Mitotic microhomology-mediated break-induced replication promotes chromoanasynthesis

Overview

Chromoanasynthesis is a form of complex chromosomal rearrangement frequently observed in cancer and congenital disorders. This study characterizes the mechanistic basis of chromoanasynthesis through single-molecule long-read DNA sequencing of ultra-complex mutational events occurring at shortened telomeres and sub-telomeric double-strand breaks in human cells. The research identifies mitotic microhomology-mediated break-induced replication (MM-BIR) as the primary generative mechanism, revealing a collaborative interaction between microhomology-mediated end-joining (MMEJ) proteins and break-induced replication (BIR) machinery.

Methods and approach

Single-molecule long-read DNA sequencing was employed to characterize ultra-complex mutational events at shortened telomeres and sub-telomeric DNA double-strand breaks in human cells. This sequencing approach enabled the detection and detailed characterization of chromoanasynthesis-consistent rearrangement patterns. The investigation specifically focused on analyzing events occurring during mitosis and examining the involvement of molecular machinery components including Polδ, PIF1, POLD3, and PCNA in the replication and rearrangement process. The experimental design allowed for the distinction between mitotic and non-mitotic pathways and the identification of template-switching propensity.

Key Findings

Chromoanasynthesis arises through a mitotic pathway involving microhomology-mediated break-induced replication, wherein MMEJ proteins initiate a Polδ-dependent BIR process. This pathway is regulated by PIF1, POLD3, and PCNA, demonstrating functional interdependence between canonical MMEJ and BIR machinery. The mitotic MM-BIR pathway exhibits marked propensity for template switching during replication, generating dramatic amplification of genomic loci within single mutational events. The sequencing data reveal the ultra-complex nature of the resulting chromosomal rearrangements and provide evidence for the mutagenic capacity of this pathway in generating the full spectrum of chromoanasynthesis complexity.

Implications

The identification of mitotic MM-BIR as a key driver of complex chromosomal rearrangements establishes a mechanistic framework for understanding chromoanasynthesis generation in both neoplastic and congenital contexts. The pathway's inherent propensity for template switching and genomic locus amplification explains the extreme mutagenic nature of chromoanasynthesis and its capacity to generate ultra-complex rearrangements in single events. These findings have direct relevance to understanding the somatic evolutionary processes underlying cancer development and the genomic mechanisms generating congenital disorders characterized by CCRs.

Scope and limitations

This summary is based on the study abstract and available metadata. It does not include a full analysis of the complete paper, supplementary materials, or underlying datasets unless explicitly stated. Findings should be interpreted in the context of the original publication.