 distinguishes """"""""Class I"""""""" MMR targets (base- mispair prototype) from """"""""Class II"""""""" (T[CPD]T/AG prototype) targets. Putative Class II targets will be tested, by surface-plasmon-resonance techniques, for abilities to form ternary complexes with (unactivated) hMutL1 and hMutS(1/2).
Aim 3 is to test PAH-purine and the prototypic T[CPD]T/AG targets for the defining Class II property-activation of the MMR-associated hExoI protein for 5'-3'excision of model nicked-circular substrates by purified-protein mixtures and cell-free extracts.
Aim 4 is to compare forward Hprt mutation induced in MMR-deficient vs. MMR-proficient cells in culture by B[a]P and B[C]Ph diol epoxides. Efficient MMR responses to DNA lesions can both reduce cancer risk and promote killing of tumor cells by some chemotherapeutic drugs. Environmental mutagens may pose unsuspected risks to individuals harboring one or (especially) multiple polymorphic partial MMR deficiencies. Additional partial deficiencies in legion-removal system may further. Future biochemical studies will analyze the efficiency and accuracy of DNA resynthesis past template lesions that have provoked 3'-5'(Aim 2) or 5'-3'(Aim 3) excision of (non-lesion) strands in extracts and recruitment (in the absence of excision) of signaling proteins in these extracts. Future genetic studies with various transgenic mice will examine effects of MMR-protein """"""""knockouts"""""""" on specific mutation pathways induced by B[a]P and B[c]Ph diol epxidos.
This exploratory work will provide the basis for more later more extensive studies. These will help identify individuals at increased risk for cancer induced by environmental agents.