Recurrent Potential G-Quadruplex Sequences in Archaeal Genomes

Abstract

Evolutionary conservation or over-representation of the potential G-quadruplex sequences (PQS) in genomes are usually considered as a sign of the functional relevance of these sequences. However, uneven base distribution (GC-content) along the genome may along the genome may result in seeming abundance of PQSs over average in the genome. Apart from this, a number of other conserved functional signals that are encoded in the GC-rich genomic regions may inadvertently result in emergence of G-quadruplex compatible sequences. Here, we analyze the genomes of archaea focusing our search to repetitive PQS (rPQS) motifs within each organism. The probability of occurrence of several identical PQSs within a relatively short archaeal genome is low and, thus, the structure and genomic location of such rPQSs may become a direct indication of their functionality. We have found that the majority of the genomes of Methanomicrobiaceae family of archaea contained multiple copies of the interspersed highly similar PQSs. Short oligonucleotides corresponding to the rPQS formed the G-quadruplex (G4) structure in presence of potassium ions as demonstrated by circular dichroism (CD) and enzymatic probing. However, further analysis of the genomic context for the rPQS revealed a 10-12 nt cytosine-rich track adjacent to 3'-end of each rPQS. Synthetic DNA fragments that included the C-rich track tended to fold into alternative structures such as hairpin structure and antiparallel triplex that were in equilibrium with G4 structure depending on the presence of potassium ions in solution. Structural properties of the found repetitive sequences, their location in the genomes of archaea, and possible functions are discussed.

Keywords: DNA; G-quadruplex; Methanomicrobiaceae; archaea; circular dichroism; nuclease probing.

Figure 1

(A) The potential quadruplex sequences (PQS) density in archaeal genomes (black circles) and a random sequence (red squares, error bars shows SD in five 10 Mb replicates for random sequence) plotted against the G/C content of the genomes. (B) PQS density in genomes of Methanomicrobiaceae family. Filled green squares are genomes containing repetitive PQSs; gray circles present the genomes lacking repetitive PQS (rPQS); red square corresponds to Methanogenium cariaci JCM 10550 genome that contains about 100 repeated sequences of the pattern GnNiGn (where n > 7, i = 1 or 2).

Figure 2

Phylogenic tree for Methanomicrobiaceae family based on alignment of 16S rRNA with ClustalW. The strains marked in blue contain the rPQS, which consensus sequences are shown on the left. Total number of each consensus rPQS is shown for each marked strain.

Figure 3

Location of rPQSs in 12 genomes of Methanomicrobiaceae family with respect to the two nearest genes. (A) Circular diagram of the rPQS distribution between the pairs of genes with head-to-head, tail-to-tail, and head-to-tail orientation. (B) The distance (in nucleotides) distribution between rPQS and the start of a CDS (left) or the end of a CDS (right).

Figure 4

Alignment of the ±100 nucleotide context of the repetitive PQSs in the genomes of Methanomicrobiaceae family revealed a C-rich motif at 3'-end of the PQS. Discovered motif in Methanomicrobiaceae’s performed with GLAM2.

Figure 5

S1 nuclease probing of the structures formed by ODNs GCA-S and GCA-L (A) and ODNs ACC-S and ACC-L (B) in the presence of Li⁺ ions or K⁺ ions before and after renaturation. L – longer ODNs ACC-L or GCA-L, S – shorter ODNs GCA-S or ACC-S. Sequencing G+A lane was performed by treatment of the ODNs with formic acid. On the right – schematic depictions of the alternative structures that the longer ODNs GCA-L or ACC-L may adopt the intramolecular triplex and G4 folding. Black arrows indicate the S1 cleavage regions common for structures regardless solution conditions; red arrows show the bands that appear after renaturation of the long ODNs in presence of potassium ions.

Figure 6

Circular dichroism (CD) spectral changes reflects potassium dependent DNA refolding. The spectra were recorded for oligonucleotides GCA-S (A), ACC-S (B), GCA-L (C), and ACC-L (D) at 1 µM concentration in 20 mM LiAc buffer pH 8.0 (black). The same sample after addition of 100 mM KCl and incubation 10 min at 20°C (blue) and subsequent fast annealing of the final solution (red).

Figure 7

Temperature dependence of CD spectra for long oligonucleotides GCA-L (A) and ACC-L (B). Temperature color code shown on insert from blue (20°C) to red (90°C).