Divergent Roles for the Two PolI-Like Organelle DNA Polymerases of Arabidopsis    

Abstract

DNA polymerases play a central role in the process of DNA replication. Yet, the proteins in charge of the replication of plant organelle DNA have not been unambiguously identified. There are however many indications that a family of proteins homologous to bacterial DNA polymerase I (PolI) is implicated in organelle DNA replication. Here, we have isolated mutant lines of the PolIA and PolIB genes of Arabidopsis (Arabidopsis thaliana) to test this hypothesis. We find that mutation of both genes is lethal, thus confirming an essential and redundant role for these two proteins. However, the mutation of a single gene is sufficient to cause a reduction in the levels of DNA in both mitochondria and plastids. We also demonstrate that polIb, but not polIa mutant lines, are hypersensitive to ciprofloxacin, a small molecule that specifically induces DNA double-strand breaks in plant organelles, suggesting a function for PolIB in DNA repair. In agreement with this result, a cross between polIb and a plastid Whirly mutant line yielded plants with high levels of DNA rearrangements and severe growth defects, indicating impairments in plastid DNA repair pathways. Taken together, this work provides further evidences for the involvement of the plant PolI-like genes in organelle DNA replication and suggests an additional role for PolIB in DNA repair.

Effects of Reduced Chloroplast Gene Copy Number on Chloroplast Gene Expression in Maize

“…These genes are predicted co-orthologs of Arabidopsis genes POL1A and POL1B (AT1G50840/AT3G20540), and they encode proteins in the DNA polymerase A superfamily. The Arabidopsis co-orthologs are each dually targeted to mitochondria and chloroplasts and have largely overlapping functions; disruption of each single gene caused no visible phenotype and only a minor reduction in organellar DNA content, whereas the double mutant is embryo lethal (Parent et al, 2011). The strong visible phenotype of w2 mutants contrasts with those findings, and shows that w2 function is essential for chloroplast biogenesis.…”

“…S3). Therefore, the 2-fold reduction in mitochondrial DNA content conditioned by strong w2 mutant alleles suggests that both w2 and its paralog contribute to mitochondrial DNA replication, similar to Pol1A and Pol1B in Arabidopsis (Parent et al, 2011). In fact, W2 has been detected in proteome analyses of mitochondria purified from Figure 3.…”

“…We found that a relatively modest decrease in chloroplast DNA content was reflected by a substantial decrease in chloroplast gene expression, with some mRNAs more sensitive to genome copy decrease than others. Our results show further that the division of labor between the pair of nucleus-encoded organelle DNA polymerases in maize and Arabidopsis (Arabidopsis thaliana) differs: in Arabidopsis, both organellar polymerases are dually targeted to chloroplasts and mitochondria and are largely redundant in function (Parent et al, 2011), whereas maize w2 is essential for virtually all chloroplast DNA replication. Finally, the severe loss of plastid DNA revealed by Southern-blot hybridization and real-time quantitative PCR (qPCR) assays of leaf DNA in w2 mutants indicates that the bulk of the signal in normal leaf tissue arises from bona fide chloroplast genomes and not from plastid gene fragments in the nuclear genome, contrasting with conclusions in several prior reports (Kumar and Bendich, 2011;Zheng et al, 2011).…”

“…Disruption of each gene causes a small reduction (20%-50%) in mitochondrial and plastid DNA copy number, whereas disruption of both genes is embryo lethal (Parent et al, 2011), implying redundant functions in DNA replication in both organelles. Although the maize genome encodes a paralog of w2, the nearly complete loss of plastid DNA in w2-mum2 mutants indicates that w2 is the primary or possibly sole source of DNA polymerase for DNA replication in the chloroplast.…”

A family portrait: structural comparison of the Whirly proteins fromArabidopsis thalianaandSolanum tuberosum

“…In the absence of the chloroplast-directed Whirly proteins, the plastid genome of both Arabidopsis thaliana and Zea mays (maize) becomes unstable and accumulates DNA rearrangements that contain microhomologies at their endpoint junction (Maré chal et al, 2009). Also, the treatment of Arabidopsis plants with ciprofloxacin, an inhibitor of DNA gyrases that induces DSBs in both plastids and mitochondria (Rowan et al, 2010;Parent et al, 2011), results in an increase in microhomologymediated DNA rearrangements in the plastids of plants lacking WHY1 and WHY3 (Cappadocia et al, 2010). WHY2, the Whirly protein directed to mitochondria in Arabidopsis, also appears to be involved in repressing the MMBIR pathway in the mitochondria, although to a lesser extent (Cappadocia et al, 2010).…”