Cell cycle checkpoints are evolutionarily conserved signaling pathways that uphold genomic integrity. Complete inactivation of the mouse checkpoint gene Hus1 results in chromosomal instability, genotoxin hypersensitivity, and embryonic lethality. To determine the functional consequences of partial Hus1 impairment, we generated an allelic series in which Hus1 expression was incrementally reduced by combining a hypomorphic The ability to accurately duplicate the genome and correctly segregate damage-free chromosomes to daughter cells is crucial to the health and longevity of all organisms. To ensure the fidelity of these processes, cells respond to genome damage by arresting the cell cycle, stabilizing replication forks, and inducing DNA repair. Cell cycle checkpoint pathways mediate these DNA damage responses and additionally can induce apoptosis if the damage is beyond repair. By preventing the accumulation of mutations that drives carcinogenesis, checkpoints can be important tumor suppressor mechanisms (3,21,27).Replication inhibitors and bulky DNA lesions activate a checkpoint pathway headed by the phosphatidylinositol kinase-like protein kinase Atr. Stalling of replication forks or processing of DNA lesions causes accumulation of singlestranded DNA that becomes coated with replication protein A (RPA) and attracts Atr in association with its binding partner Atrip (12). Atr then transmits the checkpoint signal by phosphorylating Chk1, Brca1, and other targets, which in turn regulate the cell cycle, replication, and repair machineries. Efficient DNA damage signaling through Atr also requires several accessory factors, including TopBP1/Cut5, Claspin, and the Rad9-Rad1-Hus1 (9-1-1) complex (44).With predicted structural similarity to proliferating cell nuclear antigen (PCNA), the 9-1-1 complex is believed to function as a checkpoint sliding clamp and molecular scaffold (57). Loading of the 9-1-1 trimer onto chromatin is stimulated by genotoxic stress and is mediated by a clamp loader composed of Rad17 and associated replication factor C subunits (8,41,67). Once on chromatin, the 9-1-1 complex participates in DNA damage signaling, promoting phosphorylation of Atr substrates such as Chk1, Rad17, and Rad9 itself (2,40,65,67). Signaling defects in cells lacking 9-1-1 components are associated with failure of an S-phase checkpoint that represses late origin firing in response to DNA damage (2, 40, 64). The mammalian 9-1-1 complex also directly associates with a number of DNA repair proteins, including factors involved in base excision repair (10,20,46,48,55,58,59) and translesion DNA synthesis (26, 42), and additionally is required for homologous recombinational repair (37, 61) as well as telomere maintenance (19,37). Given these key roles in checkpoint signaling and DNA repair, it is not surprising that cells defective for 9-1-1 function are hypersensitive to a wide variety of genotoxins, including replication inhibitors and DNA damaging agents (23,28,40,60,61,63,64).The mammalian 9-1-1 complex not only responds to ex...
Cells are under constant attack from genotoxins and rely on a multifaceted DNA damage response (DDR) network to maintain genomic integrity. Central to the DDR are the ATM and ATR kinases, which respond primarily to double-strand DNA breaks (DSBs) and replication stress, respectively. Optimal ATR signaling requires the RAD9A-RAD1-HUS1 (9-1-1) complex, a toroidal clamp that is loaded at damage sites and scaffolds signaling and repair factors. Whereas complete ATR pathway inactivation causes embryonic lethality, partial Hus1 impairment has been accomplished in adult mice using hypomorphic (Hus1(neo)) and null (Hus1(Δ1)) Hus1 alleles, and here we use this system to define the tissue- and cell type-specific actions of the HUS1-mediated DDR in vivo. Hus1(neo/Δ1) mice showed hypersensitivity to agents that cause replication stress, including the crosslinking agent mitomycin C (MMC) and the replication inhibitor hydroxyurea, but not the DSB inducer ionizing radiation. Analysis of tissue morphology, genomic instability, cell proliferation and apoptosis revealed that MMC treatment caused severe damage in highly replicating tissues of mice with partial Hus1 inactivation. The role of the 9-1-1 complex in responding to MMC was partially ATR-independent, as a HUS1 mutant that was proficient for ATR-induced checkpoint kinase 1 phosphorylation nevertheless conferred MMC hypersensitivity. To assess the interplay between the ATM and ATR pathways in responding to replication stress in vivo, we used Hus1/Atm double mutant mice. Whereas Hus1(neo/neo) and Atm(-/-) single mutant mice survived low-dose MMC similar to wild-type controls, Hus1(neo/neo)Atm(-/-) double mutants showed striking MMC hypersensitivity, consistent with a model in which MMC exposure in the context of Hus1 dysfunction results in DSBs to which the ATM pathway normally responds. This improved understanding of the inter-dependency between two major DDR mechanisms during the response to a conventional chemotherapeutic illustrates how inhibition of checkpoint factors such as HUS1 may be effective for the treatment of ATM-deficient and other cancers.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.