Genome Maintenance Defects in Cultured Cells and Mice following Partial Inactivation of the Essential Cell Cycle Checkpoint Gene Hus1

Abstract

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...

Crystal Structure of the Human Rad9–Hus1–Rad1 Clamp

“…[7][8][9] Deletion of Rad9, Hus1, or Rad1 in mammalian cells also causes abnormal DNA damage responses and genomic instability, and even embryonic lethality in mouse experiments, demonstrating the significance of these proteins in checkpoint controls. [10][11][12][13][14] Rad9, Rad1, and Hus1 form a heterotrimeric 9-1-1 complex, which is evolutionarily conserved in terms of both structure and function from yeasts to humans. [15][16][17] Molecular modeling, electron microscopy (EM), and biochemical analyses predicted that the structure and biochemical function of the 9-1-1 complex may be similar to those of the eukaryotic sliding clamp, proliferating cell nucleus antigen (PCNA).…”

Dual inactivation of Hus1 and p53 in the mouse mammary gland results in accumulation of damaged cells and impaired tissue regeneration

“…In addition, Atr ϩ/Ϫ mice develop various neoplasms at a low frequency (20). On the other hand, mice with a partial reduction in Hus1 expression are not prone to spontaneous tumor development, despite increased genomic instability (39). Clearly, additional studies are required to fully resolve the importance of Hus1 and the Atr checkpoint pathway in tumorigenesis.…”

Discovery of Novel Targets with High Throughput RNA Interference Screening

“…Once this threshold is crossed, many genes show a dose dependence in their ability to control phenotypes, with decreasing levels resulting to increasingly severe phenotypes [167][168][169][170][171][172][173]. Interestingly, an allelic series of the cell cycle checkpoint protein Hus1 demonstrated dose-dependent effects in vitro, but little effect in vivo [174]. For some genes the protein expression must be reduced to quite low levels before a phenotype is observed.…”