Plant organellar DNA primase-helicase synthesizes RNA primers for organellar DNA polymerases using a unique recognition sequence

Abstract

DNA primases recognize single-stranded DNA (ssDNA) sequences to synthesize RNA primers during lagging-strand replication. Arabidopsis thaliana encodes an ortholog of the DNA primase-helicase from bacteriophage T7, dubbed AtTwinkle, that localizes in chloroplasts and mitochondria. Herein, we report that AtTwinkle synthesizes RNA primers from a 5'-(G/C)GGA-3' template sequence. Within this sequence, the underlined nucleotides are cryptic, meaning that they are essential for template recognition but are not instructional during RNA synthesis. Thus, in contrast to all primases characterized to date, the sequence recognized by AtTwinkle requires two nucleotides (5'-GA-3') as a cryptic element. The divergent zinc finger binding domain (ZBD) of the primase module of AtTwinkle may be responsible for template sequence recognition. During oligoribonucleotide synthesis, AtTwinkle shows a strong preference for rCTP as its initial ribonucleotide and a moderate preference for rGMP or rCMP incorporation during elongation. RNA products synthetized by AtTwinkle are efficiently used as primers for plant organellar DNA polymerases. In sum, our data strongly suggest that AtTwinkle primes organellar DNA polymerases during lagging strand synthesis in plant mitochondria and chloroplast following a primase-mediated mechanism. This mechanism contrasts to lagging-strand DNA replication in metazoan mitochondria, in which transcripts synthesized by mitochondrial RNA polymerase prime mitochondrial DNA polymerase γ.

Figure 1.

AtTwinkle is a homolog of bacteriophage T7 primase-helicase and mitochondrial Twinkle. (A) Schematic representation of the bifunctional T7 primase-helicase in comparison to Arabidopsis and human Twinkles. T7 primase-helicase contains the six conserved motifs necessary for primase activity. Motif I is located at the ZBD and motifs II to VI in the RNAP domain. AtTwinkle conserves these motifs and contain an extra N-terminal amino acid sequence (residues 1–91) that harbors a dual organellar localization signal. Human Twinkle contains an N-terminal mitochondrial targeting sequence but lacks functional ZBD and RNAP domains. (B) Phylogenetic analysis between phage, plant and metazoan primase-helicases. The phylogenetic tree shows that plant and phage primase-helicases share a closer evolutionary relationship than plant and metazoan Twinkles. (C) Logo sequence of the amino acid region corresponding to the ZBD. The main differences between T7 Primase and AtTwinkle are the length of the ZBD that in AtTwinkle is ∼15 amino acids longer. (D) Computational homology model of the primase domain of AtPrimase-Helicase. The zinc finger is colored in blue and the RNAP domain in red. The conserved cysteines that coordinate the zinc atom are in a ball-stick representation.

Figure 2.

Recombinant AtTwinkle is an active primase. (A) Heterologous purification of recombinant primases showing the purified proteins after three chromatographic steps. The expected theoretical molecular masses for AtPrimase, AtPrimase-Helicase and T7 Primase are 36, 70, and 28 kDa, respectively. Purified protein samples were run onto a 12.5% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue. The proteins migrate near their expected theoretical molecular mass. (B) Oligoribonucleotide synthesis in a random heptameric template using [α-³²P]-ATP, [α-³²P]-CTP and [α-³²P]-GTP by AtPrimase in comparison to a synthesis reaction without added primase. The appearance of ribonucleotide products (lanes 4 and 6) in comparison to a T7 primase control (lanes 8 and 10) indicate that AtTwinkle is an active primase. (C) Identification of the 3′ nucleotide synthesized by AtPrimase-Helicase in M13 ssDNA template. The identity of each of the four labeled [α-³²P]-NTPs is indicated. In this experiment the rest of the unlabeled rNTPs were included in the reaction. After an incubation of 30 min the products were treated with alkaline phosphatase to eliminate the label corresponding to the 5′ nucleotide. (D) Identification of the 5′ nucleotide synthesized by AtPrimase-Helicase when CTP is the preferred nucleotide in the 3′ position. In this experiment individual unlabeled NTPs were incubated in the presence of [α-³²P]-CTP. The products were treated with alkaline phosphatase and run on a 27% sequencing gel.

Figure 3.

AtPrimase-Helicase is active in 4 out 64 trinucleotide sequences. Oligoribonucleotide synthesis reactions on an array of 64 possible trinucleotide combinations. The ribonucleotide complementary to the middle nucleotide dictates the identity of the radioactively labeled products. Reactions labeled with [α-³²P]-ATP, [α-³²P]-CTP, [α-³²P]-GTP and [α-³²P]-UTP, were run in panels (A), (B), (C) and (D) respectively. (E) Relative intensity of RNA products synthetized by each of the 64 trinucleotide templates by AtTwinkle. The Y-axes indicates the identity of the trinucleotide substrate and the X-ayes present the relative intensity of each product. Graphical representation of RNA synthesis indicating the percentage of each RNA product (2-mer to 8-mer) for each individual template. If the case of empty bars the intensity for the background and the reactions were equal. Oligonucleotide templates contained seven thymidines after the assayed trinucleotide sequence. Incorporation opposite those thymidines produce ribonucleotide products longer than three nucleotides. Reactions contained 5 mM of the synthetic template and 2 mM AtPrimase-Helicase.

Figure 4.

Direct comparison of the ribonucleotide synthesis activity between AtPrimase and T7 Primase. A sequencing gel showing the migration of the synthesized products by AtPrimase and T7 Primase in 5′-GGA'-3 and 5′-GTC'-3 single-stranded DNA sequences. In the case of T7 primase the lower RNA migrating product corresponds to a 5′-pppAC diribonucleotide (lanes 3 and 4) that is extended up to nine ribonucleotides according to the template thymidines. In At-Primase (lanes 1 and 2) the lower migrating product corresponds to a 5′-pppCA diribonucleotide that is extended following incorporation of AMP using the template thymidines. Reaction products were specifically labeled with [α-³²P]-ATP (lanes 1 and 3) or [α-³²P]-CTP (lanes 2 and 4). Primase reactions were carried out using 5 μM of the synthetic template and 1 μM of AtPrimase and 0.2 μM of T7 Primase.

Figure 5.

AtPrimase does not recognize bacterial and bacteriophage derived template sequences. Oligoribonucleotide synthesis by AtPrimase-Helicase on synthetic templates with canonical primase recognition sequences (S. aeurus, A. aeolicus, bacteriophage T7, E. coli) in comparison to the 5′-GGA'-3 sequence. RNA products were independently labeled with [α-³²P]-ATP, [α-³²P]-CTP, [α-³²P]-GTP or [α-³²P]-CTP. The identity of the RNA products from the 5′-pppCA'-3 diribonucleotide to the 5′-pppCAAAAAAA'-3 octaribonucleotide are indicated in the sequencing gel. Reactions contained 5 μM of the synthetic template and 2 μM of AtPrimase.

Figure 6.

Template recognition requires the cryptic 5′-GA'-3 pair. The identity of the ‘cryptic’ guanosine or adenine in the 5′-GA'-3 pair is necessary for oligoribonucleotide synthesis. Oligoribonucleotide synthesis decreases when the cryptic adenine (lanes 1–12) or guanosine (lanes 17–28) of the 5′GA'3 pair are modified with any other deoxynucleotide. Products were independently labeled with all four radioactive ribonucleotides. A control experiment with the canonical 5′-GA'-3 pair (lanes 13–16) is present and the identity of the oligoribonucleotide products are indicated.

Figure 7.

AtTwinkle is an efficient enzyme with non-selective incorporation for the 3′ rNTP base. AtTwinkle presents a preference for CTP as the 5′-NTP or initiating nucleotide. To assess the specificity for the 3′-rNTP base, the identify to a 5′-XGGA-‘-3 template sequence was systematically changed to all deoxynucleotides. The underlined GA corresponds to the cryptic element and X corresponds to the identity of the deoxynucleotide that templates for the incorporation of the 3′-rNMP. Oligoribonucleotides synthesis reactions where labeled with [α-³²P]-ATP (A) or [α-³²P]-CTP (B). The identity of the template base was either adenine (lane 1), citosine (lane 2), guanosine (lane 3) or thymidine (lane 4). The identity of the ribonucleotide products is indicated in the gel

Figure 8.

AtPrimase efficiently synthesizes RNA primers for organellar DNA polymerases. (A) Oligoribonucleotide synthesis by the AtPrimase in 5′-GGGA-3′, 5′-GGA-3′, 5′-GTA-3′, 5′-GTC-3′ synthetic templates (lanes 6–21) in comparison to the synthesis present by T7 Primase (lanes 1–5) in a 5′-GGGTC'-3 recognition sequence. AtPrimase was present from 100 to 800 nM, whereas T7 primase was present at lower protein concentrations (from 6.25 to 400 nM). Each reaction was labeled with [α-³²P]-ATP. The identity of the oligoribonucleotides is indicated in the gel. The bottom part of the figure illustrates the molar amount of oligoribonucleotide synthesis products by 100 nM of T7 and Arabidopsis primases in their preferred 5′-GGGC-3′ and 5′-GGGA-3′ ssDNA templates. To determine the molar amount of RNA, the intensity of each radioactive band was corrected with the amount of AMPs present in each ribonucleotide. (B) AtPrimase primes organellar DNA polymerases. Coupled primase-DNA polymerase reactions labeled with [α-³²P]-dATP. RNA ladder generated by T7 Primase labeled with [α-³²P]-ATP in the absence of DNA polymerases used as molecular weight markers. (lane 1). AtPolIA and AtPolIB, generate DNA:RNA products that correspond to the expected migration of a 19-mer in the presence of AtPrimase and unlabeled NTPs (lanes 2–4 and 5–7). In contrast the Klenow fragment of E. coli DNA polymerase I (lanes 8–10) and T7 DNA polymerase (lanes 11–13) are unable to perform coupled DNA synthesis. Coupled primase-DNA polymerase reactions were labeled with [α-³²P]-dATP. AtPrim was present at 150 nM, whereas DNA polymerases were used to 75 and 150 nM. The template sequence used in the coupled primase-polymerase assay was 3′(T)₁₀AGGGGGT(₉)A(T₅). Within this sequence, the AtPrimase recognition sequence is underlined

Figure 9.

Structural suggestion for the altered template recognition by AtTwinkle. (A) Multiple sequence alignment between the ZBD of T7 primase-helicase, bacterial primases, and plant Twinkles. The fours cysteines that coordinate the Zn²⁺ atom in T7 primase can be divided in two CXXC elements. In land plant primases, the first CXXC elements is conserved, whereas the second one is substituted by a CXRXKC element. Protein are designated with abbreviations as follows: Mpo, Marchantia polymorpha; Nta, Nicotiana tabacum; Zma, Zea mays; Atri, Amborella trichopoda; Cbo, Clostridium botulinum; Cdi, Clostridium difficile; Gst, geobacillus stearothermophilus. (B) Computational model of the zinc finger of AtTwinkle in comparison to the crystal structure of T7-Primase. The four β-strands that correspond to the zinc finger are colored in blue. The cysteine residues CXXC element are colored in magenta and red. The side chains of the amino acids in the second CXXC element are in a ball-stick representation. The Zn²⁺ atom is colored in black. His33 implicated in template recognition in T7 primase is in a ball-stick representation. The computational model of AtTwinkle shows the proposed structural localization of residues R166 and K168.