scholarly journals Possible roles of β-elimination and δ-elimination reactions in the repair of DNA containing AP (apurinic/apyrimidinic) sites in mammalian cells

1988 ◽  
Vol 253 (2) ◽  
pp. 553-559 ◽  
Author(s):  
V Bailly ◽  
W G Verly
Keyword(s):  
Mammalian Cells ◽  
Free Sugar ◽  
Ap Endonuclease ◽  
Phosphodiester Bond ◽  
Polymerase Beta ◽  
Beta Elimination ◽  
Elimination Reactions ◽  
The One ◽  
Ap Site

Histones and polyamines nick the phosphodiester bond 3′ to AP (apurinic/apyrimidinic) sites in DNA by inducing a beta-elimination reaction, which can be followed by delta-elimination. These beta- and delta-elimination reactions might be important for the repair of AP sites in chromatin DNA in either of two ways. In one pathway, after the phosphodiester bond 5′ to the AP site has been hydrolysed with an AP endonuclease, the 5′-terminal base-free sugar 5′-phosphate is released by beta-elimination. The one-nucleotide gap limited by 3′-OH and 5′-phosphate ends is then closed by DNA polymerase-beta and DNA ligase. We have shown in vitro that such a repair is possible. In the other pathway, the nicking 3′ to the AP site by beta-elimination occurs first. We have shown that the 3′-terminal base-free sugar so produced cannot be released by the chromatin AP endonuclease from rat liver. But it can be released by delta-elimination, leaving a gap limited by 3′-phosphate and 5′-phosphate. After conversion of the 3′-phosphate into a 3′-OH group by the chromatin 3′-phosphatase, there will be the same one-nucleotide gap, limited by 3′-OH and 5′-phosphate, as that formed by the successive actions of the AP endonuclease and the beta-elimination catalyst in the first pathway.

scholarly journals Structure of the endonuclease IV homologue fromThermotoga maritimain the presence of active-site divalent metal ions

2010 ◽  
Vol 66 (9) ◽  
pp. 1003-1012 ◽  
Author(s):  
Stephen J. Tomanicek ◽  
Ronny C. Hughes ◽  
Joseph D. Ng ◽  
Leighton Coates
Keyword(s):  
Metal Ions ◽  
Active Site ◽  
Excision Repair ◽  
Divalent Metal ◽  
Hydroxyl Group ◽  
Divalent Metal Ions ◽  
Ap Endonuclease ◽  
Phosphodiester Bond ◽  
Dna Base ◽  
Ap Site

The most frequent lesion in DNA is at apurinic/apyrimidinic (AP) sites resulting from DNA-base losses. These AP-site lesions can stall DNA replication and lead to genome instability if left unrepaired. The AP endonucleases are an important class of enzymes that are involved in the repair of AP-site intermediates during damage-general DNA base-excision repair pathways. These enzymes hydrolytically cleave the 5′-phosphodiester bond at an AP site to generate a free 3′-hydroxyl group and a 5′-terminal sugar phosphate using their AP nuclease activity. Specifically,Thermotoga maritimaendonuclease IV is a member of the second conserved AP endonuclease family that includesEscherichia coliendonuclease IV, which is the archetype of the AP endonuclease superfamily. In order to more fully characterize the AP endonuclease family of enzymes, two X-ray crystal structures of theT. maritimaendonuclease IV homologue were determined in the presence of divalent metal ions bound in the active-site region. These structures of theT. maritimaendonuclease IV homologue further revealed the use of the TIM-barrel fold and the trinuclear metal binding site as important highly conserved structural elements that are involved in DNA-binding and AP-site repair processes in the AP endonuclease superfamily.

scholarly journals The multiple activities of Escherichia coli endonuclease IV and the extreme lability of 5′-terminal base-free deoxyribose 5-phosphates

1989 ◽  
Vol 259 (3) ◽  
pp. 761-768 ◽  
Author(s):  
V Bailly ◽  
W G Verly
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Rat Liver ◽  
Half Life ◽  
Elimination Reaction ◽  
Ap Endonuclease ◽  
Exonuclease Iii ◽  
E Coli ◽  
Beta Elimination ◽  
Ap Site

Escherichia coli endonuclease IV hydrolyses the C(3′)-O-P bond 5′ to a 3′-terminal base-free deoxyribose. It also hydrolyses the C(3′)-O-P bond 5′ to a 3′-terminal base-free 2′,3′-unsaturated sugar produced by nicking 3′ to an AP (apurinic or apyrimidinic) site by beta-elimination; this explains why the unproductive end produced by beta-elimination is converted by the enzyme into a 3′-OH end able to prime DNA synthesis. The action of E. coli endonuclease IV on an internal AP site is more complex: in a first step the C(3′)-O-P bond 5′ to the AP site is hydrolysed, but in a second step the 5′-terminal base-free deoxyribose 5′-phosphate is lost. This loss is due to a spontaneous beta-elimination reaction in which the enzyme plays no role. The extreme lability of the C(3′)-O-P bond 3′ to a 5′-terminal AP site contrasts with the relative stability of the same bond 3′ to an internal AP site; in the absence of beta-elimination catalysts, at 37 degrees C the half-life of the former is about 2 h and that of the latter 200 h. The extreme lability of a 5′-terminal AP site means that, after nicking 5′ to an AP site with an AP endonuclease, in principle no 5′----3′ exonuclease is needed to excise the AP site: it falls off spontaneously. We have repaired DNA containing AP sites with an AP endonuclease (E. coli endonuclease IV or the chromatin AP endonuclease from rat liver), a DNA polymerase devoid of 5′----3′ exonuclease activity (Klenow polymerase or rat liver DNA polymerase beta) and a DNA ligase. Catalysts of beta-elimination, such as spermine, can drastically shorten the already brief half-life of a 5′-terminal AP site; it is what very probably happens in the chromatin of eukaryotic cells. E. coli endonuclease IV also probably participates in the repair of strand breaks produced by ionizing radiations: as E. coli endonuclease VI/exonuclease III, it is a 3′-phosphoglycollatase and also a 3′-phosphatase. The 3′-phosphatase activity of E. coli endonuclease VI/exonuclease III and E. coli endonuclease IV can also be useful when the AP site has been excised by a beta delta-elimination reaction.

scholarly journals The NS5A/NS5 Proteins of Viruses from Three Genera of the Family Flaviviridae Are Phosphorylated by Associated Serine/Threonine Kinases

1998 ◽  
Vol 72 (7) ◽  
pp. 6199-6206 ◽  
Author(s):  
Karen E. Reed ◽  
Alexander E. Gorbalenya ◽  
Charles M. Rice
Keyword(s):  
Mammalian Cells ◽  
Yellow Fever Virus ◽  
Diarrhea Virus ◽  
The Family ◽  
A Cell ◽  
Infected Cells ◽  
Threonine Kinases ◽  
The One ◽  
Viral Diarrhea Virus

ABSTRACT Phosphorylation of the expressed NS5A protein of hepatitis C virus (HCV), a member of the Hepacivirus genus of the familyFlaviviridae, has been demonstrated in mammalian cells and in a cell-free assay by an associated kinase activity. In this report, phosphorylation is also shown for the NS5A and NS5 proteins, respectively, of bovine viral diarrhea virus (BVDV) and yellow fever virus (YF), members of the other two established genera in this family. Phosphorylation of BVDV NS5A and YF NS5 was observed in infected cells, transient expression experiments, and a cell-free assay similar to the one developed for HCV NS5A. Phosphoamino acid analyses indicated that all three proteins were phosphorylated by serine/threonine kinases. Similarities in the properties of BVDV NS5A, YF NS5, and HCV NS5A phosphorylation in vitro further suggested that closely related kinases or the same kinase may phosphorylate these viral proteins. Conservation of this trait among three quite distantly related viruses representing three separate genera suggests that phosphorylation of the NS5A/NS5 proteins or their association with cellular kinases may play an important role in the flavivirus life cycle.

scholarly journals Strategies to Prevent Biofilm Infections on Biomaterials: Effect of Novel Naturally-Derived Biofilm Inhibitors on a Competitive Colonization Model of Titanium by Staphylococcus aureus and SaOS-2 Cells

2020 ◽  
Vol 8 (3) ◽  
pp. 345 ◽  
Author(s):  
Inés Reigada ◽  
Ramón Pérez-Tanoira ◽  
Jayendra Z. Patel ◽  
Kirsi Savijoki ◽  
Jari Yli-Kauhaluoma ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Mammalian Cells ◽  
Experimental Models ◽  
High Serum ◽  
Clinical Scenario ◽  
Implanted Medical Devices ◽  
Potential Applicability ◽  
Colonization Model ◽  
The One

Biofilm-mediated infection is a major cause of bone prosthesis failure. The lack of molecules able to act in biofilms has driven research aimed at identifying new anti-biofilm agents via chemical screens. However, to be able to accommodate a large number of compounds, the testing conditions of these screenings end up being typically far from the clinical scenario. In this study, we assess the potential applicability of three previously discovered anti-biofilm compounds to be part of implanted medical devices by testing them on in vitro systems that more closely resemble the clinical scenario. To that end, we used a competition model based on the co-culture of SaOS-2 mammalian cells and Staphylococcus aureus (collection and clinical strains) on a titanium surface, as well as titanium pre-conditioned with high serum protein concentration. Additionally, we studied whether these compounds enhance the previously proven protective effect of pre-incubating titanium with SaOS-2 cells. Out of the three, DHA1 was the one with the highest potential, showing a preventive effect on bacterial adherence in all tested conditions, making it the most promising agent for incorporation into bone implants. This study emphasizes and demonstrates the importance of using meaningful experimental models, where potential antimicrobials ought to be tested for the protection of biomaterials in translational applications.

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 Thermococcus Eurythermalis Endonuclease IV Can Cleave Various Apurinic/Apyrimidinic Site Analogues in ssDNA and dsDNA

2018 ◽  
Vol 20 (1) ◽  
pp. 69 ◽  
Author(s):  
Wei-Wei Wang ◽  
Huan Zhou ◽  
Juan-Juan Xie ◽  
Gang-Shun Yi ◽  
Jian-Hua He ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Exonuclease Activity ◽  
Endonuclease Activity ◽  
Ap Endonuclease ◽  
Phosphodiester Bond ◽  
Phosphodiester Bonds ◽  
Ap Sites ◽  
Alkane Chain ◽  
Ap Site

Endonuclease IV (EndoIV) is a DNA damage-specific endonuclease that mainly hydrolyzes the phosphodiester bond located at 5′ of an apurinic/apyrimidinic (AP) site in DNA. EndoIV also possesses 3′-exonuclease activity for removing 3′-blocking groups and normal nucleotides. Here, we report that Thermococcus eurythermalis EndoIV (TeuendoIV) shows AP endonuclease and 3′-exonuclease activities. The effect of AP site structures, positions and clustered patterns on the activity was characterized. The AP endonuclease activity of TeuendoIV can incise DNA 5′ to various AP site analogues, including the alkane chain Spacer and polyethylene glycol Spacer. However, the short Spacer C2 strongly inhibits the AP endonuclease activity. The kinetic parameters also support its preference to various AP site analogues. In addition, the efficient cleavage at AP sites requires ≥2 normal nucleotides existing at the 5′-terminus. The 3′-exonuclease activity of TeuendoIV can remove one or more consecutive AP sites at the 3′-terminus. Mutations on the residues for substrate recognition show that binding AP site-containing or complementary strand plays a key role for the hydrolysis of phosphodiester bonds. Our results provide a comprehensive biochemical characterization of the cleavage/removal of AP site analogues and some insight for repairing AP sites in hyperthermophile cells.

scholarly journals Repair of depurinated DNA with enzymes from rat liver chromatin

1984 ◽  
Vol 220 (1) ◽  
pp. 133-137 ◽  
Author(s):  
C Goffin ◽  
W G Verly
Keyword(s):  
Rat Liver ◽  
Nuclear Dna ◽  
Eukaryotic Cell ◽  
Dna Ligase ◽  
Polymerase Beta ◽  
Liver Chromatin ◽  
Dna Strand ◽  
T7 Phage ◽  
Ap Site

DNA from T7 phage containing AP (apurinic/apyrimidinic) sites was repaired by the successive actions of three chromatin enzymes [AP endodeoxyribonuclease, DNAase IV (5′----3′-exodeoxyribonuclease) and DNA polymerase-beta] prepared from rat liver and T4-phage DNA ligase. Since DNA ligase is also found in rat liver chromatin, all the activities used for the successful repair in vitro are thus present in the chromatin of a eukaryotic cell. Our results show, in particular, that the chromatin DNAase IV is capable of excising the AP site from the DNA strand nicked by the chromatin AP endodeoxyribonuclease. We did not try to combine all the enzymes, since competition between some of them might have prevented the repair; we have, for instance, shown that DNA ligase can seal the incision 5′ to the AP site made by the AP endodeoxyribonuclease. Changes in chromatin structure during repair might perhaps prevent this competition when nuclear DNA is repaired in the living cell.

scholarly journals Human Apurinic/Apyrimidinic Endonuclease (APE1) Is Acetylated at DNA Damage Sites in Chromatin, and Acetylation Modulates Its DNA Repair Activity

2016 ◽  
Vol 37 (6) ◽  
Author(s):  
Shrabasti Roychoudhury ◽  
Somsubhra Nath ◽  
Heyu Song ◽  
Muralidhar L. Hegde ◽  
Larry J. Bellot ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Excision Repair ◽  
Dna Lesions ◽  
Ap Endonuclease ◽  
Content Type ◽  
Terminal Domain ◽  
Lysine Residues ◽  
Ap Sites ◽  
Base Excision

ABSTRACT Apurinic/apyrimidinic (AP) sites, the most frequently formed DNA lesions in the genome, inhibit transcription and block replication. The primary enzyme that repairs AP sites in mammalian cells is the AP endonuclease (APE1), which functions through the base excision repair (BER) pathway. Although the mechanism by which APE1 repairs AP sites in vitro has been extensively investigated, it is largely unknown how APE1 repairs AP sites in cells. Here, we show that APE1 is acetylated (AcAPE1) after binding to the AP sites in chromatin and that AcAPE1 is exclusively present on chromatin throughout the cell cycle. Positive charges of acetylable lysine residues in the N-terminal domain of APE1 are essential for chromatin association. Acetylation-mediated neutralization of the positive charges of the lysine residues in the N-terminal domain of APE1 induces a conformational change; this in turn enhances the AP endonuclease activity of APE1. In the absence of APE1 acetylation, cells accumulated AP sites in the genome and showed higher sensitivity to DNA-damaging agents. Thus, mammalian cells, unlike Saccharomyces cerevisiae or Escherichia coli cells, require acetylation of APE1 for the efficient repair of AP sites and base damage in the genome. Our study reveals that APE1 acetylation is an integral part of the BER pathway for maintaining genomic integrity.

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

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