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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. [ABSTRACT FROM AUTHOR]

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 AtPolκ, is a homolog of Escherichia coli pol IV and human pol κ, and thus belongs to the DinB subfamily. We purified AtPolκ and found a template-directed DNA polymerase, endowed with limited processivity that is able to extend primer–terminal mispairs. The activity and processivity of AtPolκ 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 β-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. [ABSTRACT FROM AUTHOR]