RINT-1, a Novel Rad50-interacting Protein, Participates in Radiation-induced G2/M Checkpoint Control

Abstract

The RAD50 gene was first identified in Saccharomyces cerevisiae based on its role in DNA repair (1). It is a member of the RAD52 epistasis group, members of which function in recombinational DNA repair. Yeast strains carrying rad50 mutations exhibit hypersensitivity to ␥-irradiation and radiomimetic chemicals such as methyl methanesulfonate and only slight sensitivity to UV irradiation (1). In addition, rad50 mutations also lead to telomere shortening and meiotic failure (1). The 153-kDa Rad50 protein contains both an N-terminal Walker A and an C-terminal Walker B NTP binding domains, linked by two extensive coiled-coil regions that are characterized by leucine heptad repeats (2). This sequence architecture is similar to that found in other members of the structural maintenance of chromosomes (SMC) 1 protein family (3, 4). The first 35 heptad repeats span amino acids 177-421 of the Rad50 sequence, whereas the second 37 heptad repeats span amino acids 743-995. These heptad repeat sequences exhibit length and significant sequence similarity to the S2 domain of rabbit myosin (2). Mutations within the Rad50 Walker A domain that alter conserved amino acids resides within the NTP binding site confer a null phenotype, and several mutations near the nucleotide binding sites cause defects in meiosis (5).The RAD50 gene product is highly conserved among species. Human and yeast Rad50 share more than 50% sequence identity within their corresponding N-and C-terminal regions as well as a similar coiled-coil structure in between. A null mutation of the mouse Rad50 gene is associated with an embryonic lethal phenotype, and cells derived from early embryos are hypersensitive to ionizing radiation, suggesting that Rad50 is essential for normal cell proliferation and DNA double-strand break repair (6). Yeast Rad50 forms a complex with Mre11 and Xrs2 (7). Similarly, human Rad50 forms a complex with hMre11 and NBS1, the potentially functional homologue of yeast Xrs2 (8,9). It is believed that the Mre11-Rad50-NBS1 protein complex plays a central role in the cellular response to DNA damage. The NBS1 gene is mutated in Nijmegen Breakage syndrome (9, 10), which is phenotypically characterized by radiosensitivity and increased chromosomal instability upon ionizing radiation. Mre11 possesses DNA end-holding as well as endonucleolytic and exonucleolytic activities (11-15) that may be utilized during the initial steps of DNA double-strand break repair. Mouse embryonic stem cells deficient in Mre11 are not viable (16), suggesting that Mre11, like Rad50, is essential for normal proliferating cells and DNA double-strand break repair. Thus, while it is clear that these three proteins function as a triplex in DNA double-strand break repair, it is also very likely that these proteins function in association with other partners to have different biological functions.To explore other potential functions of Rad50, we have identified a novel Rad50-interacting protein, named RINT-1. These two proteins specifically interact during S and M phases of the...

Rint1 inactivation triggers genomic instability, ER stress and autophagy inhibition in the brain

“…[36][37][38][39][40][41][42][43][44][45][46][47][48][49] RINT1 is known to be involved in both pathways through interaction with p130 15 or Rad50. 14 The structure of chromosome fusion in the Rint1-deficient cells supports the hypothesis of defective DSB repair (Figures 4c and d). Notably, the frequency of mitotic defects in Rint1-deficient NSCs was significantly increased.…”

“…[10][11][12] The complex integrity is regulated by an autophagy factor UVRAG, which interacts with RINT1, demonstrating the intersection of Golgi-ER and autophagic trafficking mechanisms. 13 RINT1 was identified as a Rad50-interacting protein involved in G2/M checkpoint 14 and was shown to be involved in telomerase independent maintenance of telomeres via the interaction with p130. 15 The importance of RINT1 in Golgi-ER trafficking and thus in homeostasis of Cisand Trans-Golgi was demonstrated in vitro [16][17][18][19][20][21] where in vivo studies were limited by the early embryonic lethality (E5.5) associated with Rint1 inactivation.…”

Hec1 sequentially recruits Zwint-1 and ZW10 to kinetochores for faithful chromosome segregation and spindle checkpoint control

“…To obtain cell populations synchronized at different cell cycle stages, T24 cells were first arrested at G 0 /G 1 by growing at high-density with low-serum medium, and then released from the arrest by seeding at a lower-density with serum containing medium. Cells were collected at various time points after release as described (Chen et al, 1989;Xiao et al, 2001). To obtain cell populations synchronized at different cell cycle stages, HeLa cells were first released from thymidine (2.5 mM) block for 4 h and then treated with nocodazole for 4 h to arrest cells at mitotic phase.…”

Dsl1p, Tip20p, and the Novel Dsl3(Sec39) Protein Are Required for the Stability of the Q/t-SNARE Complex at the Endoplasmic Reticulum in Yeast

“…Three mammalian syntaxin 18-associated proteins have been known before to be involved in cell cycle checkpoint control (ZW10 and RINT-1) or apoptosis (BNip1; Chan et al, 2000;Xiao et al, 2001;Zhang et al, 2003). In mammalian cells ZW10, the putative equivalent of Dsl1p, helps to recruit dynactin and dynein to the kinetochore (Starr et al, 1998).…”