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The 3M complex maintains microtubule and genome integrity.

ABSTRACT: CUL7, OBSL1, and CCDC8 genes are mutated in a mutually exclusive manner in 3M and other growth retardation syndromes. The mechanism underlying the function of the three 3M genes in development is not known. We found that OBSL1 and CCDC8 form a complex with CUL7 and regulate the level and centrosomal localization of CUL7, respectively. CUL7 depletion results in altered microtubule dynamics, prometaphase arrest, tetraploidy, and mitotic cell death. These defects are recaptured in CUL7 mutated 3M cells and can be rescued by wild-type, but not by 3M patient-derived CUL7 mutants. Depletion of either OBSL1 or CCDC8 results in defects and sensitizes cells to microtubule damage similarly to loss of CUL7 function. Microtubule damage reduces the level of CCDC8 that is required for the centrosomal localization of CUL7. We propose that CUL7, OBSL1, and CCDC8 proteins form a 3M complex that functions in maintaining microtubule and genome integrity and normal development.

SUBMITTER: Yan J

PROVIDER: S-EPMC4165194 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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The 3M complex maintains microtubule and genome integrity.

Yan Jun J Yan Feng F Li Zhijun Z Sinnott Becky B Cappell Kathryn M KM Yu Yanbao Y Mo Jinyao J Duncan Joseph A JA Chen Xian X Cormier-Daire Valerie V Whitehurst Angelique W AW Xiong Yue Y

Molecular cell 20140501 5

CUL7, OBSL1, and CCDC8 genes are mutated in a mutually exclusive manner in 3M and other growth retardation syndromes. The mechanism underlying the function of the three 3M genes in development is not known. We found that OBSL1 and CCDC8 form a complex with CUL7 and regulate the level and centrosomal localization of CUL7, respectively. CUL7 depletion results in altered microtubule dynamics, prometaphase arrest, tetraploidy, and mitotic cell death. These defects are recaptured in CUL7 mutated 3M c ...[more]

PMID: 24793695

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Project description:The DNA damage response (DDR) orchestrates a network of cellular processes that integrates cell-cycle control and DNA repair or apoptosis, which serves to maintain genome stability. DNA-PKcs (the catalytic subunit of the DNA-dependent kinase, encoded by PRKDC), ATM (ataxia telangiectasia, mutated), and ATR (ATM and Rad3-related) are related PI3K-like protein kinases and central regulators of the DDR. Defects in these kinases have been linked to neurodegenerative or neurodevelopmental syndromes. In all cases, the key neuroprotective function of these kinases is uncertain. It also remains unclear how interactions between the three DNA damage-responsive kinases coordinate genome stability, particularly in a physiological context. Here, we used a genetic approach to identify the neural function of DNA-PKcs and the interplay between ATM and ATR during neurogenesis. We found that DNA-PKcs loss in the mouse sensitized neuronal progenitors to apoptosis after ionizing radiation because of excessive DNA damage. DNA-PKcs was also required to prevent endogenous DNA damage accumulation throughout the adult brain. In contrast, ATR coordinated the DDR during neurogenesis to direct apoptosis in cycling neural progenitors, whereas ATM regulated apoptosis in both proliferative and noncycling cells. We also found that ATR controls a DNA damage-induced G2/M checkpoint in cortical progenitors, independent of ATM and DNA-PKcs. These nonoverlapping roles were further confirmed via sustained murine embryonic or cortical development after all three kinases were simultaneously inactivated. Thus, our results illustrate how DNA-PKcs, ATM, and ATR have unique and essential roles during the DDR, collectively ensuring comprehensive genome maintenance in the nervous system.The DNA damage response (DDR) is essential for prevention of a broad spectrum of different human neurologic diseases. However, a detailed understanding of the DDR at a physiological level is lacking. In contrast to many in vitro cellular studies, here we demonstrate independent biological roles for the DDR kinases DNA-PKcs, ATM, and ATR during neurogenesis. We show that DNA-PKcs is central to DNA repair in nonproliferating cells, and restricts DNA damage accumulation, whereas ATR controls damage-induced G2 checkpoint control and apoptosis in proliferating cells. Conversely, ATM is critical for controlling apoptosis in immature noncycling neural cells after DNA damage. These data demonstrate functionally distinct, but cooperative, roles for each kinase in preserving genome stability in the nervous system.

Project description:BACKGROUND & AIMS:RNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis. METHODS:We generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2b?IEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53?IEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells, generated intestinal organoids, and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53?IEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients, measured levels of ribonuclease H2 subunit B, and associated these with patient survival times and transcriptome data. RESULTS:The H2b?IEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53?IEC mice. However, H2b/p53?IEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T>G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times. CONCLUSIONS:In analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients, we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53?IEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis.

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The 3M complex maintains microtubule and genome integrity.

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The 3M complex maintains microtubule and genome integrity.

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