Preserving genome integrity and function: the DNA damage response and histone modifications

Modulation of chromatin templates in response to cellular cues, including DNA damage, relies heavily on the post-translation modification of histones. Numerous types of histone modifications including phosphorylation, methylation, acetylation, and ubiquitylation occur on specific histone residues in response to DNA damage. These histone marks regulate both the structure and function of chromatin, allowing for the transition between chromatin states that function in undamaged condition to those that occur in the presence of DNA damage. Histone modifications play well-recognized roles in sensing, processing, and repairing damaged DNA to ensure the integrity of genetic information and cellular homeostasis. This review highlights our current understanding of histone modifications as they relate to DNA damage responses (DDRs) and their involvement in genome maintenance, including the potential targeting of histone modification regulators in cancer, a disease that exhibits both epigenetic dysregulation and intrinsic DNA damage.     

同卵双胞胎在复杂性状DNA甲基化调控机制研究中的应用

同卵双胞胎来源于同一个受精卵,DNA序列基本一致,但在某些重要表型上如复杂疾病,并不完全一样。利用表型不一致的同卵双胞胎进行研究,能在遗传背景、母体效应、年龄性别效应等一致的基础上,深入研究分析复杂性状的表观调控机制。而DNA甲基化是最为稳定的一类表观遗传修饰。在人类中,利用同卵双胞胎对印记异常疾病、精神类疾病、自身免疫病及癌症等疾病的DNA甲基化调控研究已经揭示了多个致病基因,为研究疾病的表观调控以及表观遗传学药物的应用打下了基础。本文着重对同卵双胞胎DNA甲基化状态、DNA甲基化遗传力计算以及复杂性状DNA甲基化调控的研究应用及其进展展开综述,以期为复杂性状表观调控机制研究提供借鉴和参考。     

Methylation status of Sillago japonica satellite DNA examined by bisulfite modification

A member of Sillago japonica satellite DNA contained internal subrepeats in its 174 bp unit. S. Japonica genomic DNA isolated from liver tissue was subjected to bisulfite modification, and the DNA sequences of about 40 bp flanked by both subrepeats were amplified by polymerase chain reaction (PCR). This protocol, combination of bisulfite reaction and PCR, converts cytosines in the genomic DNA to thymines in the amplified DNA, whereas 5-methylcytosines in the genomic DNA remain as cytosines. Sequence analysis of the amplified DNA fragments revealed that most of the cytosine residues at CpG were methylated in this region.     

乙酰转移酶MYST家族的生物学功能

组蛋白乙酰化是表观遗传修饰的重要方式,主要受到组蛋白乙酰转移酶（histone acetyltransferases, HATs)和组蛋白去乙酰化酶（histone deacetylase, HDACs）催化. MYST是人类HATs的4大家族之一,包括MOF(males absent on the first),TIP60 (tat interacting protein 60 kD),结合ORC1的组蛋白乙酰转移酶（histone acetyltransferase binding to ORC1, HBO1),单核细胞白血病锌指蛋白(monocytic leukemia zinc finger protein, MOZ)和MOZ相关蛋白（MOZ related factor, MORF）等,均具有典型的MYST结构域.MYST介导的乙酰化是重要的翻译后修饰,其催化底物包括组蛋白和非组蛋白,如组蛋白H3, H4, H2A, H2A突变体,以及许多参与DNA代谢、细胞增殖和发育调控的蛋白因子. MYST蛋白家族参与许多细胞的生理过程,本文主要综述其在调节基因转录、DNA损伤修复和肿瘤发生发展等方面的生物学功能.     

Cloning and expression of the Apa LI, Nsp I, Nsp HI, Sac I, Sca I, and Sap I restriction-modification systems in Escherichia coli

The genes encoding the ApaLI (5′-G^TGCAC-3′), NspI (5′-RCATG^Y-3′), NspHI (5′-RCATG^Y-3′), SacI (5′-GAGCT^C-3′), SapI (5′-GCTCTTCN1^-3′, 5′-^N4GAAGAGC-3′) and ScaI (5′-AGT^ACT-3′) restriction-modification systems have been cloned in E.␣coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5′-CATG-3′) restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5′-GCTCTTC-3′ blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene. Received: 15 April 1998 / Accepted: 3 August 1998     

Adenine methylation in eukaryotes: Apprehending the complex evolutionary history and functional potential of an epigenetic modification

While N⁶‐methyladenosine (m⁶A) is a well‐known epigenetic modification in bacterial DNA, it remained largely unstudied in eukaryotes. Recent studies have brought to fore its potential epigenetic role across diverse eukaryotes with biological consequences, which are distinct and possibly even opposite to the well‐studied 5‐methylcytosine mark. Adenine methyltransferases appear to have been independently acquired by eukaryotes on at least 13 occasions from prokaryotic restriction‐modification and counter‐restriction systems. On at least four to five instances, these methyltransferases were recruited as RNA methylases. Thus, m⁶A marks in eukaryotic DNA and RNA might be more widespread and diversified than previously believed. Several m⁶A‐binding protein domains from prokaryotes were also acquired by eukaryotes, facilitating prediction of potential readers for these marks. Further, multiple lineages of the AlkB family of dioxygenases have been recruited as m⁶A demethylases. Although members of the TET/JBP family of dioxygenases have also been suggested to be m⁶A demethylases, this proposal needs more careful evaluation. Also watch the Video Abstract .     

Synthesis of NSC 106084 and NSC 14778 and evaluation of their DNMT inhibitory activity

DNA methylation is an epigenetic modification that is performed by DNA methyltransferases (DNMTs) and that leads to the transfer of a methyl group from S-adenosylmethionine (SAM) to the C5 position of cytosine. This transformation results in hypermethylation and silencing of genes such as tumor suppressor genes. Aberrant DNA methylation has been associated with the development of many diseases, including cancer. Inhibition of DNMTs promotes the demethylation and reactivation of epigenetically silenced genes. NSC 106084 and 14778 have been reported to inhibit DNMTs in the micromolar range. We report herein the synthesis of NSC 106084 and 14778 and the evaluation of their DNMT inhibitory activity. Our results indicate that while commercial NSC 14778 is moderately active against DNMT1, 3A/3L and 3B/3L, resynthesized NSC 14778 is inactive under our assay conditions. Resynthesized 106084 was also found to be inactive.     

Rapid step-gradient purification of mitochondrial DNA

A convenient modification of the step gradient (CsCl/ethidium bomide) procedure is described. This rapid method allows isolation of covalently closed circular DNA separated from contaminating proteins, RNA and chromosomal DNA in ca. 5 h. Large scale preparations can be performed for circular DNA from eukaryotic organelles (mitochondria). The protocol uses organelle pelleting/NaCl-sarcosyl incubation steps for mitochondria followed by a CsCl step gradient and exhibits yields equal to the conventional procedures. It results in DNA sufficiently pure to be used for restriction endonuclease analysis, subcloning, 5-end labeling, gel retention assays, and various types of hybridization.     

Twenty years hunting for sulfur in DNA

Here we tell a 20-year long story. It began with an easily overlooked DNA degradation (Dnd) phenomenon during electrophoresis and eventually led to the discovery of an unprecedented DNA sulfur modification governed by five dnd genes. This unusual DNA modification, called phosphorothioation, is the first physiological modification identified on the DNA backbone, in which the nonbridging oxygen is replaced by sulfur in a sequence selective and stereo-specific manner. Homologous dnd gene clusters have been identified in diverse and distantly related bacteria and thus have drawn immediate attention of the entire microbial scientific community. Here, we summarize the progress in chemical, genetic, enzymatic, bioinformatical and analytical aspects of this novel postreplicative DNA modification. We also discuss perspectives on the physiological functions of the DNA phosphorothioate modification in bacteria and their implications.     

A Comparative Study of the Modification Efficiency of DNA Polymerases and DNA Template by the DNA Primers with Various Photoreactive Groups at Their 3"-Termini

The dependence of the modification efficiency of DNA polymerases and DNA template on the nature of photoreactive group and the length of the linker that joins the group with the heterocyclic base of the primer 3"-terminal nucleotide was studied. The primers that contained the photoreactive groups at their 3"-termini were obtained using the rat DNA polymerase or the DNA polymerase from Thermus thermophilus in the presence of one of the dTTP analogues carrying the photoreactive group in position 5 of thymidine residue. After irradiating the reaction mixture with UV light and separating the modification products, the level of covalent attachment of the [5"-³²P]primer to DNA polymerases and template was determined. The primers containing 4-azido-2,5-difluoro-3-chloropyridyl group were shown to be the most effective in the modification of DNA polymerases.     

In vitro DNA binding activity and molecular docking reveals pierisin-5 as an anti-proliferative agent against gastric cancer

Abstract

Pierisin-5 is a DNA dependent ADP ribosyltransferase (ADRT) protein from the larvae of Indian cabbage white butterfly, Pieris canidia. Interestingly, Pierisin-5 ADP-ribosylates the DNA as a substrate, but not the protein and subsequently persuades apoptotic cell death in human cancer cells. This has led to the investigation on the DNA binding activity of Pierisin-5 using in vitro and in silico approaches in the present study. However, both the structure and the mechanism of ADP-ribosylation of pierisin-5 are unknown. In silico modeled structure of the N-terminal ADRT catalytic domain interacted with the minor groove of B-DNA for ribosylation with the help of β-NAD⁺ which lead to a structural modification in DNA (DNA adduct). The possible interaction between calf thymus DNA (CT-DNA) and purified pierisin-5 protein was studied through spectral–spatial studies and the blue shift and hyperchromism in the UV–Visible spectra was observed. The DNA adduct property of pierisin-5 protein was validated by in vitro cytotoxic assay on human gastric (AGS) cancer cell lines. Our study is the first report of the mechanism of DNA binding property of pierisin-5 protein which leads to the induction of cytotoxicity and apoptotic cell death against cancer cell lines.

Communicated by Ramaswamy H. Sarma     

DNA甲基转移酶的表达调控及主要生物学功能

DNA甲基化是表观遗传学的重要部分, 同组蛋白修饰相互作用, 通过改变染色质结构, 调控基因表达。在哺乳类细胞或人体细胞中, DNA甲基化与细胞的增殖、衰老、癌变等生命现象有着重大关系。对催化DNA甲基化的DNA甲基转移酶(DNA methyltransferase, Dnmt)的研究可以揭示DNA甲基化对基因表达调控的机制, 从而研究与之相关的重要生命活动。文章以DNA甲基转移酶作为切入点, 探讨DNA甲基转移酶在基因表达调控中发挥的作用及其主要生物学功能。     

Novel, fluorescent, SSB protein chimeras with broad utility

The Escherichia coli single-stranded DNA binding protein (SSB) is a central player in DNA metabolism where it organizes genome maintenance complexes and stabilizes single-stranded DNA (ssDNA) intermediates generated during DNA processing. Due to the importance of SSB and to facilitate real-time studies, we developed a dual plasmid expression system to produce novel, chimeric SSB proteins. These chimeras, which contain mixtures of histidine-tagged and fluorescent protein(FP)-fusion subunits, are easily purified in milligram quantities and used without further modification, a significant enhancement over previous methods to produce fluorescent SSB. Chimeras retain the functionality of wild type in all assays, demonstrating that SSB function is unaffected by the FPs. We demonstrate the power and utility of these chimeras in single molecule studies providing a great level of insight into the biochemical mechanism of RecBCD. We also utilized the chimeras to show for the first time that RecG and SSB interact in vivo. Consequently, we anticipate that the chimeras described herein will facilitate in vivo, in vitro and single DNA molecule studies using proteins that do not require further modification prior to use.     

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].