Active destabilization of base pairs by a DNA glycosylase wedge initiates damage recognition

Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that Fpg, and possibly other DNA glycosylases, convert part of the binding energy into active destabilization of their substrates, using the energy differences between normal and damaged bases for fast substrate discrimination.     

Pot1 deficiency initiates DNA damage checkpoint activation and aberrant homologous recombination at telomeres

The terminal t-loop structure adopted by mammalian telomeres is thought to prevent telomeres from being recognized as double-stranded DNA breaks by sequestering the 3' single-stranded G-rich overhang from exposure to the DNA damage machinery. The POT1 (protection of telomeres) protein binds the single-stranded overhang and is required for both chromosomal end protection and telomere length regulation. The mouse genome contains two POT1 orthologs, Pot1a and Pot1b. Here we show that conditional deletion of Pot1a elicits a DNA damage response at telomeres, resulting in p53-dependent replicative senescence. Pot1a-deficient cells exhibit overall telomere length and 3' overhang elongation as well as aberrant homologous recombination (HR) at telomeres, manifested as increased telomere sister chromatid exchanges and formation of telomere circles. Telomeric HR following Pot1a loss requires NBS1. Pot1a deletion also results in chromosomal instability. Our results suggest that POT1a is crucial for the maintenance of both telomere integrity and overall genomic stability.     

DNA damage by carbonyl stress in human skin cells

Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the alpha-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N(epsilon)-(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress.     

DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts.

Cell-free extracts of Xenopus eggs will replicate plasmid DNA molecules under normal cell cycle control. We have used the neutral/neutral 2-D gel technique to map the sites at which DNA replication initiates in this system. Three different plasmids were studied: one containing the Xenopus rDNA repeat, one containing single copy Xenopus genomic DNA, and another containing the yeast 2 microns replication origin. 2-D gel profiles show that many potential sites of initiation are present on each plasmid, and are randomly situated at the level of resolution of this technique (500-1000 bp). Despite the abundance of sites capable of supporting the initiation of replication, pulse-chase experiments suggest that only a single randomly situated initiation event occurs on each DNA molecule. Once initiation has taken place, conventional replication forks appear to move away from this site at a rate of about 10nt/second, similar to the rate observed in vivo.     

Chromosomal DNA replication initiates at the same origins in meiosis and mitosis.

Autonomously replicating sequence (ARS) elements are identified by their ability to promote high-frequency transformation and extrachromosomal replication of plasmids in the yeast Saccharomyces cerevisiae. Six of the 14 ARS elements present in a 200-kb region of Saccharomyces cerevisiae chromosome III are mitotic chromosomal replication origins. The unexpected observation that eight ARS elements do not function at detectable levels as chromosomal replication origins during mitotic growth suggested that these ARS elements may function as chromosomal origins during premeiotic S phase. Two-dimensional agarose gel electrophoresis was used to map premeiotic replication origins in a 100-kb segment of chromosome III between HML and CEN3. The pattern of origin usage in premeiotic S phase was identical to that in mitotic S phase, with the possible exception of ARS308, which is an inefficient mitotic origin associated with CEN3. CEN3 was found to replicate during premeiotic S phase, demonstrating that the failure of sister chromatids to disjoin during the meiosis I division is not due to unreplicated centromeres. No origins were found in the DNA fragments without ARS function. Thus, in both mitosis and meiosis, chromosomal replication origins are coincident with ARS elements but not all ARS elements have chromosomal origin function. The efficiency of origin use and the patterns of replication termination are similar in meiosis and in mitosis. DNA replication termination occurs over a broad distance between active origins.     

Xeroderma pigmentosum and the role of UV-induced DNA damage in skin cancer

Xeroderma pigmentosum (XP) is a rare, autosomal recessive disease that is characterized by the extreme sensitivity of the skin to sunlight. Compared to normal individuals, XP patients have a more than 1000-fold increased risk of developing cancer on sun-exposed areas of the skin. Genetic and molecular analyses have revealed that the repair of ultraviolet (UV)-induced DNA damage is impaired in XP patients owing to mutations in genes that form part of a DNA-repair pathway known as nucleotide excision repair (NER). Two other diseases, Cockayne syndrome (CS) and the photosensitive form of trichothiodystrophy (TTD), are linked to a defect in the NER pathway. Strikingly, although CS and TTD patients are UV-sensitive, they do not develop skin cancer. The recently developed animal models that mimic the human phenotypes of XP, CS and TTD will contribute to a better understanding of the etiology of these diseases and the role of UV-induced DNA damage in the development of skin cancer.     

紫外线诱导的DNA损伤与皮肤癌的发生(1)

日光中紫外线(ultraviolet radiation,UV)诱导的DNA损伤是导致皮肤癌发生的一个重要因素。在皮肤癌细胞中发现,调控细胞增殖、分化和凋亡的特异性基因出现与紫外线损伤相关的变异。根据日光中紫外线波长的不同,可将其分为3种：UVA,UVB和UVC。UVB是导致DNA损伤的主要类型。目前已证实一些原癌基因和抑癌基因由于紫外线引起的DNA损伤而发生变异。UV诱导产生皮肤癌是一个复杂的过程,涉及到许多细胞和分子水平上的变化,同时还与DNA的修复作用相关。     

DNA synthesis generally initiates outside the simian virus 40 core origin in vitro.

The nucleotide positions at which DNA synthesis initiates in vitro, in the vicinity of the simian virus 40 origin, have been determined. Start sites for DNA synthesis are greatly suppressed over the simian virus 40 core origin. Relatively weak start sites are detected over the 21-bp repeats and T-antigen-binding site I; distal to these regions, stronger start sites are detected. Thus, studies using a model system for eukaryotic DNA replication indicate that DNA synthesis events initiate, in general, outside the core origin.     

Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme.

The gamma complex (gamma delta delta' chi psi) subassembly of DNA polymerase III holoenzyme transfers the beta subunit onto primed DNA in a reaction which requires ATP hydrolysis. Once on DNA, beta is a "sliding clamp" which tethers the polymerase to DNA for highly processive synthesis. We have examined beta and the gamma complex to identify which subunit(s) hydrolyzes ATP. We find the gamma complex is a DNA dependent ATPase. The beta subunit, which lacks ATPase activity, enhances the gamma complex ATPase when primed DNA is used as an effector. Hence, the gamma complex recognizes DNA and couples ATP hydrolysis to clamp beta onto primed DNA. Study of gamma complex subunits showed no single subunit contained significant ATPase activity. However, the heterodimers, gamma delta and gamma delta', were both DNA-dependent ATPases. Only the gamma delta ATPase was stimulated by beta and was functional in transferring the beta from solution to primed DNA. Similarity in ATPase activity of DNA polymerase III holoenzyme accessory proteins to accessory proteins of phage T4 DNA polymerase and mammalian DNA polymerase delta suggests the basic strategy of chromosome duplication has been conserved throughout evolution.     

After sun reversal of DNA damage: enhancing skin repair

UV-induced DNA damage has been directly linked to skin cancer, and DNA repair is an important protection against this neoplasm. This is illustrated by the genetic disease xeroderma pigmentosum wherein a serious defect in DNA repair of cyclobutane pyrimidine dimers dramatically increases the rate of skin cancer. In other instances in which skin cancer rates are elevated, deficits in DNA repair may also be one of the causal factors. For example, solid organ transplant patients have elevated rates of skin cancer that are correlated with the dose and length of exposure to immunosuppressive drugs (predominantly cyclosporine A (CsA) and ascomycin (FK506)-related tacrolimus). We have found that treatment of cultured epidermal cells with CsA or ascomycin inhibits their removal of DNA damage by about 20% at 24 h. In a further example, people with a polymorphism in the DNA repair gene 8-oxo-guanine glycosylase (OGG1) have an increased risk of skin cancer. We have found that the cells with this variant polymorphism have an increased sensitivity of about 20% to a broad range of cytotoxic agents. The DNA deficits caused by immunosuppressive drugs or the OGG1 polymorphism can be overcome by the delivery of DNA repair enzymes in liposomes. The data suggests that deficits in DNA repair, even if they are not as severe as in the case of XP, may contribute to increased rates of cancer, and that topical therapy with DNA repair enzymes may be a promising avenue for after-sun protection.     

Endogenous DNA damage clusters in human skin, 3-D model, and cultured skin cells

Clustered damages-two or more oxidized bases, abasic sites, or strand breaks on opposing DNA strands within a few helical turns-are formed in DNA by ionizing radiation. Clusters are difficult for cells to repair and thus pose significant challenges to genomic integrity. Although endogenous clusters were found in some permanent human cell lines, it was not known if clusters accumulated in human tissues or primary cells. Using high-sensitivity gel electrophoresis, electronic imaging, and number average length analysis, we determined endogenous cluster levels in DNA from human skin, a 3-D skin model, and primary cultured skin cells. DNA from dermis and epidermis of human skin contained extremely low levels of endogenous clusters (a few per gigabase). However, cultured skin fibroblasts and keratinocytes-whether in monolayer cultures or in 3-D model skin cultures-accumulated oxidized pyrimidine, oxidized purine, and abasic clusters. The levels of endogenous clusters were decreased by growing cells in the presence of selenium or by increasing cellular levels of Fpg protein, presumably by increasing processing of clustered damages. These results imply that the levels of endogenous clusters can be affected by the cells' external environment and their ability to deal with DNA damage.     

DNA replication initiates non-randomly at multiple sites near the c-myc gene in HeLa cells.

The origin of replication of the c-myc gene in HeLa cells was previously identified at low resolution within 3.5 kb 5' to the P1 promoter, based on replication fork polarity and the location of DNA nascent strands. To define the initiation events in the c-myc origin at higher resolution the template bias of nascent DNAs in a 12 kb c-myc domain has been analyzed by hybridization to strand specific probes. Strong switches in the asymmetry of nascent strand template preference confirm that replication initiates non-randomly at multiple sites within 2.4 kb 5' to the c-myc P1 promoter, and at other sites over a region of 12 kb or more. The strongest template biases occur in the 2.4 kb region 5' of the P1 promoter, shown earlier to contain sequences which allow the autonomous semiconservative replication of c-myc plasmids. An asymmetric pyrimidine heptanucleotide consensus sequence has been identified which occurs 12 times in the c-myc origin zone, and whose polarity exactly correlates with the polarity of nascent strand synthesis.     

3,3'-Dihydroxyisorenieratene and isorenieratene prevent UV-induced DNA damage in human skin fibroblasts

Skin cancer is among the most frequent neoplastic malignancies and exposure to UV irradiation is a major risk factor. In addition to topical sunscreens, photoprotection by dietary antioxidants such as carotenoids or polyphenols has been suggested as a means of prevention. Isorenieratene (IR) and dihydroxyisorenieratene (DHIR) are aromatic carotenoids with particular antioxidant properties produced by Brevibacterium linens. The aim of this study was to investigate the photoprotective and antioxidant activities of DHIR and IR in comparison to the nonaromatic carotenoid lutein in human dermal fibroblasts. Incubation of the cells with DHIR and IR significantly decreased the UV-induced formation of cyclobutane pyrimidine dimers and formation of DNA strand breaks. Lipid oxidation was lowered as determined by the formation of malondialdehyde as a biomarker. Both aromatic carotenoids also prevented oxidatively generated damage to DNA as demonstrated by a decrease in DNA strand breaks associated with the formation of oxidized DNA bases. These data highlight the multifunctional photoprotective properties of aromatic carotenoids, which may be suitable natural compounds for the prevention of skin cancer.     

Oxidative damage to DNA constituents by iron-mediated Fenton reactions: the deoxyadenosine family

The effect of exposing 2'-deoxyadenosine (dA), 5'-dAMP, 3'-dAMP, dApA, dA(pdA)(19,) and poly(dA): oligo(dT) to iron/H(2)O(2) in the presence and absence of ethanol or NADH has been studied. HPLC retention times, enzyme treatments, radio-labeled substrates, UV absorption spectra, and fast atom bombardment mass spectrometry (FABMS) have been used to distinguish 20 products arising from the reaction, of which 16 have been identified and four anomers proposed by comparison with earlier gamma radiation studies. The radical responsible for the reactions seems to be analogous to radiation-derived [Formula: see text], has many products in common, but has some novel ones probably specific for Fenton-induced damage. Two new dimeric adducts arising from the generation of hydroxylamine at N7 and its subsequent condensation with two known sugar damage products, dR-adenine-N1-oxide, and two isomers of dR-FAPy arising from radical attacks at C4 and C5, may be considered novel in the present study. Unlike radiation-derived [Formula: see text], the radical under study is difficult to eliminate due to its generation in the proximity of the substrate molecules. It is proposed that the iron binds to the phosphate group and generates the radical in its vicinity. Strand breaks in dA(pdA)(11) resulting from the Fenton reaction are of two types, spontaneous and alkali-labile. Duplex DNA is less sensitive to attack by this radical, as its various degradation products are a subset of those obtained with monomer substrates and only dR-FAPy production is relatively enhanced for poly (dA): oligo (dT) as compared to those from other substrates.     

Short interspersed DNA element-mediated detection of UVB-induced DNA damage and repair in the mouse genome, in vitro, and in vivo in skin.

We report a sensitive, SINE (Short Interspersed DNA Element)-mediated, PCR-based, DNA damage detection assay. Here, the SINE assay is used for detection of UVB-induced DNA damage and repair in cultured mouse cells and in vivo, in mouse skin. The unique feature of the SINE assay is its ability to support simultaneous amplification of multiple, random segments of genomic DNA. This can be accomplished due to the remarkable abundance, dispersion and conservation of SINEs in mammalian genomes. The most abundant SINEs in the mouse genome are the B1 elements, at a copy number of 50,000-80,000. Due to their strong sequence conservation, primers complementary to the B1 consensus sequence anneal to the majority of their targets in the genome. Consequently, long segments of genomic DNA located between pairs of B1 elements are efficiently amplified by PCR. Thus, in conjunction with the fact that many types of DNA adducts form blocks for thermostable polymerase, the B1 element anchored PCR makes a sensitive and versatile tool for assessing the overall integrity of the transcribed regions in mouse genome. We measured UVB-dose (0.1-3 kJ m-2) dependent formation of photoproducts in DNA from cultured cells, and after 20 h observed a substantial removal of damage at doses lower or equal to 0.6 kJ m-2. The sensitivity of detection of UVB-photoproducts formation and repair was compared to that of the conventional, single locus-targeting QPCR. Using the SINE assay we also have shown the distribution of UVB and UVC induced DNA adducts at a single nucleotide resolution within the B1 elements in mouse DNA. Lastly, we demonstrated that the sensitivity of the SINE assay is adequate for measurement of UVB-dose (1-6 kJ m-2) dependent formation and subsequent removal of photoproducts in vivo, in mouse skin.     

GZ17-6.02 initiates DNA damage causing autophagosome-dependent HDAC degradation resulting in enhanced anti-PD1 checkpoint inhibitory antibody efficacy

Our studies examined the molecular mechanisms by which the novel cancer therapeutic GZ17-6.02 (NCT03775525) killed GI tumor cells. TZ17-6.02 activated ATM which was responsible for increased phosphorylation of nuclear γH2AX and AMPKα T172. ATM-AMPK signaling was responsible for the subsequent inactivation of mTORC1 and mTORC2, dephosphorylation of ULK1 S757, and increased phosphorylation of ULK1 S317 and of ATG13 S318, which collectively caused enhanced autophagosome formation. GZ17-6.02 interacted with 5-fluorouracil in an additive to greater than additive fashion to kill all of the tested GI tumor cell types. This was associated with greater ATM activation and a greater mammalian target of rapamycin inactivation and autophagosome induction. As a result, autophagy-dependent degradation of multiple histone deacetylase (HDAC) proteins and chaperone proteins occurred. Loss of HDAC expression was causal in reduced expression of programed death ligand 1 (PD-L1), ornithine decarboxylase, and indole amine 2,3-dioxygenase (IDO1) and in the elevated expression of major histocompatibility complex Class IA (MHCA). Treatment with GZ17-6.02 also resulted in enhanced efficacy of a subsequently administered anti-PD1 checkpoint inhibitory antibody. Thus, the primary mode of GZ17-6.02 action is to induce a DNA damage response concomitant with ATM activation, that triggers a series of interconnected molecular events that result in tumor cell death and enhanced immunogenicity.