%0 Journal Article %J Plant Physiol %D 2011 %T Reversion-reporter transgenes to analyze all six base-substitution pathways in Arabidopsis. %A Bollmann, Stephanie R %A Tominey, Colin M %A Peter D Hoffman %A Hoffman, Taylor M C %A John B Hays %K Alleles %K Amino Acid Substitution %K Arabidopsis %K Base Sequence %K Gene Dosage %K Genes, Reporter %K Genetic Engineering %K Glucuronidase %K Immunohistochemistry %K Ions %K Metals %K Molecular Sequence Data %K Mutagenesis %K Mutant Proteins %K Mutation %K Plants, Genetically Modified %K Sequence Analysis, DNA %K Transgenes %K Ultraviolet Rays %X

To expand the repertoire of Arabidopsis (Arabidopsis thaliana) mutation-reporter transgenes, we constructed six mutant alleles in the same codon of the β-glucuronidase-encoding GUS transgene. Each allele reverts to GUS+ only via a particular one of the six transition/transversion pathways. AcV5 epitope tags, fused carboxyl terminal to the inactive GUS- proteins, enabled semiquantitative immunoassays in plant protein extracts. Spontaneous G:C→T:A transversions, previously not measured using reporter transgenes, were quite frequent. This may reflect mispairing of adenine with 8-oxoguanine in DNA attacked by endogenous oxyradicals. Spontaneous G:C→A:T was modest and other reversions were relatively low, as reported previously. Frequencies of ultraviolet C-induced TT→TC and TC→TT reversions were both high. With increased transgene copy number, spontaneous G:C→T:A reversions increased but ultraviolet C-induced reversions decreased. Frequencies of some reversion events were reduced among T4 versus T3 generation plants. Based on these and other analyses of sources of experimental variation, we propose guidelines for the employment of these lines to study genotoxic stress in planta.

%B Plant Physiol %V 155 %P 1286-300 %8 2011 Mar %G eng %N 3 %R 10.1104/pp.110.167726 %0 Journal Article %J Planta %D 2009 %T Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines. %A Curtis, Marc J %A Belcram, Katia %A Bollmann, Stephanie R %A Tominey, Colin M %A Peter D Hoffman %A Mercier, Raphael %A John B Hays %K Arabidopsis %K Arabidopsis Proteins %K Chromosomes, Plant %K DNA, Bacterial %K DNA-Directed DNA Polymerase %K Flowers %K Gene Frequency %K Genotype %K Heterozygote %K In Situ Hybridization, Fluorescence %K Models, Genetic %K Mutagenesis, Insertional %K Mutation %K Pollen %K Translocation, Genetic %X

Chromosomal rearrangements may complicate construction of Arabidopsis with multiple TDNA-insertion mutations. Here, crossing two lines homozygous for insertions in AtREV3 and AtPOLH (chromosomes I and V, respectively) and selfing F1 plants yielded non-Mendelian F2 genotype distributions: frequencies of +/++/+ and 1/1 2/2 progeny were only 0.42 and 0.25%. However, the normal development and fertility of double mutants showed AtPOLH-1 and AtREV3-2 gametes and 1/1 2/2 embryos to be fully viable. F2 distributions could be quantitatively predicted by assuming that F1 selfing produced inviable (1,2) and (+,+) gametophytes 86% of the time. Some defect intrinsic to the F1 selfing process itself thus appeared responsible. In selfing AtREV3 (+/2 ) single mutants, imaging of ovules and pollen showed arrest or abortion, respectively, of half of gametophytes; however, gametogenesis was normal in AtREV3 ( 2/2 ) homozygotes. These findings, taken together, suggested that T-DNA insertion at AtREV3 on chromosome I had caused a reciprocal I-V translocation. Spreads of meiosis I chromosomes in selfing AtREV3 (+/2 ) heterozygotes revealed the predicted cruciform four-chromosome structures, which fluorescence in situ hybridization showed to invariably include both translocated and normal chromosomes I and V. Sequencing of the two junctions of T-DNA with AtREV3 DNA and the two with gene At5g59920 suggested translocation via homologous recombination between independent inverted-repeat T-DNA insertions. Thus, when crosses between TDNA-insertion mutants yield anomalous progeny distributions, TDNA-linked translocations should be considered.

%B Planta %V 229 %P 731-45 %8 2009 Mar %G eng %N 4 %R 10.1007/s00425-008-0868-0 %0 Journal Article %J Biochemistry %D 2008 %T Biochemical evolution of DNA polymerase eta: properties of plant, human, and yeast proteins. %A Peter D Hoffman %A Curtis, Marc J %A Iwai, Shigenori %A John B Hays %K Amino Acid Sequence %K Arabidopsis %K Base Sequence %K Biochemical Phenomena %K Biochemistry %K Conserved Sequence %K DNA-Directed DNA Polymerase %K Evolution, Molecular %K Humans %K Kinetics %K Molecular Sequence Data %K Nucleotides %K Photochemistry %K Saccharomyces cerevisiae %K Sequence Alignment %X

To assess how evolution might have biochemically shaped DNA polymerase eta (Poleta) in plants, we expressed in Escherichia coli proteins from Arabidopsis thaliana (At), humans (Hs), and the yeast Saccharomyces cerevisiae (Sc), purified them to near homogeneity, and compared their properties. Consistent with the multiple divergent amino acids within mostly conserved polymerase domains, the polymerases showed modest, appreciable, and marked differences, respectively, in salt and temperature optima for activity and thermostability. We compared abilities to extend synthetic primers past template cyclobutane thymine dimers (T[CPD]T) or undamaged T-T under physiological conditions (80-110 mM salt). Specific activities for "standing-start" extension of synthetic primers ending opposite the second template nucleotide 3' to T-T were roughly similar. During subsequent "running-start" insertions past T-T and the next 5' ( N + 1) nucleotide, AtPoleta and HsPoleta appeared more processive, but DNA sequence contexts strongly affected termination probabilities. Lesion-bypass studies employed four different templates containing T[CPD]Ts, and two containing pyrimidine (6-4')-pyrimidinone photoproducts ([6-4]s). AtPoleta made the three successive insertions [opposite the T[CPD]T and (N + 1) nucleotides] that define bypass nearly as well as HsPoleta and somewhat better than ScPoleta. Again, sequence context effects were profound. Interestingly, the level of insertion opposite the ( N - 1) nucleotide 3' to T[CPD]T by HsPoleta and especially AtPoleta, but not ScPoleta, was reduced (up to 4-fold) relative to the level of insertion opposite the ( N - 1) nucleotide 3' to T-T. Evolutionary conservation of efficient T[CPD]T bypass by HsPoleta and AtPoleta may reflect a high degree of exposure of human skin and plants to solar UV-B radiation. The depressed ( N - 1) insertion upstream of T[CPD]T (but not T-T) may reduce the extent of gratuitous error-prone insertion.

%B Biochemistry %V 47 %P 4583-96 %8 2008 Apr 22 %G eng %N 16 %R 10.1021/bi701781p %0 Journal Article %J Plant J %D 2004 %T Arabidopsis thaliana AtPOLK encodes a DinB-like DNA polymerase that extends mispaired primer termini and is highly expressed in a variety of tissues. %A García-Ortiz, Maria Victoria %A Ariza, Rafael R %A Peter D Hoffman %A John B Hays %A Roldán-Arjona, Teresa %K Alternative Splicing %K Amino Acid Sequence %K Arabidopsis %K Arabidopsis Proteins %K DNA-Directed DNA Polymerase %K Gene Expression %K Gene Library %K Molecular Sequence Data %K Phylogeny %K Plants, Genetically Modified %K Recombinant Fusion Proteins %K Sequence Homology, Amino Acid %X

Cell survival after DNA damage depends on specialized DNA polymerases able to perform DNA synthesis on imperfect templates. Most of these enzymes belong to the recently discovered Y-family of DNA polymerases, none of which has been previously described in plants. We report here the isolation, functional characterization and expression analysis of a plant representative of the Y-family. This polymerase, which we have termed AtPolkappa, is a homolog of Escherichia coli pol IV and human pol kappa, and thus belongs to the DinB subfamily. We purified AtPolkappa and found a template-directed DNA polymerase, endowed with limited processivity that is able to extend primer-terminal mispairs. The activity and processivity of AtPolkappa are enhanced markedly upon deletion of 193 amino acids (aa) from its carboxy (C)-terminal domain. Loss of this region also affects the nucleotide selectivity of the enzyme, leading to the incorporation of both dCTP and dTTP opposite A in the template. We detected three cDNA forms, which result from the alternative splicing of AtPOLK mRNA and have distinct patterns of expression in different plant organs. Histochemical localization of beta-glucuronidase (GUS) activity in transgenic plants revealed that the AtPOLK promoter is active in endoreduplicating cells, suggesting a possible role during consecutive DNA replication cycles in the absence of mitosis.

%B Plant J %V 39 %P 84-97 %8 2004 Jul %G eng %N 1 %R 10.1111/j.1365-313X.2004.02112.x %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 %0 Journal Article %J Proc Natl Acad Sci U S A %D 1997 %T An Arabidopsis photolyase mutant is hypersensitive to ultraviolet-B radiation. %A Landry, L G %A Stapleton, A E %A Lim, J %A Peter D Hoffman %A John B Hays %A Walbot, V %A Last, R L %K Apoenzymes %K Arabidopsis %K Deoxyribodipyrimidine Photo-Lyase %K DNA Repair %K Dose-Response Relationship, Radiation %K Fungal Proteins %K Membrane Glycoproteins %K Mutagenesis %K Mutation %K Pyrimidine Dimers %K Radiation Tolerance %K Ultraviolet Rays %X

Photolyases are DNA repair enzymes that use energy from blue light to repair pyrimidine dimers. We report the isolation of an Arabidopsis thaliana mutant (uvr2-1) that is defective in photorepair of cyclobutylpyrimidine dimers (CPDs). Whereas uvr2-1 is indistinguishable from wild type in the absence of UV light, low UV-B levels inhibit growth and cause leaf necrosis. uvr2-1 is more sensitive to UV-B than wild type when placed under white light after UV-B treatment. In contrast, recovery in darkness or in light lacking photoreactivating blue light results in equal injury in uvr2-1 and wild type. The uvr2-1 mutant is unable to remove CPDs in vivo, and plant extracts lack detectable photolyase activity. This recessive mutation segregates as a single gene located near the top of chromosome 1, and is a structural gene mutation in the type II CPD photolyase PHR1. This mutant provides evidence that CPD photolyase is required for plant survival in the presence of UV-B light.

%B Proc Natl Acad Sci U S A %V 94 %P 328-32 %8 1997 Jan 07 %G eng %N 1 %0 Journal Article %J Mol Gen Genet %D 1996 %T PHH1, a novel gene from Arabidopsis thaliana that encodes a protein similar to plant blue-light photoreceptors and microbial photolyases. %A Peter D Hoffman %A Batschauer, A %A John B Hays %K Amino Acid Sequence %K Apoenzymes %K Arabidopsis %K Arabidopsis Proteins %K Cryptochromes %K Deoxyribodipyrimidine Photo-Lyase %K DNA Repair %K DNA, Complementary %K Drosophila Proteins %K Escherichia coli %K Eye Proteins %K Flavoproteins %K Fungal Proteins %K Genes, Plant %K Genomic Library %K Introns %K Membrane Glycoproteins %K Molecular Sequence Data %K Photoreceptor Cells, Invertebrate %K Plant Proteins %K Plasmids %K Receptors, G-Protein-Coupled %K Saccharomyces cerevisiae %K Transformation, Genetic %K Ultraviolet Rays %X

A cDNA from Arabidopsis thaliana similar to microbial photolyase genes, and designated AT-PHH1, was isolated using a photolyase-like cDNA from Sinapsis alba (SA-PHR1) as a probe. Multiple isolations yielded only PHH1 cDNAs, and a few blue-light-receptor CRY1 (HY4) cDNAs (also similar to microbial photolyase genes), suggesting the absence of any other highly similar Arabidopsis genes. The AT-PHH1 and SA-PHR1 cDNA sequences predict 89% identity at the protein level, except for an AT-PHH1 C-terminal extension (111 amino acids), also not seen in microbial photolyases. AT-PHH1 and CRY1 show less similarity (54% p4erein identity), including respective C-terminal extensions that are themselves mostly dissimilar. Analysis of fifteen AT-PHH1 genomic isolates reveals a single gene, with three introns in the coding sequence and one in the 5'-untranslated leader. Full-length AT-PHH1, and both AT-PHH1 and AT-PHH1 delta C-513 (truncated to be approximately the size of microbial photolyase genes) cDNAs, were overexpressed, respectively, in yeast and Escherichia coli mutants hypersensitive to ultraviolet light. The absence of significant effects on resistance suggests either that any putative AT-PHH1 DNA repair activity requires cofactors/chromophores not present in yeast or E. coli, or that AT-PHH1 encodes a blue-light/ultraviolet-A receptor rather than a DNA repair protein.

%B Mol Gen Genet %V 253 %P 259-65 %8 1996 Nov 27 %G eng %N 1-2