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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.

SUBMITTER: Chashchina GV

PROVIDER: S-EPMC8044849 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The human Werner and Bloom syndromes (WS and BS) are caused by deficiencies in the WRN and BLM RecQ helicases, respectively. WRN, BLM and their S. cerevisiae homologue Sgs1, are particularly active in vitro in unwinding G-quadruplex DNA (G4-DNA), a family of non-canonical nucleic acid structures formed by certain G-rich sequences. Recently, mRNA levels from loci containing potential G-quadruplex-forming sequences (PQS) were found to be preferentially altered in sgs1 mutants, suggesting that G4-DNA targeting by Sgs1 directly affects gene expression. Here, we extend these findings to human cells. Using microarrays to measure mRNAs obtained from human fibroblasts deficient for various RecQ family helicases, we observe significant associations between loci that are upregulated in WS or BS cells and loci that have PQS. No such PQS associations were observed for control expression datasets, however. Furthermore, upregulated genes in WS and BS showed no or dramatically reduced associations with sequences similar to PQS but that have considerably reduced potential to form intramolecular G4-DNA. These findings indicate that, like Sgs1, WRN and BLM can regulate transcription globally by targeting G4-DNA. Cell culture conditions and media Human fibroblast cell strains (WS: AG05229, AG12795, AG12797; BS: GM02932, GM03402, GM16891; RTS: AG18371, AG18375, AG05013; Normal/Wild-type: AG04054, AG06310, AG09975) were obtained from the Coriell Repository (Camden, NJ), from donors matched for gender and of similar ages, and were at similar passage levels. Cells were cultured in MEM supplemented with Earle’s salts, 20% fetal bovine serum, 1x penicillin/streptomycin, and 1x fungizone in 3% O2 at 37oC and harvested for RNA extraction during active growth and at ~ 80% confluence. GeneChip microarray expression Total RNA from the 12 fibroblast cell strains was isolated by extraction with TRIzol (Invitrogen) and purified using the RNeasy system (Qiagen). Total RNA was amplified by in vitro transcription using the Ovation RNA Amplification System V2 (NuGen). The resultant cDNA was fragmented and labeled using the FL-Ovation cDNA Biotin Module V2 (NuGen), and then purified using QIAquick columns (Qiagen), as specified by the Ovation System manual. Labeled probe was hybridized to Affymetrix U133A 2.0 GeneChips, and ultimately scanned using an Axon GenePix array scanner. Statistical analysis of microarray expression experiment The output files were normalized by Robust Multiarray Average (RMA), using the R package GCRMA and gene expression levels were log2-transformed.

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Recurrent Potential G-Quadruplex Sequences in Archaeal Genomes.

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