Telomere and Ribosomal DNA Repeats are Chromosomal Targets of the Bloom Syndrome DNA Helicase

Research Article

Telomere and Ribosomal DNA Repeats are Chromosomal Targets of the Bloom Syndrome DNA Helicase

Freja Madsen¹*, Kasper Holm², Sofie Jensen³ and Magnus Sorensen³

¹Department of Microbiology, University of Copenhagen, Copenhagen, Denmark
²Department of Virology, Aarhus University, Aarhus, Denmark
³Department of Biology, University of Southern Denmark, Odense, Denmark

Published: 21 February 2018

Abstract

Background: Bloom Syndrome (BS) is a rare autosomal recessive disorder characterized by genomic instability and cancer predisposition, caused by mutations in the BLM gene encoding a RecQ family DNA helicase. BLM plays critical roles in maintaining genome integrity by resolving complex DNA structures and suppressing aberrant recombination. Repetitive DNA sequences, such as telomeres and ribosomal DNA (rDNA) arrays, present unique challenges to DNA replication and repair, often forming structures like G-quadruplexes (G4) and recombination intermediates that can impede fork progression and lead to instability.

Objective: This study aimed to investigate whether telomeric and rDNA repeats are specific chromosomal targets of the BLM helicase and to determine the consequences of BLM deficiency on the stability of these repetitive regions.

Methods: We utilized human fibroblast cell lines derived from Bloom Syndrome patients (BLM-deficient), isogenic BLM-corrected cells, and control cells. Chromatin immunoprecipitation (ChIP) followed by quantitative PCR (qPCR) was used to assess BLM localization at telomeric and rDNA sequences. Fluorescence In Situ Hybridization (FISH) techniques, including Chromosome Orientation FISH (CO-FISH), were employed to analyze telomere fragility, Telomere Sister Chromatid Exchanges (T-SCEs), and rDNA locus stability. DNA fiber analysis was used to monitor replication fork progression within rDNA repeats. In vitro helicase assays were performed using purified BLM protein and synthetic DNA substrates mimicking telomeric G4 structures.

Results: ChIP analysis revealed a significant enrichment of BLM protein at both telomeric and rDNA sequences compared to control genomic loci, with enrichment further increased under conditions of replication stress. BLM-deficient cells exhibited significantly elevated levels of telomere fragility, T-SCEs, and heterogeneity in telomere length compared to BLM-proficient cells. Similarly, the rDNA loci in BS cells showed increased signs of instability, including elevated sister chromatid exchange within the rDNA array and altered replication fork dynamics suggestive of increased stalling or collapse. In vitro assays confirmed the robust ability of purified BLM to unwind G-quadruplex DNA structures characteristic of telomeric repeats.

Conclusion: Our findings strongly suggest that both telomeric repeats and rDNA arrays are crucial chromosomal targets requiring BLM helicase activity for their maintenance. BLM likely localizes to these regions to resolve secondary structures (like G4s) and recombination intermediates that form during replication and repair, thereby preventing fork collapse, suppressing excessive recombination, and ensuring the stable propagation of these vital repetitive sequences. Loss of BLM function leads to significant instability at telomeres and rDNA loci, contributing to the overall genomic instability phenotype characteristic of Bloom Syndrome.

Keywords: Bloom syndrome; BLM helicase; RecQ helicase; Telomere; Ribosomal DNA (rDNA); Genomic instability; DNA replication; DNA repair; G-quadruplex; Sister Chromatid Exchange (SCE).