The CNOT complex contributes to the maintenance of genome stability

The yeast CCR4-NOT (CNOT in mammals) complex is a large (1.0-MDa) and highly conserved multifunctional set of proteins. It is involved in many different aspects of mRNA metabolism, including repression and activation of mRNA initiation, control of mRNA elongation, and deadenylation-dependent mRNA turnover; it also has a role in ubiquitin-protein transferase activity and histone methylation. Some studies have suggested that the yeast complex may be involved in the recognition and repair of DNA damage. To investigate whether similar properties are attributable to the mammalian complex we have examined the effects of inactivation of the complex on various aspects of the DNA damage response. Inactivation was achieved by depletion of CNOT1, the largest of the CNOT proteins, which forms a scaffold to the complex. Ablation of CNOT1 expression disrupts cell cycle progression through S and G2/M phases, which subsequently arrests the cell cycle in G1, with markedly elevated levels of cyclin E, p27 and p21. At later times, the cells appear to senesce and /or undergo autophagy. As expected, depletion of CNOT1 affects global transcription and can lead to transcription-dependent replication stress and R-loop formation. CNOT1 depletion can also affect DNA replication by reducing dNTP synthesis. Activity of the RNase H2 complex decreases following loss of CNOT1, which increases the sensitivity of genomic DNA to alkaline lysis due to an increase in embedded ribonucleotides. In addition, depletion of CNOT1 results in DNA damage as seen by comet assay, and formation of micronuclei. This is accompanied by activation of Chk2 in the absence of extraneous DNA damage. In this study, we have demonstrated that the CNOT complex contributes to the maintenance of genome stability and to the response to DNA damage

Disruption of the Mammalian Ccr4–Not Complex Contributes to Transcription-Mediated Genome Instability

The mammalian Ccr4–Not complex, carbon catabolite repression 4 (Ccr4)-negative on TATA-less (Not), is a large, highly conserved, multifunctional assembly of proteins that acts at different cellular levels to regulate gene expression. It is involved in the control of the cell cycle, chromatin modification, activation and inhibition of transcription initiation, control of transcription elongation, RNA export, and nuclear RNA surveillance; the Ccr4–Not complex also plays a central role in the regulation of mRNA decay. Growing evidence suggests that gene transcription has a vital role in shaping the landscape of genome replication and is also a potent source of replication stress and genome instability. Here, we have examined the effects of the inactivation of the Ccr4–Not complex, via the depletion of the scaffold subunit CNOT1, on DNA replication and genome integrity in mammalian cells. In CNOT1-depleted cells, the elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which, together with R-loop accumulation, results in replication fork slowing, DNA damage, and senescence. Furthermore, we have shown that the stability of TBP mRNA increases in the absence of CNOT1, which may explain its elevated protein expression in CNOT1-depleted cells. Finally, we have shown the activation of mitogen-activated protein kinase signalling as evidenced by ERK1/2 phosphorylation in the absence of CNOT1, which may be responsible for the observed cell cycle arrest at the border of G1/S

Degradation of a novel DNA damage response protein, tankyrase 1 binding protein 1, following adenovirus infection

ABSTRACT Infection by most DNA viruses activates a cellular DNA damage response (DDR), which may be to the detriment or advantage of the virus. In the case of adenoviruses, they neutralize antiviral effects of DDR activation by targeting a number of proteins for rapid proteasome-mediated degradation. We have now identified a novel DDR protein, tankyrase 1 binding protein 1 (TNKS1BP1) (also known as Tab182), which is degraded during infection by adenovirus serotype 5 and adenovirus serotype 12. In both cases, degradation requires the action of the early region 1B55K (E1B55K) and early region 4 open reading frame 6 (E4orf6) viral proteins and is mediated through the proteasome by the action of cullin-based cellular E3 ligases. The degradation of Tab182 appears to be serotype specific, as the protein remains relatively stable following infection with adenovirus serotypes 4, 7, 9, and 11. We have gone on to confirm that Tab182 is an integral component of the CNOT complex, which has transcriptional regulatory, deadenylation, and E3 ligase activities. The levels of at least 2 other members of the complex (CNOT3 and CNOT7) are also reduced during adenovirus infection, whereas the levels of CNOT4 and CNOT1 remain stable. The depletion of Tab182 with small interfering RNA (siRNA) enhances the expression of early region 1A proteins (E1As) to a limited extent during adenovirus infection, but the depletion of CNOT1 is particularly advantageous to the virus and results in a marked increase in the expression of adenovirus early proteins. In addition, the depletion of Tab182 and CNOT1 results in a limited increase in the viral DNA level during infection. We conclude that the cellular CNOT complex is a previously unidentified major target for adenoviruses during infection. IMPORTANCE Adenoviruses target a number of cellular proteins involved in the DNA damage response for rapid degradation. We have now shown that Tab182, which we have confirmed to be an integral component of the mammalian CNOT complex, is degraded following infection by adenovirus serotypes 5 and 12. This requires the viral E1B55K and E4orf6 proteins and is mediated by cullin-based E3 ligases and the proteasome. In addition to Tab182, the levels of other CNOT proteins are also reduced during adenovirus infection. Thus, CNOT3 and CNOT7, for example, are degraded, whereas CNOT4 and CNOT1 are not. The siRNA-mediated depletion of components of the complex enhances the expression of adenovirus early proteins and increases the concentration of viral DNA produced during infection. This study highlights a novel protein complex, CNOT, which is targeted for adenovirus-mediated protein degradation. To our knowledge, this is the first time that the CNOT complex has been identified as an adenoviral target. </jats:p

The Regulatory Properties of the Ccr4–Not Complex

The mammalian Ccr4&ndash;Not complex, carbon catabolite repression 4 (Ccr4)-negative on TATA-less (Not), is a large, highly conserved, multifunctional assembly of proteins that acts at different cellular levels to regulate gene expression. In the nucleus, it is involved in the regulation of the cell cycle, chromatin modification, activation and inhibition of transcription initiation, control of transcription elongation, RNA export, nuclear RNA surveillance, and DNA damage repair. In the cytoplasm, the Ccr4&ndash;Not complex plays a central role in mRNA decay and affects protein quality control. Most of our original knowledge of the Ccr4&ndash;Not complex is derived, primarily, from studies in yeast. More recent studies have shown that the mammalian complex has a comparable structure and similar properties. In this review, we summarize the evidence for the multiple roles of both the yeast and mammalian Ccr4&ndash;Not complexes, highlighting their similarities

The Promotion of Genomic Instability in Human Fibroblasts by Adenovirus 12 Early Region 1B 55K Protein in the Absence of Viral Infection

The adenovirus 12 early region 1B55K (Ad12E1B55K) protein has long been known to cause non-random damage to chromosomes 1 and 17 in human cells. These sites, referred to as Ad12 modification sites, have marked similarities to classic fragile sites. In the present report we have investigated the effects of Ad12E1B55K on the cellular DNA damage response and on DNA replication, considering our increased understanding of the pathways involved. We have compared human skin fibroblasts expressing Ad12E1B55K (55K(+)HSF), but no other viral proteins, with the parental cells. Appreciable chromosomal damage was observed in 55K(+)HSFs compared to parental cells. Similarly, an increased number of micronuclei was observed in 55K(+)HSFs, both in cycling cells and after DNA damage. We compared DNA replication in the two cell populations; 55K(+)HSFs showed increased fork stalling and a decrease in fork speed. When replication stress was introduced with hydroxyurea the percentage of stalled forks and replication speeds were broadly similar, but efficiency of fork restart was significantly reduced in 55K(+)HSFs. After DNA damage, appreciably more foci were formed in 55K(+)HSFs up to 48 h post treatment. In addition, phosphorylation of ATM substrates was greater in Ad12E1B55K-expressing cells following DNA damage. Following DNA damage, 55K(+)HSFs showed an inability to arrest in cell cycle, probably due to the association of Ad12E1B55K with p53. To confirm that Ad12E1B55K was targeting components of the double-strand break repair pathways, co-immunoprecipitation experiments were performed which showed an association of the viral protein with ATM, MRE11, NBS1, DNA-PK, BLM, TOPBP1 and p53, as well as with components of the replisome, MCM3, MCM7, ORC1, DNA polymerase δ, TICRR and cdc45, which may account for some of the observed effects on DNA replication. We conclude that Ad12E1B55K impacts the cellular DNA damage response pathways and the replisome at multiple points through protein–protein interactions, causing genomic instability