%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