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. 2015 Apr 28;6:158.
doi: 10.3389/fgene.2015.00158. eCollection 2015.

R-loops and Initiation of DNA Replication in Human Cells: A Missing Link?

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Free PMC article

R-loops and Initiation of DNA Replication in Human Cells: A Missing Link?

Rodrigo Lombraña et al. Front Genet. .
Free PMC article

Abstract

The unanticipated widespread occurrence of stable hybrid DNA/RNA structures (R-loops) in human cells and the increasing evidence of their involvement in several human malignancies have invigorated the research on R-loop biology in recent years. Here we propose that physiological R-loop formation at CpG island promoters can contribute to DNA replication origin specification at these regions, the most efficient replication initiation sites in mammalian cells. Quite likely, this occurs by the strand-displacement reaction activating the formation of G-quadruplex structures that target the origin recognition complex (ORC) in the single-stranded conformation. In agreement with this, we found that R-loops co-localize with the ORC within the same CpG island region in a significant fraction of these efficient replication origins, precisely at the position displaying the highest density of G4 motifs. This scenario builds on the connection between transcription and replication in human cells and suggests that R-loop dysregulation at CpG island promoter-origins might contribute to the phenotype of DNA replication abnormalities and loss of genome integrity detected in cancer cells.

Keywords: CpG islands; DNA replication origins; G-quadruplex; ORC; R-loops.

Figures

FIGURE 1
FIGURE 1
Association between R-loops and ORC1-binding sites at CpG island-origins in human cells. (A) Venn diagrams illustrating the association between R-loops (defined as consensus DRIP peaks in Ginno et al., 2013) and ORC1-binding sites at origin regions (defined by intersecting ORC1 binding sites from Dellino et al., 2013 with SNS-Seq data from Besnard et al., 2012) at CpG island promoters in human cells (UCSC database hg19). See text for details. Alignment of the DRIP-Seq or SNS-Seq reads to the hg19 build was carried out using BWA (Li and Durbin, 2009), and peak calling was done using MACSv2 (Zhang et al., 2008). For DRIP-seq, peaks were called using all mapped reads, enforcing a greater than fivefold enrichment above input as described (Ginno et al., 2012). (B) Distribution of ORC1-binding sites (orange lines), DRIP peaks (blue lines) and G4 motifs (red lines) at the CpG-island origin set defined in (A). Composite profiles were generated by plotting hits per base over 6 kb for 485 CpG island regions centered at their TSS (defined as the 5′-end of RefSeq genes) normalized by the total hits over the whole genome. G4 positions were determined applying Quadparser on hg19 and specifying a loop size of 1–7 nucleotides between 4 tracks of GGG or CCC (Huppert and Balasubramanian, 2005). The green line represents the localization of the GC-skewed region of the analyzed CpG islands from Ginno et al. (2012).

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