Mechanisms of Genome Integrity Maintenance: Break-Induced Replication at Repetitive DNA Sequences

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. Project Number: 1R35GM162212-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Tianpeng Zhang | Institution: UNIVERSITY OF VIRGINIA, CHARLOTTESVILLE, VA | Award Amount: $444,125 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award - F Study Section[MRAF] View on NIH RePORTER: https://reporter.nih.gov/project-details/11270872