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.

Plant organellar DNA polymerases paralogs exhibit dissimilar nucleotide incorporation fidelity

“…AtPolIB plays a predominant role in DNA repair in mitochondria and chloroplasts and both DNAPs are involved in organellar DNA replication . AtPolIs are capable to bypass abasic sites and use short RNAs synthesized by the organellar DNA primase‐helicase (AtTwinkle) as primers . Recombinantly expressed AtPolIB is a low fidelity DNAP that presents higher misincorporation values than replicative mitochondrial DNAPs .…”

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

“…The organellar primase-helicases in A. thaliana (dubbed AtTwinkle) is a 709 amino acid protein with mitochondria and chloroplast localization [28] ( Figure 1A). AtTwinkle, as predicted for all plant primase-helicases, harbors both primase and helicase activities [28][29][30]. Structural studies of primase-helicase show that these enzymes assemble as heptamers or hexamers in which the helicase modules form a compact oligomeric ring to which the primase modules attach [31,32] (Figure 1B,C).…”

“…Motif I corresponds to the zinc binding domain (ZBD) necessary for template recognition, whereas regions II to VI assemble the RNA Polymerase domain ( Figure 1D). In contrast to all previously characterized primase-helicases, AtTwinkle recognizes two cryptic nucleotides within the ssDNA template [29], a biochemical property that may reduce the length of the Okazaki fragments during plant mitochondrial replication. The helicase module of AtTwinkle shares high amino acid identity with the helicase module of the T7 primase-helicase and harbors the five conserved motifs [33], including a Walker motif necessary for nucleotide hydrolysis.…”

“…The presence of an active AtTwinkle protein in Arabidopsis suggests the presence of a plant mitochondrial replisome in which a DNA polymerase replicates DNA following the unwinding of the double helix and exposing the leading-strand for continuous synthesis [34]. The primase activity suggests that a trailing DNA polymerase synthesizes the lagging-strand using primers synthesized by the primase module of AtTwinkle [29]. This model of coordinated leading and lagging strands occurs in bacteriophages T4 and T7, but not in mitochondria from metazoans and yeast [22,23,26].…”

“…Interestingly, Arabidopsis harbors a protein that contains the zinc finger and the RNA polymerase module of AtTwinkle dubbed AtTwinky [28]. This module by itself is functional in vitro [29]. An Arabidopsis insertional line in AtTwinkle shows no apparent phenotype, maybe because the T-DNA insertion occurs in an intron or because of redundant mechanisms for primer synthesis and DNA unwinding [34].…”