%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 Genes Dev %D 2004 %T Rapid accumulation of mutations during seed-to-seed propagation of mismatch-repair-defective Arabidopsis. %A Peter D Hoffman %A Leonard, Jeffrey M %A Lindberg, Gerrick E %A Bollmann, Stephanie R %A John B Hays %K Arabidopsis %K Arabidopsis Proteins %K Base Pair Mismatch %K DNA Repair %K Genomic Instability %K Microsatellite Repeats %K Mutation %K MutS Homolog 2 Protein %K Plants, Genetically Modified %K Reproduction, Asexual %K Seeds %X

During the many cell divisions that precede formation of plant gametes, their apical-meristem and floral antecedents are continually exposed to endogenous and environmental mutagenic threats. Although some deleterious recessive mutations may be eliminated during growth of haploid gametophytes and functionally haploid early embryos ("haplosufficiency quality-checking"), the multiplicity of plant genome-maintenance systems suggests aggressive quality control during prior diploid growth. To test in Arabidopsis a hypothesis that prior mismatch repair (MMR) is paramount in defense of plant genetic fidelity, we propagated in parallel 36 MMR-defective (Atmsh2-1) and 36 wild-type lines. The Atmsh2-1 lines rapidly accumulated a wide variety of mutations: fifth-generation (G5) plants showed abnormalities in morphology and development, fertility, germination efficiency, seed/silique development, and seed set. Only two Atmsh2-1, but all 36 wild-type lines, appeared normal at G5. Analyses of insertion/deletion mutation at six repeat-sequence (microsatellite) loci showed each Atmsh2-1 line to have evolved its own "fingerprint," the results of as many as 10 microsatellite mutations in a single line. Thus, MMR during diploid growth is essential for plant genomic integrity.

%B Genes Dev %V 18 %P 2676-85 %8 2004 Nov 01 %G eng %N 21 %R 10.1101/gad.1217204