%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 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 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 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 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