Mechanisms of genome integrity maintenance: break-induced replication at repetitive DNA sequences

The National Institute of General Medical Sciences awarded the University of Virginia $444,125 on March 1, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to investigate the mechanisms of genome integrity maintenance and break-induced replication at repetitive DNA sequences.

The research focuses on elucidating how break-induced replication drives genome instability in repetitive DNA regions, particularly telomere maintenance through the alternative lengthening of telomeres mechanism in ALT-positive cancers. The investigator's prior work established quantitative methodologies to monitor telomere replication and repair, identifying that spontaneous single-strand breaks trigger replication fork collapse leading to break-induced replication, and revealing that BLM helicase amplifies replication stress at ALT telomeres. The work leverages an error-prone repair mechanism specific to ALT-positive cancers as a potential therapeutic vulnerability. Under this award, the independent laboratory will investigate regulatory mechanisms underlying ALT-associated break-induced replication, building on recent discoveries that this process induces DNA damage tolerance marked by PCNA ubiquitination and other uncharacterized post-translational modifications. The project will functionally characterize how these PCNA modifications orchestrate DNA damage tolerance and alternative lengthening.

Performance occurs in Charlottesville, Virginia, with a period of performance from March 1, 2026 through February 28, 2031. The award mechanism is a Project Grant, supporting basic research that advances understanding of biological processes foundational to disease diagnosis, treatment, and prevention.

Abstract

This proposal aims to elucidate the mechanisms underlying genome instability driven by break-induced replication (BIR), with a focus on repetitive DNA sequences, particularly telomere maintenance mechanism in alternative lengthening of telomeres (ALT)-positive cancers. Repetitive DNA sequences, like telomeres, centromeres, rdna, short tandem repeats, etc., constituting over 50% of the human genome, represent regions of replication difficulty and genomic instability. Genome instability of the repetitive DNA sequences is associated with diseases such as cancers, neurodegenerative disorders, and age-related syndromes. My previous research focuses on ALT-mediated telomere maintenance mechanism, a telomerase-independent mechanism, which is utilized in approximately 15% of human cancers. ALT-positive cancer cells, elongate telomeres via a specialized homology-directed repair mechanism, termed BIR, independent from telomerase. By establishing quantitative methodologies to monitor telomere replication and repair, my work showed that spontaneous single strand breaks-induced replication fork collapse triggers BIR; unexpectedly, we uncovered that BLM helicase amplifies replication stress at ALT telomeres; notably, complex DNA repair mechanisms are activated to enable ALT telomere length and rearrangement, at the expense of exacerbating genome instability. The unique error-prone repair mechanism represents a potential vulnerability for ALT-positive cancers. In my independent laboratory, we will further investigate regulatory mechanism underlying ALT-associated BIR. We recently discovered that ALT- associated BIR induces significant DNA damage tolerance marked by pcna ubiquitination, and other uncharacterized pcna post-translational modifications. We will functionally characterize how these post- translational modifications of pcna orchestrate DNA damage tolerance and ALT-associated BIR. Additionally, BIR (or BIR-like mechanism) occurs not only at ALT telomeres, but also other repetitive DNA sequences. We will explore the genetic requirement and molecular mechanisms of BIR-mediated chromosome rearrangements in repetitive DNA sequences. Altogether, this research will advance our understanding of genome integrity maintenance mechanisms, offer insights into repetitive DNA sequences instability in cancers and other diseases. The findings will provide critical resources for developing therapeutic strategies by targeting BIR-related vulnerabilities.