Plant organellar DNA polymerases paralogs exhibit dissimilar nucleotide incorporation fidelity

Abstract

The coding sequences of plant mitochondrial and chloroplast genomes present a lower mutation rate than the coding sequences of animal mitochondria. However, plant mitochondrial genomes frequently rearrange and present high mutation rates in their noncoding sequences. DNA replication in plant organelles is carried out by two DNA polymerases (DNAP) paralogs. In Arabidopsis thaliana at least one DNAP paralog (AtPolIA or AtPolIB) is necessary for plant viability, suggesting that both genes are partially redundant. To understand how AtPolIs replicate genomes that present low and high mutation rates, we measured their nucleotide incorporation for all 16-base pair combinations in vitro. AtPolIA presents an error rate of 7.26 × 10 , whereas AtPolIB has an error rate of 5.45 × 10 . Thus, AtPolIA and AtPolIB are 3.5 and 26-times less accurate than human mitochondrial DNAP γ. The 8-fold difference in fidelity between both AtPolIs results from a higher catalytic efficiency in AtPolIA. Both AtPolIs extend from mismatches and the fidelity of AtPolIs ranks between high fidelity and lesion bypass DNAPs. The different nucleotide incorporation fidelity between AtPolIs predicts a prevalent role of AtPolIA in DNA replication and AtPolIB in DNA repair. We hypothesize that in plant organelles, DNA mismatches generated during DNA replication are repaired via recombination-mediated or DNA mismatch repair mechanisms that selectively target the coding region and that the mismatches generated by AtPolIs may result in the frequent expansion and rearrangements present in plant mitochondrial genomes.

MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes

“…mitochondrial and nuclear DNA, as evidenced by increased oxidative damage in the double mutant damaged bases in plant cytoplasmic genomes (9,(14)(15)(16)(17). In both mitochondria and plastids, there is often above 10 -3 errors per bp and much worse in certain sequence contexts (35) -setting a problematic noise threshold for accurate detection of rare variants.…”

“…Therefore, this 298 aspect of the mutation spectrum is more likely explained by polymerase misincorporations during 299 DNA replication, although direct measurements of the spectrum of misincorporations by PolIA and 300 PolIB would be needed to help test this hypothesis. We reasoned that disrupting the POLIA gene 301 would increase mutation rates because of higher misincorporation rates for PolIB (9). The failure to 302 find such an effect suggests a general insensitivity to polymerase errors when MSH1 is intact,…”

“…presumably because of its ability to recognize and repair these errors. This proposed role of MSH1 304 would similarly explain why sequence evolution is so slow in these genomes despite polymerases 305 with unusually high misincorporation rates (9). Disrupting genes involved in the repair of uracil damage.…”

“…However, the presence of a conventional 259 MMR pathway would not explain how plant mitochondria and plastids maintain mutation rates 260 substantially lower than in most eukaryotic genomes. An alternative, non-conventional pathway 261 could involve use of the GIY-YIG endonuclease domain to introduce a DSB near sites identified by 262 the mismatch recognition domain, followed by HDR of the DSB (9,14,16). This previously proposed 263 model could lead to unusually high repair accuracy because it does not require use of a heuristic to 264 determine which strand carries the error at mismatched sites, instead employing homologous 265 recombination with an unaffected genome copy to "break the tie".…”

Structure–Function Analysis Reveals the Singularity of Plant Mitochondrial DNA Replication Components: A Mosaic and Redundant System

“…Flowering plants harbor two paralogous POP genes with chloroplast and mitochondrial localization. In Arabidopsis, one POP is a high-fidelity DNAP (AtPolIA), whereas the other, AtPolIB, is a low-fidelity enzyme [132]. From a structural point of view, the most distinctive elements in POPs are the presence of three unique insertions in their polymerization domain, two of those insertions are located in the thumb subdomain (Ins1 and Ins2), whereas the third insertion is placed in the fingers subdomain [122][123][124][125].…”