Acetyltransferase p300 regulates NBS1-mediated DNA damage response

The role of p300 in DNA damage response is unclear. To understand how ATM-dependent phosphorylation of p300 affects its function in response to DNA damage, we present evidence that S106 of p300, which is phosphorylated by ATM, regulates stability of NBS1 and recruitment into damaged DNA, possibly leading to regulation of DNA repair. Non-phosphorylatable p300 (S106A) destabilized NBS1 and decreased NBS1–p300 interaction. The recruitment of NBS1 into damaged DNA was impaired in the presence of S106A. Also, a dominant negative p300 lacking enzymatic activity induced destabilization of NBS1, suggesting that its acetyltransferase is required for NBS1 stability. These results indicate that phosphorylation of p300 can regulate NBS1-mediated DNA damage response, and that these events occur in an acetylation-dependent manner.

Structured summary

MINT-8058074, MINT-8058083: p300 (uniprotkb:Q09472) physically interacts (MI:0915) with NBS1 (uniprotkb:O60934) by anti bait coimmunoprecipitation (MI:0006)MINT-8058111: p300 (uniprotkb:Q09472) and NBS1 (uniprotkb:O60934) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-8058657: p300 (uniprotkb:Q09472) physically interacts (MI:0915) with NBS1 (uniprotkb:O60934) by two hybrid (MI:0018)MINT-8058093: p300 (uniprotkb:Q09472) physically interacts (MI:0915) with NBS1 (uniprotkb:O60934) by anti tag coimmunoprecipitation (MI:0007)     

DNA base repair – recognition and initiation of catalysis

Endogenous DNA damage induced by hydrolysis, reactive oxygen species and alkylation modifies DNA bases and the structure of the DNA duplex. Numerous mechanisms have evolved to protect cells from these deleterious effects. Base excision repair is the major pathway for removing base lesions. However, several mechanisms of direct base damage reversal, involving enzymes such as transferases, photolyases and oxidative demethylases, are specialized to remove certain types of photoproducts and alkylated bases. Mismatch excision repair corrects for misincorporation of bases by replicative DNA polymerases. The determination of the 3D structure and visualization of DNA repair proteins and their interactions with damaged DNA have considerably aided our understanding of the molecular basis for DNA base lesion repair and genome stability. Here, we review the structural biochemistry of base lesion recognition and initiation of one-step direct reversal (DR) of damage as well as the multistep pathways of base excision repair (BER), nucleotide incision repair (NIR) and mismatch repair (MMR).     

Autophagy regulates DNA repair through SQSTM1/p62

Macroautophagy/autophagy is primarily a degradative pathway that clears malfunctioning cellular components in response to various types of stress. Recent studies have indicated that autophagy also plays an important role in maintaining genome stability. Loss of autophagy is associated with increased damage to DNA, inappropriate amplification of genomic regions and abnormal chromosome number. In a recent paper by Wang et al. the authors uncover a mechanism through which autophagy regulates the ubiquitination of chromatin. In particular, the autophagy receptor and substrate SQSTM1/p62 inhibits the E3 ligase RNF168-dependent ubiquitination of histone in response to DNA double-strand breaks. Dysregulation of this process leads to a reduced ability to repair DNA and a corresponding increase in the sensitivity of cells to radiation-induced damage.     

Discrimination and versatility in mismatch repair

Evolutionarily-conserved mismatch-repair (MMR) systems correct all or almost all base-mismatch errors from DNA replication via excision-resynthesis pathways, and respond to many different DNA lesions. Consideration of DNA polymerase error rates and possible consequences of excess gratuitous excision of perfectly paired (homoduplex) DNA in vivo suggests that MMR needs to discriminate against homoduplex DNA by three to six orders of magnitude. However, numerous binding studies using MMR base-mispair-recognition proteins, bacterial MutS or eukaryotic MSH2.MSH6 (MutSalpha), have typically shown discrimination factors between mismatched and homoduplex DNA to be 5-30, depending on the binding conditions, the particular mismatches, and the DNA-sequence contexts. Thus, downstream post-binding steps must increase MMR discrimination without interfering with the versatility needed to recognize a large variety of base-mismatches and lesions. We use a complex but highly MMR-active model system, human nuclear extracts mixed with plasmid substrates containing specific mismatches and defined nicks 0.15 kbp away, to measure the earliest quantifiable committed step in mismatch correction, initiation of mismatch-provoked 3'-5' excision at the nicks. We compared these results to binding of purified MutSalpha to synthetic oligoduplexes containing the same mismatches in the same sequence contexts, under conditions very similar to those prevailing in the nuclear extracts. Discrimination against homoduplex DNA, only two-to five-fold in the binding studies, increased to 60- to 230-fold or more for excision initiation, depending on the particular mismatches. Remarkably, the mismatch-preference order for excision initiation was substantially altered from the order for hMutSalpha binding. This suggests that post-binding steps not only strongly discriminate against homoduplex DNA, but do so by mechanisms not tightly constrained by initial binding preferences. Pairs of homoduplexes (40, 50, and 70 bp) prepared from synthetic oligomers or cut out of plasmids showed virtually identical hMutSalpha binding affinities, suggesting that high hMutSalpha binding to homoduplex DNA is not the result of misincorporations or lesions introduced during chemical synthesis. Intrinsic affinities of MutS homologs for perfectly paired DNA may help these proteins efficiently position themselves to carry out subsequent mismatch-specific steps in MMR pathways.     

Direct involvement of p53 in the base excision repair pathway of the DNA repair machinery

The p53 tumor suppressor that plays a central role in the cellular response to genotoxic stress was suggested to be associated with the DNA repair machinery which mostly involves nucleotide excision repair (NER). In the present study we show for the first time that p53 is also directly involved in base excision repair (BER). These experiments were performed with p53 temperature-sensitive (ts) mutants that were previously studied in in vivo experimental models. We report here that p53 ts mutants can also acquire wild-type activity under in vitro conditions. Using ts mutants of murine and human origin, it was observed that cell extracts overexpressing p53 exhibited an augmented BER activity measured in an in vitro assay. Depletion of p53 from the nuclear extracts abolished this enhanced activity. Together, this suggests that p53 is involved in more than one DNA repair pathway.     

NEIL2-initiated, APE-independent repair of oxidized bases in DNA: Evidence for a repair complex in human cells

DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3' phospho alpha,beta-unsaturated aldehyde (3' PUA) or 3' phosphate (3' P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3' blocking groups (3' PUA and 3' P) to generate the 3'-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3' PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3' phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3' phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase beta (Pol beta), DNA ligase IIIalpha (Lig IIIalpha) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol beta and Lig IIIalpha. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol beta, Lig IIIalpha and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.     

Active DNA demethylation and DNA repair

DNA methylation on cytosine is an epigenetic modification and is essential for gene regulation and genome stability in vertebrates. Traditionally DNA methylation was considered as the most stable of all heritable epigenetic marks. However, it has become clear that DNA methylation is reversible by enzymatic “active” DNA demethylation, with examples in plant cells, animal development and immune cells. It emerges that “pruning” of methylated cytosines by active DNA demethylation is an important determinant for the DNA methylation signature of a cell. Work in plants and animals shows that demethylation occurs by base excision and nucleotide excision repair. Far from merely protecting genomic integrity from environmental insult, DNA repair is therefore at the heart of an epigenetic activation process.     

Role of excision mechanisms of DNA repair in induction of apoptosis

DNA repair and apoptosis lead to principally different final results: the first mechanism removes damages from DNA, restoring genome integrity; the second mechanism eliminates potentially dangerous cells harboring DNA lesions. The cells deficient in mismatch repair (MMR) demonstrate inceased resistance (viability) to DNA-damaging agents due to decreased ability to undergo apoptosis. This means that mechanism of MMR both restores structure of DNA and generates a signal for apoptosis. DNA breaks and single strand gaps, which are temporarily produced by excison mechanism during DNA repair, are suggested to be the initial signals for apoptosis. However pathway involved in such signaling at least partially is independent of p53 function.     

Ligation of newly replicated DNA controls the timing of DNA mismatch repair

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Spontaneous DNA repair in human mononuclear cells is calcium-dependent

Spontaneous DNA repair in peripheral blood mononuclear cells (PBMC) has been recently described. The aim of this study was to evaluate whether spontaneous DNA repair is Ca(2+)-dependent, as in vitro-stimulated DNA repair. Spontaneous DNA repair in PBMC was measured in a 1mM Ca2+ medium. The effect of extracellular Ca2+ chelation by EGTA, intracellular Ca2+ chelation by bapta-AM, and Ca2+ loading by the ionophore A23187 was examined. The signal transduction pathway was evaluated by inhibiting protein tyrosine kinase with genistein, calmodulin with W7, and calcineurin with cyclosporin A and tacrolimus. Extracellular Ca2+ chelation had no effect on spontaneous DNA repair, while both intracellular chelation and calcium overloading inhibited the DNA repair. Inhibition of protein tyrosine kinase, calmodulin or calcineurin reduced DNA repair. In conclusion, spontaneous DNA repair is mainly Ca(2+)-dependent at a narrow range of intracellular Ca2+ concentrations. The signal transduction cascade includes protein tyrosine kinase, calmodulin, and calcineurin.     

Checkpoint mechanisms at the intersection between DNA damage and repair

In response to genomic insults cells trigger a signal transduction pathway, known as DNA damage checkpoint, whose role is to help the cell to cope with the damage by coordinating cell cycle progression, DNA replication and DNA repair mechanisms. Accumulating evidence suggests that activation of the first checkpoint kinase in the cascade is not due to the lesion itself, but it requires recognition and initial processing of the lesion by a specific repair mechanism. Repair enzymes likely convert a variety of physically and chemically different lesions to a unique common structure, a ssDNA region, which is the checkpoint triggering signal. Checkpoint kinases can modify the activity of repair mechanisms, allowing for efficient repair, on one side, and modulating the generation of the ssDNA signal, on the other. This strategy may be important to allow the most effective repair and a prompt recovery from the damage condition. Interestingly, at least in some cases, if the damage level is low enough the cell can deal with the lesions and it does not need to activate the checkpoint response. On the other hand if damage level is high or if the lesions are not rapidly repairable, checkpoint mechanisms become important for cell survival and preservation of genome integrity.     

Large conformational changes in MutS during DNA scanning, mismatch recognition and repair signalling

MutS protein recognizes mispaired bases in DNA and targets them for mismatch repair. Little is known about the transient conformations of MutS as it signals initiation of repair. We have used single-molecule fluorescence resonance energy transfer (FRET) measurements to report the conformational dynamics of MutS during this process. We find that the DNA-binding domains of MutS dynamically interconvert among multiple conformations when the protein is free and while it scans homoduplex DNA. Mismatch recognition restricts MutS conformation to a single state. Steady-state measurements in the presence of nucleotides suggest that both ATP and ADP must be bound to MutS during its conversion to a sliding clamp form that signals repair. The transition from mismatch recognition to the sliding clamp occurs via two sequential conformational changes. These intermediate conformations of the MutS:DNA complex persist for seconds, providing ample opportunity for interaction with downstream proteins required for repair.     

重离子辐照酿酒酵母DNA损伤修复途径研究进展

重离子辐照通过直接和间接作用导致生物体DNA产生损伤,包括DNA的链断裂、碱基的插入或丢失以及氧化损伤等.DNA损伤直接影响复制、转录和蛋白质合成,同时还是突变的重要原因,因此,DNA损伤修复系统尤为重要.在酿酒酵母中,这些损伤主要是通过同源重组修复(homologous recombination repair,HRR)、碱基错配修复(mismatch repair,MMR)和碱基切除修复(base excision repair,BER)等途径来修复的.作为真核生物研究的模式生物,对于酿酒酵母DNA损伤修复的HRR、MMR和BER途径研究颇多,也不断有一些新的成果出现,特别是对于相关途径的完善和相关蛋白的深化更是研究热点,在此对近年来有关重离子辐照酿酒酵母DNA损伤修复途径方面的研究做一综述.     

DNA repair in murine embryonic stem cells and differentiated cells

Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.     

Acetylation regulates the stability of glutamate carboxypeptidase II protein in human astrocytes

Glutamate carboxypeptidase II (GCPII) is known to be implicated in brain diseases such as schizophrenia and bipolar disorder, and dramatically increases in prostate cancer. Here, we investigated the regulation of GCPII expression in astrocytes and examined whether GCPII is epigenetically regulated through histone modification. In this study, valproic acid (VPA), a drug used for bipolar disorder and epilepsy and a known histone deacetylase (HDAC) inhibitor was used. We found that acute exposure of VPA for 4–6 h increased the GCPII protein level in human astrocyte U87MG cells but did not have a similar effect after 12–24 h exposure. Real-time polymerase chain reaction analysis revealed that VPA did not affect the GCPII mRNA expression. In contrast, decrease in GCPII protein level by cycloheximide treatment was blocked by VPA, indicating that VPA increases GCPII protein stability. Treatment with MG132, a proteasome inhibitor, suggested that the VPA-induced increase of GCPII protein level is dependent on the ubiquitin/proteasome pathway. In addition, immunoprecipitation analysis revealed that VPA increased the acetylation of GCPII protein at the lysine residues and facilitated a decrease of the poly-ubiquitinated GCPII level. Similarly, M344, a specific HDAC 1/6 inhibitor, also increased the GCPII protein level. In contrast, treatment with C646, a histone acetyltransferase inhibitor of p300/CBP, significantly reduced the level of GCPII protein. Taken together, this study demonstrated that the increase in GCPII induced by VPA is not due to the classical epigenetic mechanism, but via enhanced acetylation of lysine residues in GCPII.