Regulation of Cellular Senescence by Polycomb Chromatin Modifiers through Distinct DNA Damage- and Histone Methylation-Dependent Pathways

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Structural Basis for EPC1-Mediated Recruitment of MBTD1 into the NuA4/TIP60 Acetyltransferase Complex

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细胞衰老与衰老细胞

细胞衰老(cellular senescence)是一个应激导致细胞生长停滞的生理过程．一部分发生衰老的细胞会被机体自身清除,但另一些衰老的细胞会随着时间的推移在体内积累增多,并分泌一些免疫刺激因子,导致低水平炎症发生,引起周围组织衰老或癌变,这类具有特殊生物学特征和功能的细胞就是衰老细胞(senescent cell)．实验揭示,衰老细胞不仅是衰老过程的产物,也可能是组织器官进一步衰退的重要原因．近日,Baker等的一项研究成果(Nature,2011,479(7372)：232-236)表明,清除衰老细胞可延缓小鼠的衰老进程,该成果有望开辟出一条对抗衰老的新途径．     

NCAPH Stabilizes GEN1 in Chromatin to Resolve Ultra-Fine DNA Bridges and Maintain Chromosome Stability

Repairing damaged DNA and removing all physical connections between sister chromosomes is important to ensure proper chromosomal segregation by contributing to chromosomal stability. Here, we show that the depletion of non-SMC condensin I complex subunit H (NCAPH) exacerbates chromosome segregation errors and cytokinesis failure owing to sister-chromatid intertwinement, which is distinct from the ultra-fine DNA bridges induced by DNA inter-strand crosslinks (DNA-ICLs). Importantly, we identified an interaction between NCAPH and GEN1 in the chromatin involving binding at the N-terminus of NCAPH. DNA-ICL activation, using ICL-inducing agents, increased the expression and interaction between NCAPH and GEN1 in the soluble nuclear and chromatin, indicating that the NCAPH–GEN1 interaction participates in repairing DNA damage. Moreover, NCAPH stabilizes GEN1 within chromatin at the G2/M-phase and is associated with DNA-ICL-induced damage repair. Therefore, NCAPH resolves DNA-ICL-induced ultra-fine DNA bridges by stabilizing GEN1 and ensures proper chromosome separation and chromosome structural stability.     

The NuA4 Complex Promotes Translesion Synthesis (TLS)-Mediated DNA Damage Tolerance

Lesions in DNA can block replication fork progression, leading to its collapse and gross chromosomal rearrangements. To circumvent such outcomes, the DNA damage tolerance (DDT) pathway becomes engaged, allowing the replisome to bypass a lesion and complete S phase. Chromatin remodeling complexes have been implicated in the DDT pathways, and here we identify the NuA4 remodeler, which is a histone acetyltransferase, to function on the translesion synthesis (TLS) branch of DDT. Genetic analyses in Saccharomyces cerevisiae showed synergistic sensitivity to MMS when NuA4 alleles, esa1-L254P and yng2Δ, were combined with the error-free bypass mutant ubc13Δ. The loss of viability was less pronounced when NuA4 complex mutants were disrupted in combination with error-prone/TLS factors, such as rev3Δ, suggesting an epistatic relationship between NuA4 and error-prone bypass. Consistent with cellular viability measurements, replication profiles after exposure to MMS indicated that small regions of unreplicated DNA or damage were present to a greater extent in esa1-L254P/ubc13Δ mutants, which persist beyond the completion of bulk replication compared to esa1-L254P/rev3Δ. The critical role of NuA4 in error-prone bypass is functional even after the bulk of replication is complete. Underscoring this observation, when Yng2 expression is restricted specifically to G2/M of the cell cycle, viability and TLS-dependent mutagenesis rates were restored. Lastly, disruption of HTZ1, which is a target of NuA4, also resulted in mutagenic rates of reversion on level with esa1-L254P and yng2Δ mutants, indicating that the histone variant H2A.Z functions in vivo on the TLS branch of DDT.     

衰老过程中大白鼠脾细胞的DNA单链断裂与重接能力的变化

 DNA被紫外线损伤后,由DNA切除修复酶切除嘧啶二聚体,随之以另一条正常的DNA链为模板修复合成DNA片段,最后由DNA连接酶将新合成的DNA片与原有的DNA链连接。本文用荧光法测定DNA修复过程中DNA单链的断裂及重接能力与衰老的关系。结果表明,不同年龄大鼠脾细胞均具有修复DNA单链断裂的能力,DNA单链断裂重接的能力与年龄有相关性,断乳鼠及青年鼠的脾细胞当保温至30min时,即开始了DNA链的重接,保温90min后则恢复到原有水平;而老年鼠脾细胞保温至90min时才开始DNA链的重接,保温150min,尚未恢复到原有水平。还发现,断乳鼠及老年鼠脾细胞的单链DNA含量高于青年鼠。     

DNA损伤监测及修复相关酶与细胞衰老

衰老是细胞的重要生命现象之一,衰老假说之一认为细胞中残留DNA损伤的积累可加速细胞的衰老.因此,细胞内DNA损伤监测及修复系统的正常运行与细胞衰老调控密切相关,DNA损伤监测及修复相关酶如PARP、DNA-PK、ATM、p53等在细胞衰老中的调控作用日益受到广泛关注.研究这些蛋白质分子间的相互作用及其在细胞衰老过程中的调控功能,有利于揭示DNA损伤应激、损伤修复调控与细胞衰老之间的内在联系,为抗衰老研究及从衰老角度治疗肿瘤提供新的思路.     

Nuclear DNA Ligase and Its Action on Chromatin DNA in Neuronal, Glial and Liver Nuclei Isolated from Adult Guinea Pigs

Abstract: DNA ligase activities were measured in neuron-rich and glial nuclear preparations and liver nuclei isolated from adult guinea pigs. The enzymatic properties of cerebral and liver nuclear DNA ligases were studied with isolated nuclei and nuclear extracts. ATP (K_m= 46–48 μM) and bivalent cation (Mg²⁺ or Mn²⁺) were required for the maximal activities in cerebral and liver nuclei. β-Mercaptoethanol did not affect the activities, but N-ethylmaleimide and p-chloromercuribenzoate completely inhibited the activities. Deoxyadenosine-5′-triphosphate partially inhibited the activities in both cerebral and liver nuclei. An interdependent effect of Na⁺ and Mg²⁺ on the enzyme activities was observed. A high concentration (200 mM) of Na⁺ activated both enzymes and shifted to the acid side the optimal pH for both enzymes. DNA ligase was more easily extracted with lower concentrations of NaCl from liver nuclei than from cerebral nuclei, but the extraction curves from both nuclear species reached a plateau level (92% of total activities of nuclear enzymes) at 200 mM-NaCl. Apparent K_m for the substrate [³²P]phosphoryl DNA was determined according to a modification of the Michaelis-Menten equation, which was applied for the case where an unknown amount of substrate nicks in chromatin DNA coexisted with the nicks in exogenous substrate DNA. Neuronal and glial nuclear enzymes had similar K_m values (about 20 μg of [³²P]phosphoryl DNA/ml), but the liver nuclear enzyme had a higher K_m value (54 μg of [³²P]phosphoryl DNA/ml). The modified Michaelis-Menten equation provided the amounts of nicks available as substrate in chromatin DNA of isolated nuclei. Neuronal and glial nuclei contained 1.5 and 0.29 pmol of nicks/μg of nuclear DNA, respectively, in contrast to an intermediate amount of nicks in liver nuclei (0.63 pmol/μg of nuclear DNA). DNA ligase activity in neuronal nuclei [312 units (fmol of 5′-phosphomonoester converted into a phosphatase-resistant form per min at 37°C) per μg of nuclear DNA] was 11-fold higher than that in glial nuclei [28.7 units/μg of nuclear DNA]. Liver nuclei contained an intermediate activity [54.7 units/μg of nuclear DNA].     

NAD^+与细胞衰老

烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD^+)作为糖酵解、三羧酸循环和氧化磷酸化中关键酶的辅助因子,参与了细胞的物质代谢、能量合成、损伤DNA的修复等多种生理病理过程。近年来越来越多的研究发现,细胞内NAD^+水平在机体或细胞衰老过程中呈明显下降趋势,而补充NAD^+能延缓细胞/机体的衰老,使NAD^+及其前体物质在细胞衰老中的作用受到广泛关注。该文就NAD^+及其前体物质与细胞代谢、衰老的关系及相关分子机制研究的最新进展进行综述,以期深入认识NAD^+与细胞衰老的内在联系,为细胞衰老相关的基础及应用研究提供理论参考。     

染色质改构因子BRG1降低UV照射引起的细胞凋亡

生命体的遗传物质基础是DNA分子,多种因素可以作用于细胞内的DNA分子,导致多种类型的DNA损伤。若受损的DNA得不到及时和有效的修复,细胞将走向凋亡或发生变异。染色质改构复合物(chromatin remodeling complex)在基因表达调控和DNA复制等方面扮演着重要角色。依赖ATP的染色质改构复合物SWI/SNF的核心亚基Brahma Related Gene1(BRG1)在染色质结构调整和基因转录调控等多个细胞进程中具有重要作用,仅有有限的文献报道BRG1参与到DNA的损伤修复过程。因此,进一步研究与验证BRG1在调控DNA的损伤修复进而挽救细胞凋亡中的作用十分重要。本文通过利用不同强度的UV照射检测细胞凋亡的情况,初步建立了DNA损伤修复的实验体系。将BRG1表达质粒瞬时转染到SW13(BRG1-/-)细胞系中,并利用30J/m2的UV照射,分别在0h、6h和24h检测细胞早期凋亡程度。结果表明,SW13(BRG1-/-)细胞中瞬时表达BRG1可以明显降低由UV照射引起的细胞凋亡,其中UV照射后24h的细胞表现最明显。我们进一步在HeLa细胞中通过瞬时表达BRG1验证了上述结果。由于BRG1通过染色质改构在基因的转录调控、复制和重组等方面起着重要的作用,我们推测BRG1可能通过染色质改构参与了DNA的损伤修复过程,进而影响了细胞凋亡。     

Cellular osmoregulation: beyond ion transport and cell volume

All cells are characterized by the expression of osmoregulatory mechanisms, although the degree of this expression is highly variable in different cell types even within a single organism. Cellular osmoregulatory mechanisms constitute a conserved set of adaptations that offset antagonistic effects of altered extracellular osmolality/environmental salinity on cell integrity and function. Cellular osmoregulation includes the regulation of cell volume and ion transport but it does not stop there. We know that organic osmolyte concentration, protein structure, cell turnover, and other cellular parameters are osmoregulated as well. In this brief review two important aspects of cellular osmoregulation are emphasized: 1) maintenance of genomic integrity, and 2) the central role of protein phosphorylation. Novel insight into these two aspects of cellular osmoregulation is illustrated based on two cell models, mammalian kidney inner medullary cells and teleost gill epithelial cells. Both cell types are highly hypertonicity stress-resistant and, therefore, well suited for the investigation of osmoregulatory mechanisms. Damage to the genome is discussed as a newly discovered aspect of hypertonic threat to cells and recent insights on how mammalian kidney cells deal with such threat are presented. Furthermore, the importance of protein phosphorylation as a core mechanism of osmosensory signal transduction is emphasized. In this regard, the potential roles of the 14-3-3 family of phospho-protein adaptor molecules for cellular osmoregulation are highlighted primarily based on work with fish gill epithelial cells. These examples were chosen for the reader to appreciate the numerous and highly specific interactions between stressor-specific and non-specific pathways that form an extensive cellular signaling network giving rise to adaptive compensation of hypertonicity. Furthermore, the example of 14-3-3 proteins illustrates that a single protein may participate in several pathways that are non-specific with regard to the type of stress and, at the same time, in stress-specific pathways to promote cell integrity and function during hypertonicity.     

Werner syndrome protein associates with gamma H2AX in a manner that depends upon Nbs1

The WRN protein is mutated in the chromosomally unstable Werner syndrome (WS) and the Nbs1 protein is mutated in Nijmegen breakage syndrome (NBS). The Nbs1 protein is an integral component of the M/R/N complex. Although WRN is known to interact with this complex in response to gamma-irradiation, the mechanism of action is unclear. Here, we show that WRN co-localizes and associates with gamma H2AX, a marker protein of DNA double strand breaks (DSBs), after cellular exposure to gamma-irradiation. While the DNA damage-inducible Nbs1 foci formation is normal in WS cells, WRN focus formation is defective in NBS cells. Consistent with this, gamma H2AX colocalizes with Nbs1 in WS cells but not with WRN in NBS cells. The defective WRN-gamma H2AX association in NBS cells can be complemented with wild-type Nbs1, but not with an Nbs1 S343A point mutant that lacks an ATM phosphorylation site. WRN associates with H2AX in a manner dependent upon the M/R/N complex. Our results suggest a novel pathway in which Nbs1 may recruit WRN to the site of DNA DSBs in an ATM-dependent manner.     

Exonuclease 1 and its versatile roles in DNA repair

Exonuclease 1 (EXO1) is a multifunctional 5′ → 3′ exonuclease and a DNA structure-specific DNA endonuclease. EXO1 plays roles in DNA replication, DNA mismatch repair (MMR) and DNA double-stranded break repair (DSBR) in lower and higher eukaryotes and contributes to meiosis, immunoglobulin maturation, and micro-mediated end-joining in higher eukaryotes. In human cells, EXO1 is also thought to play a role in telomere maintenance. Mutations in the human EXO1 gene correlate with increased susceptibility to some cancers. This review summarizes recent studies on the enzymatic functions and biological roles of EXO1, its possible protective role against cancer and aging, and regulation of EXO1 by posttranslational modification.     

A comprehensive approach to determining BER capacities and their change with aging in Drosophila melanogaster mitochondria by oligonucleotide microarray

DNA repair mechanisms are key components for the maintenance of the essential mitochondrial genome. Among them, base excision repair (BER) processes, dedicated in part to oxidative DNA damage, are individually well known in mitochondria. However, no large view of these systems in differential physiological conditions is available yet. Combining the use of pure mitochondrial fractions and a multiplexed oligonucleotide cleavage assay on a microarray, we demonstrated that a large range of glycosylase activities were present in Drosophila mitochondria. Most of them were quantitatively different from their nuclear counterpart. Moreover, these activities were modified during aging.