Specialized replication mechanisms maintain genome stability at human centromeres.

The high incidence of whole-arm chromosome aneuploidy and translocations in tumors suggests instability of centromeres, unique loci built on repetitive sequences and essential for chromosome separation. The causes behind this fragility and the mechanisms preserving centromere integrity remain elusive. We show that replication stress, hallmark of pre-cancerous lesions, promotes centromeric breakage in mitosis, due to spindle forces and endonuclease activities. Mechanistically, we unveil unique dynamics of the centromeric replisome distinct from the rest of the genome. Locus-specific proteomics identifies specialized DNA replication and repair proteins at centromeres, highlighting them as difficult-to-replicate regions. The translesion synthesis pathway, along with other factors, acts to sustain centromere replication and integrity. Prolonged stress causes centromeric alterations like ruptures and translocations, as observed in ovarian cancer models experiencing replication stress. This study provides unprecedented insights into centromere replication and integrity, proposing mechanistic insights into the origins of centromere alterations leading to abnormal cancerous karyotypes.

From rest to repair: Safeguarding genomic integrity in quiescent cells.

Quiescence is an important non-pathological state in which cells pause cell cycle progression temporarily, sometimes for decades, until they receive appropriate proliferative stimuli. Quiescent cells make up a significant proportion of the body, and maintaining genomic integrity during quiescence is crucial for tissue structure and function. While cells in quiescence are spared from DNA damage associated with DNA replication or mitosis, they are still exposed to various sources of endogenous DNA damage, including those induced by normal transcription and metabolism. As such, it is vital that cells retain their capacity to effectively repair lesions that may occur and return to the cell cycle without losing their cellular properties. Notably, while DNA repair pathways are often found to be downregulated in quiescent cells, emerging evidence suggests the presence of active or differentially regulated repair mechanisms. This review aims to provide a current understanding of DNA repair processes during quiescence in mammalian systems and sheds light on the potential pathological consequences of inefficient or inaccurate repair in quiescent cells.

exo-FISH: Protocol for detecting DNA breaks in repetitive regions of mammalian genomes.

Detecting DNA breaks in defined regions of the genome is critical to advancing our understanding of genome stability maintenance. Here, we present exo-FISH, a protocol to label exposed single-stranded DNA in defined repetitive regions of mammalian genomes by combining in vitro restriction enzyme digestion on fixed cells with fluorescence in situ hybridization (FISH). We describe steps for cell harvesting and fixation, slide treatments, and FISH probe hybridization. We then detail procedures for imaging and analysis. For complete details on the use and execution of this protocol, please refer to Saayman et al. (2023).1.