Increased tRNA modification and gene-specific codon usage regulate cell cycle progression during the DNA damage response

S-phase and DNA damage promote increased ribonucleotide reductase (RNR) activity. Translation of RNR1 has been linked to the wobble uridine modifying enzyme tRNA methyltransferase 9 (Trm9). We predicted that changes in tRNA modification would translationally regulate RNR1 after DNA damage to promote cell cycle progression. In support, we demonstrate that the Trm9-dependent tRNA modification 5-methoxycarbonylmethyluridine (mcm?U) is increased in hydroxyurea (HU)-induced S-phase cells, relative to G? and G?, and that mcm?U is one of 16 tRNA modifications whose levels oscillate during the cell cycle. Codon-reporter data matches the mcm?U increase to Trm9 and the efficient translation of AGA codons and RNR1. Further, we show that in trm9Δ cells reduced Rnr1 protein levels cause delayed transition into S-phase after damage. Codon re-engineering of RNR1 increased the number of trm9Δ cells that have transitioned into S-phase 1 h after DNA damage and that have increased Rnr1 protein levels, similar to that of wild-type cells expressing native RNR1. Our data supports a model in which codon usage and tRNA modification are regulatory components of the DNA damage response, with both playing vital roles in cell cycle progression.     

DNA damage signaling triggers the cytoplasm-to-vacuole pathway of autophagy to regulate cell cycle progression

Budding yeast cells suffering a single unrepaired DNA double-strand break (DSB) trigger the ATR (Mec1)-dependent DNA damage checkpoint and arrest prior to anaphase for 12–15 h, following which they adapt and resume cell division. When the DNA lesion can be repaired, the checkpoint is extinguished and cells “recover” and resume mitosis. In this autophagic punctum, we report that hyperactivation of autophagy—specifically via the cytoplasm-to-vacuole targeting (Cvt) pathway—prevents both adaptation to, and recovery from, DNA damage, resulting in the permanent arrest of cells in G₂/M. We show that Saccharomyces cerevisiae deleted for genes encoding the Golgi-associated retrograde protein transport (GARP) complex are both adaptation- and recovery-defective. GARP mutants such as vps51Δ exhibit mislocalization of the key mitotic regulator, securin (Pds1), and its degradation by the vacuolar protease Prb1. In addition, separase (Esp1), is excluded from the nucleus, accounting for pre-anaphase arrest. Pds1 is degraded via the Cvt pathway. Many of the same defects seen by deleting GARP genes can be mimicked by hyperactivation of the Cvt pathway by overexpressing an unphosphorylatable form of ATG13 or by adding the TORC1 inhibitor rapamycin. These results suggest that nuclear events such as DNA damage can have profound effects on cytoplasmic processes and further expand the burgeoning connections between DNA damage and autophagy.     

APC/CCdh1 controls CtIP stability during the cell cycle and in response to DNA damage

Human cells have evolved elaborate mechanisms for responding to DNA damage to maintain genome stability and prevent carcinogenesis. For instance, the cell cycle can be arrested at different stages to allow time for DNA repair. The APC/C^Cdh1 ubiquitin ligase mainly regulates mitotic exit but is also implicated in the DNA damage‐induced G₂ arrest. However, it is currently unknown whether APC/C^Cdh1 also contributes to DNA repair. Here, we show that Cdh1 depletion causes increased levels of genomic instability and enhanced sensitivity to DNA‐damaging agents. Using an integrated proteomics and bioinformatics approach, we identify CtIP, a DNA‐end resection factor, as a novel APC/C^Cdh1 target. CtIP interacts with Cdh1 through a conserved KEN box, mutation of which impedes ubiquitylation and downregulation of CtIP both during G₁ and after DNA damage in G₂. Finally, we find that abrogating the CtIP–Cdh1 interaction results in delayed CtIP clearance from DNA damage foci, increased DNA‐end resection, and reduced homologous recombination efficiency. Combined, our results highlight the impact of APC/C^Cdh1 on the maintenance of genome integrity and show that this is, at least partially, achieved by controlling CtIP stability in a cell cycle‐ and DNA damage‐dependent manner.     

Break dosage, cell cycle stage and DNA replication influence DNA double strand break response

DNA double strand breaks (DSBs) can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HR). HR requires nucleolytic degradation of 5' DNA ends to generate tracts of single-stranded DNA (ssDNA), which are also important for the activation of DNA damage checkpoints. Here we describe a quantitative analysis of DSB processing in the budding yeast Saccharomyces cerevisiae. We show that resection of an HO endonuclease-induced DSB is less extensive than previously estimated and provide evidence for significant instability of the 3' ssDNA tails. We show that both DSB resection and checkpoint activation are dose-dependent, especially during the G1 phase of the cell cycle. During G1, processing near the break is inhibited by competition with NHEJ, but extensive resection is regulated by an NHEJ-independent mechanism. DSB processing and checkpoint activation are more efficient in G2/M than in G1 phase, but are most efficient at breaks encountered by DNA replication forks during S phase. Our findings identify unexpected complexity of DSB processing and its regulation, and provide a framework for further mechanistic insights.     

The tRNA cycle and its relation to the rate of protein synthesis

With the aid of a kinetic model, we have investigated how the adaptation between the various components of the tRNA cycle and the codon frequencies affects the rate of protein synthesis. Depending on the relative amounts of total tRNA, synthetase and ribosomes, the optimal correlations vary between a situation where all tRNA species are either present in equal amounts or are present in amounts proportional to the square-root of the corresponding codon frequencies, and a situation where the amounts of the different tRNA species present are linearly proportional to the codon frequencies.Abbreviations EFTu Elongation factor Tu     

Genes adopt non‐optimal codon usage to generate cell cycle‐dependent oscillations in protein levels

The cell cycle is a temporal program that regulates DNA synthesis and cell division. When we compared the codon usage of cell cycle‐regulated genes with that of other genes, we discovered that there is a significant preference for non‐optimal codons. Moreover, genes encoding proteins that cycle at the protein level exhibit non‐optimal codon preferences. Remarkably, cell cycle‐regulated genes expressed in different phases display different codon preferences. Here, we show empirically that transfer RNA (tRNA) expression is indeed highest in the G2 phase of the cell cycle, consistent with the non‐optimal codon usage of genes expressed at this time, and lowest toward the end of G1, reflecting the optimal codon usage of G1 genes. Accordingly, protein levels of human glycyl‐, threonyl‐, and glutamyl‐prolyl tRNA synthetases were found to oscillate, peaking in G2/M phase. In light of our findings, we propose that non‐optimal (wobbly) matching codons influence protein synthesis during the cell cycle. We describe a new mathematical model that shows how codon usage can give rise to cell‐cycle regulation. In summary, our data indicate that cells exploit wobbling to generate cell cycle‐dependent dynamics of proteins.     

Graded requirement for the spliceosome in cell cycle progression

Genome stability is ensured by multiple surveillance mechanisms that monitor the duplication, segregation, and integrity of the genome throughout the cell cycle. Depletion of components of the spliceosome, a macromolecular machine essential for mRNA maturation and gene expression, has been associated with increased DNA damage and cell cycle defects. However, the specific role for the spliceosome in these processes has remained elusive, as different cell cycle defects have been reported depending on the specific spliceosome subunit depleted. Through a detailed cell cycle analysis after spliceosome depletion, we demonstrate that the spliceosome is required for progression through multiple phases of the cell cycle. Strikingly, the specific cell cycle phenotype observed after spliceosome depletion correlates with the extent of depletion. Partial depletion of a core spliceosome component results in defects at later stages of the cell cycle (G2 and mitosis), whereas a more complete depletion of the same component elicits an early cell cycle arrest in G1. We propose a quantitative model in which different functional dosages of the spliceosome are required for different cell cycle transitions.     

Chromatin modulation and the DNA damage response

The ability to sense and respond appropriately to genetic lesions is vitally important to maintain the integrity of the genome. Emerging evidence indicates that various modulations to chromatin structure are centrally important to many aspects of the DNA damage response (DDR). Here, we discuss recently described roles for specific post-translational covalent modifications to histone proteins, as well as ATP-dependent chromatin remodelling, in DNA damage signalling and repair of DNA double strand breaks.     

组蛋白翻译后修饰与DNA损伤响应蛋白招募的调控

组蛋白翻译后修饰是细胞DNA损伤早期应答反应的重要内涵,一方面是松弛、开放染色质结构的必要分子调节事件,以便DNA损伤响应蛋白能接近DNA损伤位点;另一方面直接参与DNA损伤修复蛋白招募过程的调控。综述了在DNA损伤信号激发下,发生的组蛋白主要修饰类型,异组蛋白H2AX、H2A.Z在DNA损伤部位与组蛋白置换,及其对DNA损伤响应蛋白招募的调节作用和机制。     

Optimization protein productivity of human interleukin-2 through codon usage,gene copy number and intracellular tRNA concentration in CHO cells

Transfer RNA (tRNA) abundance is one of the critical factors for the enhancement of protein productivity in prokaryotic and eukaryotic hosts. Gene copy number of tRNA and tRNA codon usage bias are generally used to match tRNA abundance of protein-expressing hosts and to optimize the codons of recombinant proteins. Because sufficient concentration of intracellular tRNA and optimized codons of recombinant proteins enhanced translation efficiency, we hypothesized that sufficient supplement of host’s tRNA improved protein productivity in mammalian cells. First, the small tRNA sequencing results of CHO-K1 cells showed moderate positive correlation with gene copy number and codon usage bias. Modification of human interleukin-2 (IL-2) through codons with high gene copy number and high codon usage bias (IL-2 HH, modified on Leu, Thr, Glu) significantly increased protein productivity in CHO-K1 cells. In contrast, modification through codons with relatively high gene copy number and low codon usage bias (IL-2 HL, modified on Ala, Thr, Val), or relatively low gene copy number and low codon usage bias (IL-2 LH, modified on Ala, Thr, Val) did not increase IL-2 productivity significantly. Furthermore, supplement of the alanine tRNA or threonine tRNA increased IL-2 productivity of IL-2 HL. In summary, we revealed a potential strategy to enhance productivity of recombinant proteins, which may be applied in production of protein drug or design of DNA vaccine.     

The heterogenic final cell cycle of chicken retinal Lim1 horizontal cells is not regulated by the DNA damage response pathway

Cells with aberrations in chromosomal ploidy are normally removed by apoptosis. However, aneuploid neurons have been shown to remain functional and active both in the cortex and in the retina. Lim1 horizontal progenitor cells in the chicken retina have a heterogenic final cell cycle, producing some cells that enter S-phase without proceeding into M-phase. The cells become heteroploid but do not undergo developmental cell death. This prompted us to investigate if the final cell cycle of these cells is under the regulation of an active DNA damage response. Our results show that the DNA damage response pathway, including γ-H2AX and Rad51 foci, is not triggered during any phase of the different final cell cycles of horizontal progenitor cells. However, chemically inducing DNA adducts or double-strand breaks in Lim1 horizontal progenitor cells activated the DNA damage response pathway, showing that the cells are capable of a functional response to DNA damage. Moreover, manipulation of the DNA damage response pathway during the final cell cycle using inhibitors of ATM/ATR, Chk1/2, and p38MAPK, neither induced apoptosis nor mitosis in the Lim1 horizontal progenitor cells. We conclude that the DNA damage response pathway is functional in the Lim1 horizontal progenitor cells, but that it is not directly involved in the regulation of the final cell cycle that gives rise to the heteroploid horizontal cell population.     

SOCS3 regulates p21 expression and cell cycle arrest in response to DNA damage

Genotoxic agents such as ionizing radiation trigger cell cycle arrest at the G1/S and G2/M checkpoints, allowing cells to repair damaged DNA before entry into mitosis. DNA damage-induced G1 arrest involves p53-dependent expression of p21 (Cip1/Waf-1), which inhibits cyclin-dependent kinases and blocks S phase entry. While much of the core DNA damage response has been well-studied, other signaling proteins that intersect with and modulate this response remain uncharacterized. In this study, we identify Suppressor of Cytokine Signaling (SOCS)-3 as an important regulator of radiation-induced G1 arrest. SOCS3-deficient fibroblasts fail to undergo G1 arrest and accumulate in the G2/M phase of the cell cycle. SOCS3 knockout cells phosphorylate p53 and H2AX normally in response to radiation, but fail to upregulate p21 expression. In addition, STAT3 phosphorylation is elevated in SOCS3-deficient cells compared to WT cells. Normal G1 arrest can be restored in SOCS3 KO cells by retroviral transduction of WT SOCS3 or a dominant-negative mutant of STAT3. Our results suggest a novel function for SOCS3 in the control of genome stability by negatively regulating STAT3-dependent radioresistant DNA synthesis, and promoting p53-dependent p21 expression.     

Ubiquitin and the DNA damage response

Comment on: Gatti M, et al. Cell Cycle 2012; 11:2538-44.     

Listeria monocytogenes induces host DNA damage and delays the host cell cycle to promote infection

Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation.     

Studying the DNA damage response using in vitro model systems

Exogenous and endogenous insults continuously damage DNA. DNA damage must be detected in order to prevent loss of vital genetic information. Cells respond to DNA damage by activating checkpoint pathways that delay the progression through the cell cycle, promote DNA repair or induce cell death. A regulatory network of proteins has been identified that participate in DNA damage checkpoint pathways. Central to this network are ATM, ATR and the Mre11/Rad50/Nbs1 (MRN) complex. Detailed biochemical analysis of ATM, ATR and the MRN dependent DNA damage responses has taken advantage of several in vitro model systems to understand the detailed mechanisms underlying their function. Here we describe some recent findings obtained analysing these pathways using in vitro model systems. In particular we focus on the studies performed in the Xenopus laevis egg cell free extract, which recapitulates the DNA damage response in the context of the cell cycle.     

DNA damage response by single-strand breaks in terminally differentiated muscle cells and the control of muscle integrity

DNA single-strand breaks (SSB) formation coordinates the myogenic program, and defects in SSB repair in post-mitotic cells have been associated with human diseases. However, the DNA damage response by SSB in terminally differentiated cells has not been explored yet. Here we show that mouse post-mitotic muscle cells accumulate SSB after alkylation damage, but they are extraordinarily resistant to the killing effects of a variety of SSB-inducers. We demonstrate that, upon SSB induction, phosphorylation of H2AX occurs in myotubes and is largely ataxia telangiectasia mutated (ATM)-dependent. However, the DNA damage signaling cascade downstream of ATM is defective as shown by lack of p53 increase and phosphorylation at serine 18 (human serine 15). The stabilization of p53 by nutlin-3 was ineffective in activating the cell death pathway, indicating that the resistance to SSB inducers is due to defective p53 downstream signaling. The induction of specific types of damage is required to activate the cell death program in myotubes. Besides the topoisomerase inhibitor doxorubicin known for its cardiotoxicity, we show that the mitochondria-specific inhibitor menadione is able to activate p53 and to kill effectively myotubes. Cell killing is p53-dependent as demonstrated by full protection of myotubes lacking p53, but there is a restriction of p53-activated genes. This new information may have important therapeutic implications in the prevention of muscle cell toxicity.