Plant Organellar DNA Polymerases Evolved Multifunctionality through the Acquisition of Novel Amino Acid Insertions

Abstract

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.

Keywords: DNA repair; DNA replication; chloroplast; mitochondria; plant organellar DNA polymerases.

Figure 1

Structural architecture of AtPOLIs. (A) AtPOLIs, like all POPs, are modular polymerases with exonuclease (editing) and polymerization domains. AtPOLIs harbor a dual targeting sequence (DTS) for mitochondria and plastid localization and a N terminal disordered region of approximately 200 amino acids. (B) Homology model of AtPOLIBs: The three unique amino acid insertions in the polymerization domain of AtPOLIs are represented in a ball stick representation and colored in orange, green, and cyan.

Figure 2

Plant mitochondria harbor proteins similar to the T7-like replisome. (A) Representation of the putative T7-like mitochondrial replisome of A. thaliana. The model predicts that in A. thaliana, a plant mitochondrial RNA polymerase (AtmtRNAP) may be involved in primer synthesis. AtTWINKLE is proposed to be involved in the coordination of leading and lagging DNA synthesis by specific interactions with the replicative AtPOLIs. The single-stranded segments are predicted to be covered by AtSSB1 and AtSSB2. (B) Amino acid sequence alignment of primases-helicases from plants in comparison to T7 primase-helicase. In both primase-helicases, the C-terminal part is highly acidic, suggesting that AtTWINKLE and AtPOLIs may exert physical interaction via charged residues as in the T7 replisome [28].

Figure 3

Schematic illustration of the role of insertions 1 and 3 of AtPOLIB in TLS. Homology model showing a close view of AtPOLIB in complex with a dsDNA substrate. This homology model was constructed using the molecular operating environment (MOE) software and as a template, the crystal the structure of Bacillus DNAP bound to dsDNA (PDB: 4DSE) [34,65]. This model shows that insertions 1 and 3 are in an optimal position to interact with dsDNA and highlights the relative positions of insert 1 (red-colored) and insert 3 (pink-colored) poised to clamp DNA. The localization of residues K593 and K866 located in insertions 1 and 3 respectively, are labeled.

Figure 4

Schematic representation of the organellar BER pathway in A. thaliana. BER starts with a DNA glycosylase that recognizes and hydrolyzes the N-glycosidic bond of a damaged base leaving an AP site. Monofunctional glycosylases only harbor hydrolytic activity, while bifunctional glycosylases harbor both hydrolytic and lyase activities. Bifunctional DNA glycosylases, like NTH/EndoIII, cleaves the phosphodiester bond at the 3′ position of the AP site, producing a 3′-OH and a 3′-deoxyribose phosphate (3′-dRP). An AP endonuclease (ARP) processes AP sites produced by monofunctional glycosylases generating 3′-OH and 5′-dRP ends. ARP also process 3′-dRP ends produced by bifunctional glycosylases generating 3′-OH and 5′-P ends. From this point, the BER pathway is bifurcated into two sub-pathways, dubbed short (SP) and long patch (LP). During the BER-LP, the strand-displacement activity of AtPOLIs generates a 5′-flap that is cleaved by a putative FEN1 endonuclease (OEX1 and OEX2). The lyase activity of AtPOLIA or AtPOLIB is required to process the 5′-dRP moiety and incorporate a single nucleotide during BER-SP. In both sub-pathways, a DNA ligase seals the remaining nick. Alternatively, a bifunctional glycosylase, such as AtFpg, excises a DNA lesion with concomitant processing of the AP site by β, δ-elimination, creating 3′-P and 5′-P ends. The 3′-P end blocks replication and it is putatively removed by AtZDP phosphatase to leave a 3′-OH end, which is subsequently resolved by BER-SP.

Figure 5

Plant mitochondria employ different mechanisms to repair DSBs. (A) DNA repair by HR. Repair of double-strand breaks is initiated by resection enzymes that generate a 3′-OH end. The length of the 3′OH end can be long or short. In plant mitochondria, a long 3′-OH (right panel) is protected by single-stranded binding proteins like SSBs, WHY, or OSBs. AtRECAs, with the help of accessory proteins, displace the single-stranded binding proteins and initiate the search for homologous sequences. After the strand invasion, DNA synthesis is initiated by an organellar DNA polymerase. Finally, a ligation step is necessary to seal the break. (B) DNA repair by MMEJ. In short 3′-OH (left panel) that contain micro-homologous sequences, DNA polymerase anneals two strands by trans complementation. This mechanism is known as MMEJ. These short sequences require a minimum of two base pairs to bind and extend them by the same DNA polymerase. In the organelles of A. thaliana, MMEJ is executed by AtPOLIA and AtPOLIB.