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Occurrence of potential cruciform and H-DNA forming sequences in genomic DNA.

ABSTRACT: We have used computer-assisted methods to search large amounts of the human, yeast and Escherichia coli genomes for inverted repeat (IR) and mirror repeat (MR) DNA sequence patterns. In highly supercoiled DNA some IRs can form cruciforms, while some MRs can form intramolecular triplexes, or H-DNA. We find that total IR and MR sequences are highly enriched in both eukaryotic genomes. In E. coli, however, only total IRs are enriched, while total MRs only occur as frequently as in random sequence DNA. We then used a set of experimentally derived criteria to predict which of the total IRs and MRs are most likely to form cruciforms or H-DNA in supercoiled DNA. We show that strong cruciform forming sequences occur at a relatively high frequency in yeast (1/19 700 bp) and humans (1/41 800 bp), but that H-DNA forming sequences are abundant only in humans (1/49 400 bp). Strong cruciform and H-DNA forming sequences are not abundant in the E.coli genome. These results suggest that cruciforms and H-DNA may have a functional role in eukaryotes, but probably not prokaryotes.

SUBMITTER: Schroth GP

PROVIDER: S-EPMC306972 | biostudies-other | 1995 Jun

REPOSITORIES: biostudies-other

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Occurrence of potential cruciform and H-DNA forming sequences in genomic DNA.

Schroth G P GP Ho P S PS

Nucleic acids research 19950601 11

We have used computer-assisted methods to search large amounts of the human, yeast and Escherichia coli genomes for inverted repeat (IR) and mirror repeat (MR) DNA sequence patterns. In highly supercoiled DNA some IRs can form cruciforms, while some MRs can form intramolecular triplexes, or H-DNA. We find that total IR and MR sequences are highly enriched in both eukaryotic genomes. In E. coli, however, only total IRs are enriched, while total MRs only occur as frequently as in random sequence D ...[more]

PMID: 7596826

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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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Occurrence of potential cruciform and H-DNA forming sequences in genomic DNA.

Publications

Occurrence of potential cruciform and H-DNA forming sequences in genomic DNA.

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