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Plant Organellar DNA Primase-Helicase Synthesizes RNA 10764–10774 Nucleic Acids Research, 2017, Vol. 45, No. 18 Published online 30 August 2017 doi: 10.1093/nar/gkx745 Plant organellar DNA primase-helicase synthesizes RNA primers for organellar DNA polymerases using a unique recognition sequence Antol´ın Peralta-Castro, Noe Baruch-Torres and Luis G. Brieba* Laboratorio Nacional de Genomica´ para la Biodiversidad, Centro de Investigacion´ y de Estudios Avanzados del IPN, Apartado Postal 629, Irapuato, Guanajuato, CP 36821, Mexico´ Downloaded from https://academic.oup.com/nar/article/45/18/10764/4097637 by guest on 29 September 2021 Received June 01, 2017; Revised August 09, 2017; Editorial Decision August 10, 2017; Accepted August 24, 2017 ABSTRACT human and yeast mitochondria share an evolutionary ori- gin with the replication apparatus of T-odd bacteriophages DNA primases recognize single-stranded DNA (ss- (1–3). In the T7 replisome, three bacteriophage-encoded DNA) sequences to synthesize RNA primers dur- proteins and one host protein coordinate DNA synthe- ing lagging-strand replication. Arabidopsis thaliana sis: T7 DNA polymerase (gp5), T7 primase-helicase (T7 encodes an ortholog of the DNA primase-helicase primase-helicase or gp4), single-stranded DNA binding from bacteriophage T7, dubbed AtTwinkle, that lo- protein (T7 SSB or gp2.5) (4,5)andEscherichia coli´s thiore- calizes in chloroplasts and mitochondria. Herein, we doxin. T7 primase-helicase is a modular protein that in- report that AtTwinkle synthesizes RNA primers from cludes a primase module that synthesizes primers for the a5-(G/C)GGA-3 template sequence. Within this lagging-strand T7 DNA polymerase and a helicase mod- sequence, the underlined nucleotides are cryptic, ule that processively unwinds dsDNA (4,6,7). T7 primase meaning that they are essential for template recog- belongs to the family of prokaryotic primases that is com- posed of a RNA polymerization domain (RPD) that cat- nition but are not instructional during RNA synthe- alyzes ribonucleotide addition and a zinc-binding domain sis. Thus, in contrast to all primases characterized to (ZBD) involved in ssDNA template recognition (8,9). Or- date, the sequence recognized by AtTwinkle requires ganellar proteins derived from T7-odd bacteriophages have two nucleotides (5 -GA-3 ) as a cryptic element. The undergone evolutionary changes with respect to the ances- divergent zinc finger binding domain (ZBD) of the tral bacteriophage proteins (10–12). An example of these primase module of AtTwinkle may be responsible for changes occurs in the metazoan replicative mitochondrial template sequence recognition. During oligoribonu- DNA helicase TWINKLE (T7 gp4-like protein with intra- cleotide synthesis, AtTwinkle shows a strong prefer- mitochondrial nucleoid localization). Metazoan TWIN- ence for rCTP as its initial ribonucleotide and a mod- KLE lacks the cysteines necessary for zinc coordination erate preference for rGMP or rCMP incorporation dur- in the region homologous to the ZBD of T7 primase and ing elongation. RNA products synthetized by AtTwin- the conserved residues in the RPD-like region required for RNA synthesis (13,14). In metazoans, transcripts generated kle are efficiently used as primers for plant organellar by mitochondrial RNA polymerase are used as primers dur- DNA polymerases. In sum, our data strongly suggest ing leader and lagging strand-synthesis (14). Furthermore, that AtTwinkle primes organellar DNA polymerases fungi are devoid of a TWINKLE homologue, and short during lagging strand synthesis in plant mitochon- RNA transcripts synthesized by mitochondrial RNA poly- dria and chloroplast following a primase-mediated merases may be used as primers (10,15–18). mechanism. This mechanism contrasts to lagging- Prokaryotic primases recognize a variety of template se- strand DNA replication in metazoan mitochondria, in quences. For example, bacterial primases from Staphylococ- which transcripts synthesized by mitochondrial RNA cus aeurus, Aquifex aeolicus, E. coli, and primases-helicases polymerase prime mitochondrial DNA polymerase ␥. from bacteriophages T4, T7 and SP6 recognize 5 -CTA-3 , 5-CCC-3,5-CTG-3,5-GTT-3,5-GTC-3 and 5-GCA- 3, respectively, as ssDNA template sequences (19–25). In a INTRODUCTION primase, the identity of the middle base of a minimal trinu- cleotide recognition sequence determines the identity of the Organelles are products of early bacterial endosymbiotic events. Phylogenetic analyses, however, reveal that the gene initial or 5 NTP, and the 5 base determines 3 NMP incor- products responsible for transcription and replication in poration specificity (9,26). Despite the diversity in template *To whom correspondence should be addressed. Tel: +52 462 166 3007; Fax: +52 462 624 5846; Email: [email protected] C The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2017, Vol. 45, No. 18 10765 recognition sequences, in all primases studied to date the ing fractions were dialyzed in 20 mM Tris–HCl pH 7.5, 25 3-nucleotide of the initiation triplet sequence is not tran- mM NaCl and 10% glycerol. Proteins then were loaded in a scribed. It is required, however, for template recognition, HiTrap HP heparin column and washed with a linear gra- and for this reason it is called cryptic. dient from 0 to 1000 mM of NaCl. Fractions containing Seminal work predicted that TWINKLE harbors both pure protein were pooled and concentrated. Protein sam- primase and helicase activities in most eukaryotes, except in ples were further purified by gel filtration, using a Superdex metazoans where this enzyme is predicted only have helicase 75 10/300 for AtPrimase and a Superdex 200 10/300 for the acitivity (2,27). The nuclear-encoded TWINKLE homolog AtPrimase-Helicase. Both gel filtration columns were equi- from the flowering plant Arabidopsis thaliana (dubbed At- librated in 20 mM Tris–HCl pH 7.5, 25 mM NaCl and 10% Twinkle) is a 709-residue protein that is translocated into glycerol. T7 primase was expressed and purified as previ- mitochondria and chloroplasts owing to its dual N-terminal ously described (33). targeting sequence (28–32). The module with a predicted To determine the molar amount of oligoribonucleotide primase activity contains the six conserved regions present synthesis product by T7 and Arabidopsis primase, the ra- Downloaded from https://academic.oup.com/nar/article/45/18/10764/4097637 by guest on 29 September 2021 in prokaryotic primases (2,27). Motif I corresponds to the dioactive [␣-32P]-ATP substrate and the oligonucleotide ZBD necessary for template recognition (26), whereas re- RNAs were quantified with ImageQuant. The processed in- gions II to VI are located in the RNAP domain. In vitro tensity was subjected to the following equation, previously characterization of AtTwinkle demonstrated that this en- derived by the Patel group (34): zyme has both primase and helicase activities (28,29,32). [RNA ribonucleotide(μM)] = (R/(R + A) ∗ ([ATP])(μM) However, its preferred ssDNA template sequence, its puta- tive role in organellar replication, and its interactions with where R refers to the band intensity observed for each ri- plant organellar DNA polymerases remain unclear. bonucleotide, A is the intensity of the radioactively label Herein, we found that AtTwinkle requires for template ATP substrate and [ATP] refers to the total molar concen- recognition a ssDNA template sequence that consists of two tration of ATP (labeled and unlabeled). The molar amount cryptic nucleotides and that RNA products generated by of RNA products is obtained by dividing the number of AtTwinkle are used as primers for plant organellar DNA ATPs in the RNA chain. polymerases. Our data suggests that the plant organellar replisome functionally resembles the replisome of T-odd Template-directed oligoribonucleotide synthesis bacteriophages. Primase reactions were assayed in a buffer containing 40 mM Tris–HCl pH 7.5, 50 mM potassium glutamate, 10 MATERIALS AND METHODS mM MgCl2 and 10 mM DTT. Primase reactions contained Expression and purification of recombinant proteins 100 ␮M of the indicated NTPs and 10 ␮Ci of [␣-32P]-NTP (3,000 Ci/mM). After incubation at 30◦C for 60 min, re- The open reading frame corresponding to the predicted pro- actions were terminated by 15 mM of EDTA. Loading cessed AtTwinkle (residues 92–709) was codon optimized buffer (95% formamide, 0.1% xylene cyanol) was immedi- for bacterial expression and cloned into a pUC19 vector. ately added to the reaction and the products were separated Open reading frames of AtPrimase-Helicase and AtPrimase on a 27% denaturing polyacrylamide gel containing 3 M were PCR amplified using the cloning vector as a tem- urea. Each primase reaction contained varied amounts of plate. The primase domain of bacteriophage T7 primase- recombinant protein or single-stranded DNA template as helicase (residues 1–262) was PCR amplified from genomic indicated in the figure legends. The ribonucleotide products T7 DNA. AtPrimase-Helicase (residues 92–709) and AtPri- were analyzed by phosphorimaging on a Storm 860 Phos- mase (residues 92–410) gene products were subcloned into phorImager. a modified pET19 vector
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