%0 Journal Article %J Environ Mol Mutagen %D 2006 %T Testing excision models for responses of mismatch-repair systems to UV photoproducts in DNA. %A Wang, Huxian %A Peter D Hoffman %A Christopher W Lawrence %A John B Hays %K Animals %K Base Pair Mismatch %K CHO Cells %K Cricetinae %K Cricetulus %K DNA %K DNA Repair %K HeLa Cells %K Humans %K Plasmids %K Pyrimidine Dimers %K Ultraviolet Rays %X

Mismatch-repair (MMR) systems correct DNA replication errors and respond to a variety of DNA lesions. Previous observations that MMR antagonizes UV mutagenesis, and that the mismatch-recognition protein heterodimer MSH2*MSH6 (MutSalpha) selectively binds DNA containing "mismatched" photoproducts (T[CPD]T/AG, T[6-4]T/AG) but not "matched" photoproducts (T[CPD]T/AA, T[6-4]T/AA), suggested that mismatched photoproducts would provoke MMR excision similar to mismatched bases. Excision of incorrect nucleotides inserted opposite template photoproducts might then prevent UV-induced mutation. We tested T[CPD]T/AG DNA, in a sequence context in which it is bound substantially by hMutSalpha and in three other contexts, for stimulation of 3' MMR excision in mammalian nuclear extracts. T[CPD]T/AG was inactive in HeLa extracts, or in extracts deficient in the photoproduct-binding proteins DDB or XPC* hHR23B, arguing against interference from the nucleotide excision repair pathway. Prior incubation with hMutSalpha and MLH2.PMS2 (hMutLalpha) did not increase excision relative to homoduplex controls. T[6-4]T/AG also failed to provoke excision. T/G, C/A, and T/T substrates, even though bound by hMutSalpha no better than T[CPD]T/AG substrates, efficiently provoked excision. Even a substrate containing three T[CPD]T/AG photoproducts (in different contexts) did not significantly provoke excision. Thus, MMR may suppress UV mutagenesis by non-excisive mechanisms.

%B Environ Mol Mutagen %V 47 %P 296-306 %8 2006 May %G eng %N 4 %R 10.1002/em.20206 %0 Journal Article %J DNA Repair (Amst) %D 2005 %T Binding of MutS mismatch repair protein to DNA containing UV photoproducts, "mismatched" opposite Watson--Crick and novel nucleotides, in different DNA sequence contexts. %A Peter D Hoffman %A Wang, Huixian %A Christopher W Lawrence %A Iwai, Shigenori %A Hanaoka, Fumio %A John B Hays %K Adenosine Triphosphatases %K Amino Acid Sequence %K Bacterial Proteins %K Base Pair Mismatch %K Base Sequence %K DNA %K DNA Repair %K DNA-Binding Proteins %K Electrophoretic Mobility Shift Assay %K Molecular Sequence Data %K Mutagenesis %K MutS DNA Mismatch-Binding Protein %K Nucleotides %K Protein Binding %K Ultraviolet Rays %X

Mismatch-repair (MMR) systems suppress mutation via correction of DNA replication errors (base-mispairs) and responses to mutagenic DNA lesions. Selective binding of mismatched or damaged DNA by MutS-homolog proteins-bacterial MutS, eukaryotic MSH2.MSH6 (MutSalpha) and MSH2.MSH3-initiates mismatch-correction pathways and responses to lesions, and may cumulatively increase discrimination at downstream steps. MutS-homolog binding selectivity and the well-known but poorly understood effects of DNA-sequence contexts on recognition may thus be primary determinants of MMR specificity and efficiency. MMR processes that modulate UV mutagenesis might begin with selective binding by MutS homologs of "mismatched" T[CPD]T/AG and T[6--4]T/AG photoproducts, reported previously for hMutSalpha and described here for E. coli MutS protein. If MMR suppresses UV mutagenesis by acting directly on pre-mutagenic products of replicative bypass, mismatched photoproducts should be recognized in most DNA-sequence contexts. In three of four contexts tested here (three substantially different), T[CPD]T/AG was bound only slightly better by MutS than was T[CPD]T/AA or homoduplex DNA; only one of two contexts tested promoted selective binding of T[6--4]T/AG. Although the T:G pairs in T[CPD]T/AG and T/G both adopt wobble conformations, MutS bound T/G well in all contexts (K(1/2) 2.1--2.9 nM). Thus, MutS appears to select the two mismatches by different mechanisms. NMR analyses elsewhere suggest that in the (highly distorted) T[6--4]T/AG a forked H-bond between O2 of the 3' thymine and the ring 1-imino and exocyclic 2-amino guanine protons stabilizes a novel planar structure not possible in T[6--4]T/AA. Replacement of G by purines lacking one (inosine, 2-aminopurine) or both (nebularine) protons markedly reduced or eliminated selective MutS binding, as predicted. Previous studies and the work here, taken together, suggest that in only about half of DNA sequence contexts could MutS (and presumably MutSalpha) selectively bind mismatched UV photoproducts and directly suppress UV mutagenesis.

%B DNA Repair (Amst) %V 4 %P 983-93 %8 2005 Aug 15 %G eng %N 9 %R 10.1016/j.dnarep.2005.04.018 %0 Journal Article %J DNA Repair (Amst) %D 2005 %T Discrimination and versatility in mismatch repair. %A John B Hays %A Peter D Hoffman %A Wang, Huixian %K Base Pair Mismatch %K DNA %K DNA Repair %K HeLa Cells %K Humans %K MutS DNA Mismatch-Binding Protein %K Protein Binding %K Substrate Specificity %X

Evolutionarily-conserved mismatch-repair (MMR) systems correct all or almost all base-mismatch errors from DNA replication via excision-resynthesis pathways, and respond to many different DNA lesions. Consideration of DNA polymerase error rates and possible consequences of excess gratuitous excision of perfectly paired (homoduplex) DNA in vivo suggests that MMR needs to discriminate against homoduplex DNA by three to six orders of magnitude. However, numerous binding studies using MMR base-mispair-recognition proteins, bacterial MutS or eukaryotic MSH2.MSH6 (MutSalpha), have typically shown discrimination factors between mismatched and homoduplex DNA to be 5-30, depending on the binding conditions, the particular mismatches, and the DNA-sequence contexts. Thus, downstream post-binding steps must increase MMR discrimination without interfering with the versatility needed to recognize a large variety of base-mismatches and lesions. We use a complex but highly MMR-active model system, human nuclear extracts mixed with plasmid substrates containing specific mismatches and defined nicks 0.15 kbp away, to measure the earliest quantifiable committed step in mismatch correction, initiation of mismatch-provoked 3'-5' excision at the nicks. We compared these results to binding of purified MutSalpha to synthetic oligoduplexes containing the same mismatches in the same sequence contexts, under conditions very similar to those prevailing in the nuclear extracts. Discrimination against homoduplex DNA, only two-to five-fold in the binding studies, increased to 60- to 230-fold or more for excision initiation, depending on the particular mismatches. Remarkably, the mismatch-preference order for excision initiation was substantially altered from the order for hMutSalpha binding. This suggests that post-binding steps not only strongly discriminate against homoduplex DNA, but do so by mechanisms not tightly constrained by initial binding preferences. Pairs of homoduplexes (40, 50, and 70 bp) prepared from synthetic oligomers or cut out of plasmids showed virtually identical hMutSalpha binding affinities, suggesting that high hMutSalpha binding to homoduplex DNA is not the result of misincorporations or lesions introduced during chemical synthesis. Intrinsic affinities of MutS homologs for perfectly paired DNA may help these proteins efficiently position themselves to carry out subsequent mismatch-specific steps in MMR pathways.

%B DNA Repair (Amst) %V 4 %P 1463-74 %8 2005 Dec 08 %G eng %N 12 %R 10.1016/j.dnarep.2005.09.002 %0 Journal Article %J Methods Mol Biol %D 1999 %T Measurement of activities of cyclobutane-pyrimidine-dimer and (6-4)-photoproduct photolyases. %A John B Hays %A Peter D Hoffman %K Chromatography, Thin Layer %K Deoxyribodipyrimidine Photo-Lyase %K DNA %K DNA Damage %K DNA Primers %K DNA Repair %K Polymerase Chain Reaction %K Pyrimidine Dimers %K Substrate Specificity %K Ultraviolet Rays %B Methods Mol Biol %V 113 %P 133-46 %8 1999 %G eng %R 10.1385/1-59259-675-4:133 %0 Journal Article %J Photochem Photobiol %D 1996 %T Developmental responses of amphibians to solar and artificial UVB sources: a comparative study. %A John B Hays %A Andrew R Blaustein %A Kiesecker, J M %A Peter D Hoffman %A Pandelova, I %A Coyle, D %A Richardson, T %K Amphibians %K Animals %K Deoxyribodipyrimidine Photo-Lyase %K DNA %K DNA Repair %K Female %K Ovum %K Radiation Tolerance %K Sunlight %K Ultraviolet Rays %X

Many amphibian species, in widely scattered locations, currently show population declines and/or reductions in range, but other amphibian species show no such declines. There is no known single cause for these declines. Differential sensitivity to UVB radiation among species might be one contributing factor. We have focused on amphibian eggs, potentially the most UVB-sensitive stage, and compared their resistance to UVB components of sunlight with their levels of photolyase, typically the most important enzyme for repair of the major UV photoproducts in DNA, cyclobutane pyrimidine dimers. Photolyase varied 100-fold among eggs/oocytes of 10 species. Among three species-Hyla regilla, Rana cascadae, and Bufo boreas-for which resistance of eggs to solar UVB irradiance in their natural locations was measured, hatching success correlated strongly with photolyase. Two additional species, Rana aurora and Ambystoma gracile, now show similar correlations. Among the low-egg-photolyase species, R. cascadae and B. boreas are showing declines, and the status of A. gracile is not known. Of the two high-photolyase species, populations of H. regilla remain robust, but populations of R. aurora are showing declines. To determine whether levels of photolyase or other repair activities are affected by solar exposures during amphibian development, we have initiated an extended study of H. regilla and R. cascadae, and of Xenopus laevis, laboratory-reared specimens of which previously showed very low photolyase levels. Hyla regilla and R. cascadae tadpoles are being reared to maturity in laboratories supplemented with modest levels of UV light or light filtered to remove UVB wavelengths. Young X. laevis females are being reared indoors and outdoors. Initial observations reveal severe effects of both UVA and UVB light on H. regilla and R. cascadae tadpoles and metamorphs, including developmental abnormalities and high mortalities. Assays of photolyase levels in the skins of young animals roughly parallel previous egg/oocyte photolyase measurements for all three species.

%B Photochem Photobiol %V 64 %P 449-56 %8 1996 Sep %G eng %N 3 %0 Journal Article %J Proc Natl Acad Sci U S A %D 1994 %T UV repair and resistance to solar UV-B in amphibian eggs: a link to population declines? %A Andrew R Blaustein %A Peter D Hoffman %A Hokit, D G %A Kiesecker, J M %A Walls, S C %A John B Hays %K Animals %K Anura %K Bufonidae %K Deoxyribodipyrimidine Photo-Lyase %K DNA %K DNA Damage %K DNA Repair %K Female %K Models, Biological %K Ovum %K Population Dynamics %K Radiation Tolerance %K Ranidae %K Species Specificity %K Ultraviolet Rays %X

The populations of many amphibian species, in widely scattered habitats, appear to be in severe decline; other amphibians show no such declines. There is no known single cause for the declines, but their widespread distribution suggests involvement of global agents--increased UV-B radiation, for example. We addressed the hypothesis that differential sensitivity among species to UV radiation contributes to these population declines. We focused on species-specific differences in the abilities of eggs to repair UV radiation damage to DNA and differential hatching success of embryos exposed to solar radiation at natural oviposition sites. Quantitative comparisons of activities of a key UV-damage-specific repair enzyme, photolyase, among oocytes and eggs from 10 amphibian species were reproducibly characteristic for a given species but varied > 80-fold among the species. Levels of photolyase generally correlated with expected exposure of eggs to sunlight. Among the frog and toad species studied, the highest activity was shown by the Pacific treefrog (Hyla regilla), whose populations are not known to be in decline. The Western toad (Bufo boreas) and the Cascades frog (Rana cascadae), whose populations have declined markedly, showed significantly lower photolyase levels. In field experiments, the hatching success of embryos exposed to UV radiation was significantly greater in H. regilla than in R. cascadae and B. boreas. Moreover, in R. cascadae and B. boreas, hatching success was greater in regimes shielded from UV radiation compared with regimes that allowed UV radiation. These observations are thus consistent with the UV-sensitivity hypothesis.

%B Proc Natl Acad Sci U S A %V 91 %P 1791-5 %8 1994 Mar 01 %G eng %N 5