No abstract

Plant mitochondria have very active DNA recombination activities that are responsible for its plastic structures and that should be involved in the repair of double-strand breaks in the mitochondrial genome. Little is still known on plant mitochondrial DNA repair, but repair by recombination is believed to be a major determinant in the rapid evolution of plant mitochondrial genomes. In flowering plants, mitochondria possess at least two eubacteria-type RecA proteins that should be core components of the mitochondrial repair mechanisms. We have performed functional analyses of the two Arabidopsis (Arabidopsis thaliana) mitochondrial RecAs (RECA2 and RECA3) to assess their potential roles in recombination-dependent repair. Heterologous expression in Escherichia coli revealed that RECA2 and RECA3 have overlapping as well as specific activities that allow them to partially complement bacterial repair pathways. RECA2 and RECA3 have similar patterns of expression, and mutants of either display the same molecular phenotypes of increased recombination between intermediate-size repeats, thus suggesting that they act in the same recombination pathways. However, RECA2 is essential past the seedling stage and should have additional important functions. Treatment of plants with several DNA-damaging drugs further showed that RECA3 is required for different recombination-dependent repair pathways that significantly contribute to plant fitness under stress. Replication repair of double-strand breaks results in the accumulation of crossovers that increase the heteroplasmic state of the mitochondrial DNA. It was shown that these are transmitted to the plant progeny, enhancing the potential for mitochondrial genome evolution.

The mitochondria of flowering plants have considerably larger and more complex genomes than the mitochondria of animals or fungi, mostly due to recombination activities that modulate their genomic structures. These activities most probably participate in the repair of mitochondrial DNA (mtDNA) lesions by recombination-dependent processes. Rare ectopic recombination across short repeats generates new genomic configurations that contribute to mtDNA heteroplasmy, which drives rapid evolution of the sequence organization of plant mtDNAs. We found that Arabidopsis thaliana RECG1, an ortholog of the bacterial RecG translocase, is an organellar protein with multiple roles in mtDNA maintenance. RECG1 targets to mitochondria and plastids and can complement a bacterial recG mutant that shows defects in repair and replication control. Characterization of Arabidopsis recG1 mutants showed that RECG1 is required for recombination-dependent repair and for suppression of ectopic recombination in mitochondria, most likely because of its role in recovery of stalled replication forks. The analysis of alternative mitotypes present in a recG1 line and of their segregation following backcross allowed us to build a model to explain how a new stable mtDNA configuration, compatible with normal plant development, can be generated by stoichiometric shift.

No abstract

No abstract

Z-DNA-forming sequences are shown to elicit a biological response in Escherichia coli. Plasmids containing sequences capable of adopting the Z conformation (GC and CA/GT) are shown to be hot spots for spontaneous deletions.All the deletions involve an even number of base pairs. The distribution of the deletion events shows that the process ends when the Z-DNA-forming sequence has been reduced to a size no longer capable of adopting the Z conformation at natural superhelical density. Lac-->Lac+ (3n+1) bp insert: the loss of (3p+1) bp or the addition of (3p+2) bp are detected (3n+2) bp insert: the loss of (3p+2) bp or the addition of (3p+ 1) bp are detected Lac + ->Lac -(3n) bp insert: the loss or the addition of (3p+ 1) bp or (3p+2) bp are detected FIG. 1. Strategy for the frameshift mutation assay. Various lengths of alternating purine-pyrimidine bases (such as GC, GT, or AT) have been cloned in the early part of the lacZ gene of plasmid pUC8. As shown, these plasmids were used in the f-galactosidase a complementation assay to monitor the frameshift mutation frequency within the inserted sequence.intensities were used to calculate the frequencies of the different mutational events.Biochemical analysis. About 104 transformants containing plasmid pUC-(GC)13 were pooled, and their plasmid DNA was analyzed on sequencing gels as described for the phenotypic analysis. The autoradiogram revealed a high intensity band of starting plasmid and several lighter bands. We have only quantified the bands corresponding to the "long deletion events." Two-Dimensional Agarose Gel Technologies. The topoisomers were run on a 2% agarose gel, 10 cm long, in TAE buffer (30). The first direction was run without intercalating agent at 3 V/cm for 17 hr. In the second direction, the gel (turned 900) Abbreviations: SADE, small addition/deletion event; LDE, long deletion event; AAF, 2-acetylaminofluorene. 7465The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

No abstract

SummaryIn Escherichia coli, bulky DNA lesions are repaired primarily by nucleotide excision repair (NER). Unrepaired lesions encountered by DNA polymerase at the replication fork create a blockage which may be relieved through RecF-dependent recombination. We have designed an assay to monitor the different mechanisms through which a DNA polymerase blocked by a single AAF lesion may be rescued by homologous double-stranded DNA sequences. Monomodified single-stranded plasmids exhibit low survival in non-SOS induced E. coli cells; we show here that the presence of a homologous sequence enhances the survival of the damaged plasmid more than 10-fold in a RecA-dependent way. Remarkably, in an NER proficient strain, 80% of the surviving colonies result from the UvrA-dependent repair of the AAF lesion in a mechanism absolutely requiring RecA and RecF activity, while the remaining 20% of the surviving colonies result from homologous recombination mechanisms. These results uncover a novel mechanism -RecA-mediated excision repair -in which RecA-dependent pairing of the mono-modified singlestranded template with a complementary sequence allows its repair by the UvrABC excinuclease.