The signaling cascade initiated in response to DNA double-strand breaks (DSBs) has been extensively investigated in interphase cells. Here, we show that mitotic cells treated with DSB-inducing agents activate a “primary” DNA damage response (DDR) comprised of early signaling events, including activation of the protein kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), histone H2AX phosphorylation together with recruitment of mediator of DNA damage checkpoint 1 (MDC1), and the Mre11–Rad50–Nbs1 (MRN) complex to damage sites. However, mitotic cells display no detectable recruitment of the E3 ubiquitin ligases RNF8 and RNF168, or accumulation of 53BP1 and BRCA1, at DSB sites. Accordingly, we found that DNA-damage signaling is attenuated in mitotic cells, with full DDR activation only ensuing when a DSB-containing mitotic cell enters G1. Finally, we present data suggesting that induction of a primary DDR in mitosis is important because transient inactivation of ATM and DNA-PK renders mitotic cells hypersensitive to DSB-inducing agents.

Mutations in CDCA7, the SNF2 family protein HELLS (LSH), or the DNA methyltransferase DNMT3b cause immunodeficiency–centromeric instability–facial anomalies (ICF) syndrome. While it has been speculated that DNA methylation defects cause this disease, little is known about the molecular function of CDCA7 and its functional relationship to HELLS and DNMT3b. Systematic analysis of how the cell cycle, H3K9 methylation, and the mitotic kinase Aurora B affect proteomic profiles of chromatin in Xenopus egg extracts revealed that HELLS and CDCA7 form a stoichiometric complex on chromatin, in a manner sensitive to Aurora B. Although HELLS alone fails to remodel nucleosomes, we demonstrate that the HELLS–CDCA7 complex possesses nucleosome remodeling activity. Furthermore, CDCA7 is essential for loading HELLS onto chromatin, and CDCA7 harboring patient ICF mutations fails to recruit the complex to chromatin. Together, our study identifies a unique bipartite nucleosome remodeling complex where the functional remodeling activity is split between two proteins and thus delineates the defective pathway in ICF syndrome.

Significance: The centromere is a highly specialized genomic locus playing a critical role in chromosome segregation during cell division and is made of characteristic repetitive DNA sequences. Due to their repetitive nature, it has been difficult to study the organization of human centromeres. Here, we present a way to monitor stability of the human centromeres using chromosome-orientation fluorescent in situ hybridization and superresolution microscopy. We show that the centromere-specific histone variant CENP-A and the centromere-associated proteins CENP-C and CENP-T contribute to maintenance of centromere integrity and present evidence of centromere disregulation in cancer and during cellular senescence, which is related to aging. Our study reveals the existence of a centromere-specific mechanism to organize the repetitive structure and prevent human centromeres from suffering illegitimate rearrangements.

Maintenance of an intact genome is essential for cellular and organismal homeostasis. The centromere is a specialized chromosomal locus required for faithful genome inheritance at each round of cell division. Human centromeres are composed of large tandem arrays of repetitive alpha-satellite DNA, which are often sites of aberrant rearrangements that may lead to chromosome fusions and genetic abnormalities. While the centromere has an essential role in chromosome segregation during mitosis, the long and repetitive nature of the highly identical repeats has greatly hindered in-depth genetic studies, and complete annotation of all human centromeres is still lacking. Here, we review our current understanding of human centromere genetics and epigenetics as well as recent investigations into the role of centromere DNA in disease, with a special focus on cancer, aging, and human immunodeficiency–centromeric instability–facial anomalies (ICF) syndrome. We also highlight the causes and consequences of genomic instability at these large repetitive arrays and describe the possible sources of centromere fragility. The novel connection between alpha-satellite DNA instability and human pathological conditions emphasizes the importance of obtaining a truly complete human genome assembly and accelerating our understanding of centromere repeats’ role in physiology and beyond.

Significance: CENP-A, the histone H3 variant that forms a unique centromeric chromatin, is essential for faithful chromosome segregation during mitosis. Inability to connect the centromere to the mitotic spindle causes aneuploidy, a hallmark of many cancers. In addition to chromosome missegregation, chromosome fusions at (peri)centromeres are prevalent in cancers, but how such rearrangements arise remains unclear. Here, we identified a role for CENP-A in maintaining the integrity of centromere-associated repetitive sequences by ensuring their effective replication in human cells. In the absence of CENP-A, generation of DNA–RNA hybrids due to transcription–replication conflicts causes delayed DNA replication, centromere breakage, recombination, and chromosome translocations at centromeres. Centromeres thus possess a special mechanism to facilitate their replication and suppress chromosome translocations.