RESUMO

PrimPol is a novel Primase-Polymerase that synthesizes RNA and DNA primers de novo and extents from these primers as a DNA polymerase. Animal PrimPol is involved in nuclear and mitochondrial DNA replication by virtue of its translesion DNA synthesis (TLS) and repriming activities. Here we report that the plant model Arabidopsis thaliana encodes a functional PrimPol (AtPrimPol). AtPrimPol is a low fidelity and a TLS polymerase capable to bypass DNA lesions, like thymine glycol and abasic sites, by incorporating directly across these lesions or by skipping them. AtPrimPol is also an efficient primase that preferentially recognizes the single-stranded 3'-GTCG-5' DNA sequence, where the 3'-G is cryptic. AtPrimPol is the first DNA polymerase that localizes in three cellular compartments: nucleus, mitochondria, and chloroplast. In vitro, AtPrimPol synthesizes primers that are extended by the plant organellar DNA polymerases and this reaction is regulated by organellar single-stranded binding proteins. Given the constant exposure of plants to endogenous and exogenous DNA-damaging agents and the enzymatic capabilities of lesion bypass and re-priming of AtPrimPol, we postulate a predominant role of this enzyme in avoiding replication fork collapse in all three plant genomes, both as a primase and as a TLS polymerase.

Assuntos

RESUMO

The majority of DNA polymerases (DNAPs) are specialized enzymes with specific roles in DNA replication, translesion DNA synthesis (TLS), or DNA repair. The enzymatic characteristics to perform accurate DNA replication are in apparent contradiction with TLS or DNA repair abilities. For instance, replicative DNAPs incorporate nucleotides with high fidelity and processivity, whereas TLS DNAPs are low-fidelity polymerases with distributive nucleotide incorporation. Plant organelles (mitochondria and chloroplast) are replicated by family-A DNA polymerases that are both replicative and TLS DNAPs. Furthermore, plant organellar DNA polymerases from the plant model Arabidopsis thaliana (AtPOLIs) execute repair of double-stranded breaks by microhomology-mediated end-joining and perform Base Excision Repair (BER) using lyase and strand-displacement activities. AtPOLIs harbor three unique insertions in their polymerization domain that are associated with TLS, microhomology-mediated end-joining (MMEJ), strand-displacement, and lyase activities. We postulate that AtPOLIs are able to execute those different functions through the acquisition of these novel amino acid insertions, making them multifunctional enzymes able to participate in DNA replication and DNA repair.

Assuntos

RESUMO

Plant organelles cope with endogenous DNA damaging agents, byproducts of respiration and photosynthesis, and exogenous agents like ultraviolet light. Plant organellar DNA polymerases (DNAPs) are not phylogenetically related to yeast and metazoan DNAPs and they harbor three insertions not present in any other DNAPs. Plant organellar DNAPs from Arabidopsis thaliana (AtPolIA and AtPolIB) are translesion synthesis (TLS) DNAPs able to bypass abasic sites, a lesion that poses a strong block to replicative polymerases. Besides abasic sites, reactive oxidative species and ionizing radiation react with thymine resulting in thymine glycol (Tg), a DNA adduct that is also a strong block to replication. Here, we report that AtPolIA and AtPolIB bypass Tg by inserting an adenine opposite the lesion and efficiently extend from a Tg-A base pair. The TLS ability of AtPolIB is mapped to two conserved lysine residues: K593 and K866. Residue K593 is situated in insertion 1 and K866 is in insertion 3. With basis on the location of both insertions on a structural model of AtPolIIB, we hypothesize that the two positively charged residues interact to form a clamp around the primer-template. In contrast with nuclear and bacterial replication, where lesion bypass involves an interplay between TLS and replicative DNA polymerases, we postulate that plant organellar DNAPs evolved to exert replicative and TLS activities.

Assuntos

RESUMO

In plants, the ancestral cyanobacterial triosephosphate isomerase (TPI) was replaced by a duplicated version of the cytosolic TPI. This isoform acquired a transit peptide for chloroplast localization and functions in the Calvin-Benson cycle. To gain insight into the reasons for this gene replacement in plants, we characterized the TPI from the photosynthetic bacteria Synechocystis (SyTPI). SyTPI presents typical TPI enzyme kinetics profiles and assembles as a homodimer composed of two subunits that arrange in a (ß-α)8 fold. We found that oxidizing agents diamide (DA) and H2O2, as well as thiol-conjugating agents such as oxidized glutathione (GSSG) and methyl methanethiosulfonate (MMTS), do not inhibit the catalytic activity of SyTPI at concentrations required to inactivate plastidic and cytosolic TPIs from the plant model Arabidopsis thaliana (AtpdTPI and AtcTPI, respectively). The crystal structure of SyTPI revealed that each monomer contains three cysteines, C47, C127, and C176; however only the thiol group of C176 is solvent exposed. While AtcTPI and AtpdTPI are redox-regulated by chemical modifications of their accessible and reactive cysteines, we found that C176 of SyTPI is not sensitive to redox modification in vitro. Our data let us postulate that SyTPI was replaced by a eukaryotic TPI, because the latter contains redox-sensitive cysteines that may be subject to post-translational modifications required for modulating TPI's enzymatic activity.

RESUMO

Plant mitochondrial and chloroplast genomes encode essential proteins for oxidative phosphorylation and photosynthesis. For proper cellular function, plant organelles must ensure genome integrity. Although plant organelles repair damaged DNA using the multi-enzyme Base Excision Repair (BER) pathway, the details of this pathway in plant organelles are largely unknown. The initial enzymatic steps in BER produce a 5'-deoxyribose phosphate (5'-dRP) moiety that must be removed to allow DNA ligation and in plant organelles, the enzymes responsible for the removal of a 5'-dRP group are unknown. In metazoans, DNA polymerases (DNAPs) remove the 5'-dRP moiety using their intrinsic lyase and/or strand-displacement activities during short or long-patch BER sub-pathways, respectively. The plant model Arabidopsis thaliana encodes two family-A DNAPs paralogs, AtPolIA and AtPolIB, which are the sole DNAPs in plant organelles identified to date. Herein we demonstrate that both AtPolIs present 5'-dRP lyase activities. AtPolIB performs efficient strand-displacement on a BER-associated 1-nt gap DNA substrate, whereas AtPolIA exhibits only moderate strand-displacement activity. Both lyase and strand-displacement activities are dependent on an amino acid insertion that is exclusively present in plant organellar DNAPs. Within this insertion, we identified that residue AtPollB-Lys593 acts as nucleophile for lyase activity. Our results demonstrate that AtPolIs are functionally equipped to play a role in short-patch BER and suggest a major role of AtPolIB in a predicted long-patch BER sub-pathway. We propose that the acquisition of insertion 1 in the polymerization domain of AtPolIs was a key component in their evolution as BER associated and replicative DNAPs.

Assuntos

RESUMO

In plants triosephosphate isomerase (TPI) interconverts glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) during glycolysis, gluconeogenesis, and the Calvin-Benson cycle. The nuclear genome of land plants encodes two tpi genes, one gene product is located in the cytoplasm and the other is imported into the chloroplast. Herein we report the crystal structures of the TPIs from the vascular plant Arabidopsis thaliana (AtTPIs) and address their enzymatic modulation by redox agents. Cytoplasmic TPI (cTPI) and chloroplast TPI (pdTPI) share more than 60% amino acid identity and assemble as (ß-α)8 dimers with high structural homology. cTPI and pdTPI harbor two and one accessible thiol groups per monomer respectively. cTPI and pdTPI present a cysteine at an equivalent structural position (C13 and C15 respectively) and cTPI also contains a specific solvent accessible cysteine at residue 218 (cTPI-C218). Site directed mutagenesis of residues pdTPI-C15, cTPI-C13, and cTPI-C218 to serine substantially decreases enzymatic activity, indicating that the structural integrity of these cysteines is necessary for catalysis. AtTPIs exhibit differential responses to oxidative agents, cTPI is susceptible to oxidative agents such as diamide and H2O2, whereas pdTPI is resistant to inhibition. Incubation of AtTPIs with the sulfhydryl conjugating reagents methylmethane thiosulfonate (MMTS) and glutathione inhibits enzymatic activity. However, the concentration necessary to inhibit pdTPI is at least two orders of magnitude higher than the concentration needed to inhibit cTPI. Western-blot analysis indicates that residues cTPI-C13, cTPI-C218, and pdTPI-C15 conjugate with glutathione. In summary, our data indicate that AtTPIs could be redox regulated by the derivatization of specific AtTPI cysteines (cTPI-C13 and pdTPI-C15 and cTPI-C218). Since AtTPIs have evolved by gene duplication, the higher resistance of pdTPI to redox agents may be an adaptive consequence to the redox environment in the chloroplast.