BLM helicase suppresses recombination at G-quadruplex motifs in transcribed genes

Abstract

Bloom syndrome is a cancer predisposition disorder caused by mutations in the BLM helicase gene. Cells from persons with Bloom syndrome exhibit striking genomic instability characterized by excessive sister chromatid exchange events (SCEs). We applied single-cell DNA template strand sequencing (Strand-seq) to map the genomic locations of SCEs. Our results show that in the absence of BLM, SCEs in human and murine cells do not occur randomly throughout the genome but are strikingly enriched at coding regions, specifically at sites of guanine quadruplex (G4) motifs in transcribed genes. We propose that BLM protects against genome instability by suppressing recombination at sites of G4 structures, particularly in transcribed regions of the genome.

Fig. 1

High-resolution mapping of SCEs and common fragile site hotspots. a, b Representative Strand-seq libraries generated from a a WT fibroblast and b a BS fibroblast. Mapped DNA template strand reads are plotted on directional chromosome ideograms; reads mapping to the Crick (positive) strand of the reference genome are shown in green, those mapping to the Watson (negative) strand are shown in orange. SCEs are identified as a switch in template strand state, indicated by arrowheads. c, d Number of SCEs detected during a single cell cycle in c primary fibroblasts and d EBV-transformed B-lymphocytes obtained from healthy donor and BS patients. Each grey point represents number of SCEs detected in a single-cell Strand-seq library, red lines indicate mean ± SD. p-values were calculated using ANOVA. e SCE mapping resolutions across all eight cell lines. Lines represent percentage of the total number of SCEs mapped at resolutions below indicated values. f, g Correlations between average numbers of SCEs/chromosome/library and chromosome size for f WT and g BS cells. R²-values are color-matched to the cell lines. h Example of SCE hotspot detected within FRA3B (FHIT). Mapped SCE regions for each cell line were uploaded onto the UCSC Genome Browser. Black bars represent genomic locations of SCE regions; size indicates mapping resolution using the BAIT program. Red box indicates the location of the SCE hotspot as detected by Strand-seq

Fig. 2

Bloom syndrome SCEs are enriched at G4 motifs in active genes. Relative SCE enrichments (red points) over random distributions (violin plots) for SCEs that overlap with one or multiple a genes; b G4 motifs (G₃₊N_1–7G₃₊N_1-7G₃₊N_1-7G₃₊); c active genes containing one or more G4 motifs; d active genes without G4 motifs; e silent genes containing one or more G4 motifs; and f silent genes without G4 motifs. All values were normalized to the median permuted value for overlap of SCEs with FOIs (out of 1,000 permutations) and relative SCE enrichments over these values were plotted on the y-axis. p-values indicate the fraction of permuted overlaps (out of 1,000 permutations) equal to or higher than overlap with observed SCE regions. Significant p-values are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Bloom syndrome SCE enrichments occur at transcribed G4 motifs. a Intragenic G4 motifs can occur on either the transcribed strand (RNA shown in red), or on the non-transcribed strand; b–h relative SCE enrichments (red points) over random distributions (violin plots) for SCEs overlapping with h for intergenic G4 motifs, and G4 motifs occurring on c intragenic transcribed strands; d non-transcribed strands; e transcribed strands of active genes; f non-transcribed strands for active genes; g transcribed strands for silent genes; and h non-transcribed strands for silent genes. p-values indicate the fraction of permuted overlaps (out of 1,000 permutations) equal to or higher than overlap with observed SCE regions. Significant p-values are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Confirmation of SCE enrichments at G4 motifs in Blm^−/− mouse ES cells. a SCE rates detected WT, Blm^−/−, and Blm^+/− mouse ES cells. Each grey point represents number of SCEs detected in a single-cell Strand-seq library, red lines indicate mean ± SD. p-values were calculated using t-test and ANOVA. b–g Relative SCE enrichments (red points) over random distributions (violin plots) for SCEs overlapping one or more (b) genes; c G4 motifs; and G4 motifs occurring on d transcribed strands of active genes; e non-transcribed strands for active genes; f transcribed strands for silent genes; and g non-transcribed strands for silent genes. p-values indicate the fraction of permuted overlaps (out of 1,000 permutations) equal to or higher than overlap with observed SCE regions. Significant p-values are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. h Frequency of observed SCE regions occurred on the same homolog as allele-specific G4 motifs. Indicated is the homolog containing G4 motif, concordant indicates SCE occurred on same homolog, discordant indicates SCE occurred on opposite homolog. Number of allelic G4 motifs included in analysis is shown above each bar. p-values were calculated using binomial distributions based on a 50% chance of SCE and G4 motif occurring on the same homolog

Fig. 5

Low levels of loss of heterozygosity in Blm^−/− mouse ES cells. Frequency of a unique LOH regions; b aneuploidy; and c local copy number variations detected at in single-cell whole genome sequencing libraries at different passages in WT, Blm^−/−, and Blm^+/− mouse ES cells. The number of single-cell sequencing libraries included in the analysis is shown above each bar. p-values for LOH events were calculated using binomial distributions, for aneuploidy and CNVs by ANOVA

Fig. 6

BLM helicase suppresses recombination at G4 structures. Model for the role of BLM in suppressing recombination at sites of G4 structures. G4 structures are more likely to form or persist in the absence of unwinding by BLM. They can form at G4 motifs throughout the genome, but formation is promoted by transcription, especially if the G4 motif is present on the transcribed strand. In BLM proficient cells, BLM unwinds the G4 structure before the genomic region is replicated, ensuring smooth DNA replication. In the absence of BLM, G4 structures are not unwound, preventing replication fork progression, and leading to replication fork stalling. Stalled forks require homologous recombination (HR)-mediated repair, leading to formation of a double Holliday junction (dHJ). In the absence of BLM, dHJs cannot be dissolved by the BLM-TOPO3α-RMI1-RMI2 (BTRR) complex and must be resolved by MUS81-EME1 or GEN1, leading to frequent formation of sister chromatid exchanges event, and potentially loss of heterozygosity, and other types of mutations