scholarly journals δ-elimination in the repair of AP (apurinic/apyrimidinic) sites in DNA

1989 ◽  
Vol 261 (3) ◽  
pp. 707-713 ◽  
Author(s):  
V Bailly ◽  
M Derydt ◽  
W G Verly
Keyword(s):  
Mammalian Cells ◽  
Nuclear Dna ◽  
Replicative Form ◽  
Dna Polymerization ◽  
E Coli ◽  
Polynucleotide Kinase ◽  
Ap Sites ◽  
Beta Elimination ◽  
Repair Pathways ◽  
Hydrolysis Of

[5′-32P]pdT8d(-)dT7, containing an AP (apurinic/apyrimidinic) site in the ninth position, and [d(-)-1′,2′-3H, 5′-32P]DNA, containing AP sites labelled with 3H in the 1′ and 2′ positions of the base-free deoxyribose [d(-)] and with 32P 5′; to this deoxyribose, were used to investigate the yields of the beta-elimination and delta-elimination reactions catalysed by spermine, and also the yield of hydrolysis, by the 3′-phosphatase activity of T4 polynucleotide kinase, of the 3′-phosphate resulting from the beta delta-elimination. Phage-phi X174 RF (replicative form)-I DNA containing AP (apurinic) sites has been repaired in five steps: beta-elimination, delta-elimination, hydrolysis of 3′-phosphate, DNA polymerization and ligation. Spermine, in one experiment, and Escherichia coli formamidopyrimidine: DNA glycosylase, in another experiment, were used to catalyse the first and second steps (beta-elimination and delta-elimination). These repair pathways, involving a delta-elimination step, may be operational not only in E. coli repairing its DNA containing a formamido-pyrimidine lesion, but also in mammalian cells repairing their nuclear DNA containing AP sites.

scholarly journals Deoxyribonuclease IV from rat liver chromatin and the excision of apurinic sites from depurinated DNA

1985 ◽  
Vol 225 (2) ◽  
pp. 535-542 ◽  
Author(s):  
G Grondal-Zocchi ◽  
W G Verly
Keyword(s):  
Rat Liver ◽  
Liver Cells ◽  
Ap Endonuclease ◽  
Phosphodiester Bond ◽  
Double Stranded Dna ◽  
Rat Liver Cells ◽  
Liver Chromatin ◽  
Ap Sites ◽  
Beta Elimination ◽  
Hydrolysis Of

Deoxyribonuclease IV, a 5′-3′ exonuclease degrading double-stranded DNA from intra-strand nicks, has been purified from the chromatin of rat liver cells. The enzyme, which has an Mr of 58000, excises the apurinic (AP) sites from a depurinated DNA nicked 5′ to these AP sites with the chromatin AP endonuclease. The excision is not the result of hydrolysis of the phosphodiester bond 3′ to the AP sites since the excision product does not behave as deoxyribose 5-phosphate but as its 2,3-unsaturated derivative. This result suggests that, to remove the AP sites from the DNA nicked by an AP endonuclease, the chromatin deoxyribonuclease IV rather acts as a catalyst of beta-elimination.

scholarly journals The effect of APOBEC3B deaminase on double-stranded DNA

2019 ◽  
Author(s):  
Joseph Chapman ◽  
Michael Custance ◽  
Birong Shen ◽  
Anthony V. Furano
Keyword(s):  
Mammalian Cells ◽  
Chemotherapeutic Agents ◽  
Double Stranded Dna ◽  
Cancer Initiation ◽  
Development Accounting ◽  
Elevated Expression ◽  
Ap Sites ◽  
Hydrolysis Of ◽  
Ap Site

ABSTRACTMutations mediated by the APOBEC3 (A3) family of single-strand specific cytosine deaminases can accumulate in various cancers, as strand-coordinated clusters and isolated lesions. A3-mediated mutations also occur during normal development, accounting for ~20% of heritable mutations. A3B is an archetypical member of this family and is thought to contribute to both cancer initiation and progression. A3B has a strong preference for C in a TC context and catalyzes hydrolysis of the primary amine of un-paired C to generate U. Subsequent repair generates a distinctive pattern of C-substitutions, which along with their context signify their A3B origin. Although single-stranded DNA is the preferred A3B substrate, we report here that in some instances A3B can deaminate the C of TC in a double-stranded DNA context in vitro. These include C paired to O6-methylguanine (O6meG), to an abasic (AP) site, or to a G adjacent to an AP site. AP sites are the most common lesion in DNA, and O6meG levels increase under alkylating conditions caused by environmental nitrosamines and some chemotherapeutic agents. We also show that elevated expression of A3B can enhance double-stranded breaks induced by the alkylating agent MNNG in mammalian cells, but this effect does not require A3B deaminase activity.

scholarly journals Escherichia coli endonuclease III is not an endonuclease but a β-elimination catalyst

1987 ◽  
Vol 242 (2) ◽  
pp. 565-572 ◽  
Author(s):  
V Bailly ◽  
W G Verly
Keyword(s):  
Dna Polymerase ◽  
Exonuclease Activity ◽  
Alkaline Ph ◽  
Sugar Phosphate ◽  
Exonuclease Iii ◽  
E Coli ◽  
Endonuclease Iii ◽  
Ap Sites ◽  
Beta Elimination ◽  
Ap Site

The oligonucleotide [5′-32P]pdT8d(-)dTn, containing an apurinic/apyrimidinic (AP) site [d(-)], yields three radioactive products when incubated at alkaline pH: two of them, forming a doublet approximately at the level of pdT8dA when analysed by polyacrylamide-gel electrophoresis, are the result of the beta-elimination reaction, whereas the third is pdT8p resulting from beta delta-elimination. The incubation of [5′-32P]pdT8d(-)dTn, hybridized with poly(dA), with E. coli endonuclease III yields two radioactive products which have the same electrophoretic behaviour as the doublet obtained by alkaline beta-elimination. The oligonucleotide pdT8d(-) is degraded by the 3′-5′ exonuclease activity of T4 DNA polymerase as well as pdT8dA, showing that a base-free deoxyribose at the 3′ end is not an obstacle for this activity. The radioactive products from [5′-32P]pdT8d(-)dTn cleaved by alkaline beta-elimination or by E. coli endonuclease III are not degraded by the 3‘-5’ exonuclease activity of T4 DNA polymerase. When DNA containing AP sites labelled with 32P 5′ to the base-free deoxyribose labelled with 3H in the 1′ and 2′ positions is degraded by E. coli endonuclease VI (exonuclease III) and snake venom phosphodiesterase, the two radionuclides are found exclusively in deoxyribose 5-phosphate and the 3H/32P ratio in this sugar phosphate is the same as in the substrate DNA. When DNA containing these doubly-labelled AP sites is degraded by alkaline treatment or with Lys-Trp-Lys, followed by E. coli endonuclease VI (exonuclease III), some 3H is found in a volatile compound (probably 3H2O) whereas the 3H/32P ratio is decreased in the resulting sugar phosphate which has a chromatographic behaviour different from that of deoxyribose 5-phosphate. Treatment of the DNA containing doubly-labelled AP sites with E. coli endonuclease III, then with E. coli endonuclease VI (exonuclease III), also results in the loss of 3H and the formation of a sugar phosphate with a lower 3H/32P ratio that behaves chromatographically as the beta-elimination product digested with E. coli endonuclease VI (exonuclease III). From these data, we conclude that E. coli endonuclease III cleaves the phosphodiester bond 3′ to the AP site, but that the cleavage is not a hydrolysis leaving a base-free deoxyribose at the 3′ end as it has been so far assumed. The cleavage might be the result of a beta-elimination analogous to the one produced by an alkaline pH or Lys-Trp-Lys. Thus it would seem that E. coli ‘endonuclease III’ is, after all, not an endonuclease.

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

Cell-Specific Chemical Delivery Using a Selective Nitroreductase–Nitroaryl Pair

2018 ◽  
Author(s):  
Todd D. Gruber ◽  
Chithra Krishnamurthy ◽  
Jonathan B. Grimm ◽  
Michael R. Tadross ◽  
Laura M. Wysocki ◽  
...  
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Target Cells ◽  
Specific Chemical ◽  
E Coli ◽  
Enzyme Substrate ◽  
Cell Specificity ◽  
General Chemical ◽  
Increased Activity ◽  
Enzyme Variant

<p>The utility of<b> </b>small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. ‘Masking’ the activity of small molecules using a general chemical modification and ‘unmasking’ it only within target cells could overcome this limitation. To this end, we have developed a selective enzyme–substrate pair consisting of engineered variants of <i>E. coli</i> nitroreductase (NTR) and a 2‑nitro-<i>N</i>-methylimidazolyl (NM) masking group. To discover and optimize this NTR–NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.</p>

scholarly journals Nucleoside Analogues Are Potent Inducers of Pol V-mediated Mutagenesis

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 843
Author(s):  
Balagra Kasim Sumabe ◽  
Synnøve Brandt Ræder ◽  
Lisa Marie Røst ◽  
Animesh Sharma ◽  
Eric S. Donkor ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mutation Frequency ◽  
Nucleoside Analogues ◽  
Cancer Therapies ◽  
E Coli ◽  
Replicative Stress ◽  
Pol V ◽  
Dna And Rna ◽  
Sos Induction ◽  
Anti Cancer

Drugs targeting DNA and RNA in mammalian cells or viruses can also affect bacteria present in the host and thereby induce the bacterial SOS system. This has the potential to increase mutagenesis and the development of antimicrobial resistance (AMR). Here, we have examined nucleoside analogues (NAs) commonly used in anti-viral and anti-cancer therapies for potential effects on mutagenesis in Escherichia coli, using the rifampicin mutagenicity assay. To further explore the mode of action of the NAs, we applied E. coli deletion mutants, a peptide inhibiting Pol V (APIM-peptide) and metabolome and proteome analyses. Five out of the thirteen NAs examined, including three nucleoside reverse transcriptase inhibitors (NRTIs) and two anti-cancer drugs, increased the mutation frequency in E. coli by more than 25-fold at doses that were within reported plasma concentration range (Pl.CR), but that did not affect bacterial growth. We show that the SOS response is induced and that the increase in mutation frequency is mediated by the TLS polymerase Pol V. Quantitative mass spectrometry-based metabolite profiling did not reveal large changes in nucleoside phosphate or other central carbon metabolite pools, which suggests that the SOS induction is an effect of increased replicative stress. Our results suggest that NAs/NRTIs can contribute to the development of AMR and that drugs inhibiting Pol V can reverse this mutagenesis.

scholarly journals The Saccharomyces cerevisiae RAD6 Group Is Composed of an Error-Prone and Two Error-Free Postreplication Repair Pathways

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1633-1641 ◽  
Author(s):  
Wei Xiao ◽  
Barbara L Chow ◽  
Stacey Broomfield ◽  
Michelle Hanna
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Signal Transducer ◽  
Repair Pathway ◽  
Polyubiquitin Chain ◽  
Postreplication Repair ◽  
Translesion Dna Synthesis ◽  
Radiation Repair ◽  
Repair Pathways ◽  
High Degree

Abstract The RAD6 postreplication repair and mutagenesis pathway is the only major radiation repair pathway yet to be extensively characterized. It has been previously speculated that the RAD6 pathway consists of two parallel subpathways, one error free and another error prone (mutagenic). Here we show that the RAD6 group genes can be exclusively divided into three rather than two independent subpathways represented by the RAD5, POL30, and REV3 genes; the REV3 pathway is largely mutagenic, whereas the RAD5 and the POL30 pathways are deemed error free. Mutants carrying characteristic mutations in each of the three subpathways are phenotypically indistinguishable from a single mutant such as rad18, which is defective in the entire RAD6 postreplication repair/tolerance pathway. Furthermore, the rad18 mutation is epistatic to all single or combined mutations in any of the above three subpathways. Our data also suggest that MMS2 and UBC13 play a key role in coordinating the response of the error-free subpathways; Mms2 and Ubc13 form a complex required for a novel polyubiquitin chain assembly, which probably serves as a signal transducer to promote both RAD5 and POL30 error-free postreplication repair pathways. The model established by this study will facilitate further research into the molecular mechanisms of postreplication repair and translesion DNA synthesis. In view of the high degree of sequence conservation of the RAD6 pathway genes among all eukaryotes, the model presented in this study may also apply to mammalian cells and predicts links to human diseases.

scholarly journals Phosphorylation: The Molecular Switch of Double-Strand Break Repair

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
K. C. Summers ◽  
F. Shen ◽  
E. A. Sierra Potchanant ◽  
E. A. Phipps ◽  
R. J. Hickey ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Genomic Stability ◽  
Molecular Switches ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Brca1 And Brca2 ◽  
Damage Response ◽  
Repair Mechanisms ◽  
Repair Pathways

Repair of double-stranded breaks (DSBs) is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ), homologous recombination (HR), or the inclusive DNA damage response (DDR). These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.

scholarly journals Repair of abasic sites by mammalian cell extracts

1994 ◽  
Vol 304 (3) ◽  
pp. 699-705 ◽  
Author(s):  
G Frosina ◽  
P Fortini ◽  
O Rossi ◽  
F Carrozzino ◽  
A Abbondandolo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Excision Repair ◽  
Chinese Hamster ◽  
Pyrimidine Dimer ◽  
Repair Synthesis ◽  
Repair Patch ◽  
Cell Extracts ◽  
Pyrimidine Dimers ◽  
Ap Sites ◽  
Ap Site

Hamster cell extracts that perform repair synthesis on covalently closed circular DNA containing pyrimidine dimers, were used to study the repair of apurinic/apyrimidinic (AP) sites and methoxyamine (MX)-modified AP sites. Plasmid molecules were heat-treated at pH 5 and incubated with MX when required. The amount of damage introduced ranged from 0.2 to 0.9 AP sites/kb. Extracts were prepared from the Chinese hamster ovary CHO-9 cell line and from its derivative, 43-3B clone which is mutated in the nucleotide excision repair (NER) ERCC1 gene. AP and MX-AP sites stimulated repair synthesis by CHO-9 cell extracts. The level of synthesis correlated with the number of lesions and was of similar magnitude to the repair stimulated by 4.3 u.v. photoproducts/kb. Repair of AP and MX-AP sites was faster than the repair of u.v. damage and was independent of ERCC1 gene product. The high level of repair replication was due to a very efficient and rapid incision of plasmids carrying AP or MX-AP sites, performed by abundant AP endonucleases present in the extract. The calculated average repair patch sizes were: 7 nucleotides per AP site; 10 nucleotides per MX-AP site; 28 nucleotides per (6-4) u.v. photoproduct or cyclobutane pyrimidine dimer. The data indicate that AP and MX-AP sites are very efficiently repaired by base-excision repair in mammalian cells and suggest that MX-AP sites may also be processed via alternative repair mechanisms.

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