Protist, Vol. 170, 141–152, April 2019 http://www.elsevier.de/protis Published online date 13 February 2019 ORIGINAL PAPER Mapping the Single Origin of Replication in the Naegleria gruberi Extrachromosomal DNA Element 1,2 John C. Mullican , Nora M. Chapman, and Steven Tracy Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA Submitted October 25, 2018; Accepted February 6, 2019 Monitoring Editor: C. Graham Clark The genes encoding the ribosomal RNA (rRNA) subunits of the amoeba Naegleria gruberi are encoded in a relatively uncommon arrangement: on a circular extrachromosomal DNA element with each organism carrying about 4,000 copies of the element. As complete sequence analysis of the N. gruberi chromo- somal DNA revealed no copy of the rRNA genes, these extrachromosomal elements must therefore replicate autonomously. We reported elsewhere the molecular cloning and the complete sequence analysis of the entire rRNA gene-containing element of N. gruberi (strain EGB). Using neutral/neutral two-dimensional agarose electrophoresis, the region in the element enclosing the single replication origin using DNA from asynchronous and axenically propagated N. gruberi populations was localized within a 2.1 kbp fragment located approximately 2,300 bp from the 18S rRNA gene and 3,700 bp from the 28S rRNA gene. The results indicate that replication occurs from a single origin via a theta-type mode of replication rather than by a rolling circle mode. Further, G-quadruplex elements, often located near DNA replication origins, occur in and near this fragment in a repeated sequence. © 2019 Elsevier GmbH. All rights reserved. Key words: Amoeba; Naegleria gruberi; ribosomal RNA; extrachromosomal DNA element; origin of replication; G-quadruplex. 1 Corresponding author. 2 Current address: Department of Biology, Washburn University, Topeka, Kansas 66621, USA. e-mail [email protected] (J.C. Mullican). Abbreviations: TBE, Tris-borate-EDTA; ARS, Autonomous replication sequence; ORC, Origin Replication Complex; ori, origin; mtDNA, mitochondrial DNA; NRS, Non-Ribosomal Sequence; G4, G-quadruplex; pG4, Potential G-quadruplex; rDNA, ribosomal DNA; RDE, Ribosomal DNA-containing Element; rRNA, ribosomal RNA. https://doi.org/10.1016/j.protis.2019.02.001 1434-4610/© 2019 Elsevier GmbH. All rights reserved. 142 J.C. Mullican et al. Introduction chromosomal, RDE and mtDNA) of the N. gruberi organism. This is considerably higher than the ear- Free-living amoebae, such as the 47 characterized lier estimation of 17% (Clark and Cross 1987), a species of the Naegleria genus (De Jonckheere discrepancy that can be attributed to more precise 2014), are ubiquitous in soil and fresh water world- measurements of both the genome and element wide (Denet et al. 2017; Rodriguez-Zaragoza 1994; sizes (Fritz-Laylin et al. 2010). As the mass of Tyml et al. 2016). Naegleria fowleri is the sole the RDE population’s DNA represents a significant known Naegleria species that is pathogenic for fraction of the total DNA in a Naegleria organism, it humans and is infamous due to its ability, fol- will be important to understand the mechanism by lowing ingestion into the nasal cavity, to induce which this extrachromosomal element replicates. the nearly universally fatal disease, primary amoe- With thousands of copies of the RDE per cell bic meningoencephalitis (Grace et al. 2015). The (Clark and Cross 1987) and no ‘master’ copy of non-pathogenic Naegleria gruberi, on the other rDNA or the RDE itself in the chromosomal DNA hand, represents a useful research model organism (Clark and Cross 1987; Fritz-Laylin et al. 2010), (Ahmed Khan et al. 2015; Opperdoes et al. 2011). Clark and Cross (1987) inferred that the RDE must From bacteria to humans, all cells contain replicate autonomously. There have been no pub- repetitive rRNA genes (rDNA) (Long and Dawid lished studies to date designed to map the origin(s) 1980). Examining the NEG-M strain of N. gruberi, of replication (ori) of the RDE in Naegleria. Stud- Clark and Cross (1987) first demonstrated that ies of eukaryotic DNA replication ori have focused this Naegleria species carries its ribosomal RNA on chromosomes in the yeast, human and murine (rRNA) genes on an extrachromosomal element; genomes (Parker et al. 2017). It is noteworthy later, Maruyama and Nozaki (2007) sequenced that the primary structure requirements of eukary- the extrachromosomal element of this particu- otic DNA ori are not well understood, save for lar strain. Complete sequence analysis of the yeast; indeed, there is a better understanding of N. gruberi organism’s nuclear DNA (Fritz-Laylin the proteins involved in the ori replication complex et al. 2010) confirmed earlier work based on (ORC) than of the requirements of the DNA site(s) Southern blot hybridization analysis (Clark and that is/are recognized [reviewed in (Leonard and Cross 1987) that there is no copy of the ribo- Mechali 2013; O’Donnell et al. 2013)]. somal DNA in the chromosomal DNA. While Nonetheless, the search for specific primary DNA this arrangement is uncommon, the placement structure correlates of DNA ori has identified certain of rDNA only on an extrachromosomal element genetic elements often associated with replication has also been demonstrated for other members initiation. The discovery that GC enrichment of of the order Schizopyrenida (Clark and Cross regions where origins are found in metazoan DNA 1988). Entamoeba histolytica and E. invadens also has led to the recognition of a role for a structural lodge the rDNA on an extrachromosomal element component called the G-quadruplex (G4) (Prioleau (Bhattacharya et al. 1989, 1998) as do some other 2017). The G4s derive from primary sequences eukaryotic microbial species, several of which pos- containing four separated repeats of two or more sess circular rDNA (Torres-Machorro et al. 2010). G nucleotides. They can form structures, based Earlier work estimated the number of the ribo- on the non-canonical interactions [Hoogsteen base somal DNA-containing extrachromosomal element pairing; (Hoogsteen 1963)] of the G nucleotides, to (RDE) molecules at about 4,000 per cell based on form 2 or more planar structures. These are termed visual analysis of slot-blot data (Clark and Cross G tetrads [(Sen and Gilbert 1988); reviewed and 1987). Complete sequence analysis of the entire depicted in (Maizels 2006)]. Such tetrads can stack genome of N. gruberi (strain NEG-M) (Fritz-Laylin upon each other to form the G-quadruplex (Sup- et al. 2010) revealed the mass of the nuclear DNA plementary Material Fig. S1). Recognition of these 6 × per organism to be 0.045 pg DNA (41 10 bp sequence motifs associated with DNA ori (Cayrou per genome, calculated at 660 Da/bp). Each organ- et al. 2012) has prompted mutational analyses, ism (Clark 1990) also contains approximately 420 which demonstrate that while these components copies of mitochondrial DNA (mtDNA) which, at are not sufficient for an origin of DNA replication, 50 kbp per copy (Fritz-Laylin et al. 2010), yields they are necessary at least for some defined sites about 0.023 pg mtDNA per cell. Thus, at about (Valton et al. 2014). 4 × 4,000 copies of the 1.4 10 bp RDE per cell (Clark Autonomous replication sequences (ARS) with and Cross 1987; Maruyama and Nozaki 2007) or conserved origin sequence motifs have been iden- about 0.061 pg per cell, the RDE population repre- tified in yeast species. And, just as for the G4s, sents about 47% of the total DNA mass (nuclear these appear to be necessary for ori function (Peng N. gruberi Extrachromosomal rDNA Origin 143 et al. 2015). Interestingly, two sequences with clone revealed the location of the 5.8S, 18S homology to the conserved ori sequence motifs and 28S rRNA genes as well as numerous of Saccharomyces cerevisiae were noted in the repeat sequences [(Mullican et al. 2018); anno- N. gruberi ribosomal DNA extrachromosomal ele- tated in GenBank Accession MG699123]. We found ment (Mullican et al. 2018). The nonribosomal (Mullican et al. 2018), as had Maruyama and region of the RDE is largely composed of directly Nozaki (2007) earlier, that the 18S rDNA of the repeated DNA sequences ranging in size up to two different strains of N. gruberi lacked the type 315 nucleotides and include both interspersed and I intron reported to be in most Naegleria species’ tandemly repeated sequences. Due to the high RDEs (Wikmark et al. 2006). The two sequences copy number of these RDEs in Naegleria cells as derived from two closely related N. gruberi strains well as the considerable evolutionary divergence of are very similar with most differences contained these Excavata protists from metazoans and yeast, within a region of 120 bp tandem repeats in the and the repeated observations that DNA primary non-rDNA coding region. The RDEs of the 2 dif- structure in ori is quite variable, it is possible that ferent amoebic strains differ at only 4 sites (with the primary (as well as higher order) structure of nucleotide numbering referring to the pNgrubEGB the ori in the Naegleria RDE may differ from other sequence positions; GenBank MG699123): a sin- eukaryotic origins studied (Leonard and Mechali gle nucleotide (nt) deletion (nt8581-8582), a single 2013; Parker et al. 2017). nucleotide variation (nt8703) and a 119 bp deletion As the mechanism of the Naegleria RDE replica- (relative to the sequence for strain NEG-M; Gen- tion and regulation remains unexplored, we initiated Bank AB298288) of part of the 4th and 5th copy of studies toward defining the RDE ori. We hypothe- the 120 bp tandem repeat (nt8748-8749) of the non- sized that each RDE possesses at least one ori and ribosomal sequence (NRS). There is also a single we sought to map the location of such a domain nt deletion in the EGB strain’s RDE at nt13736- within a Naegleria RDE.