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.

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 Molecular basis of abasic site sensing in single-stranded DNA by the SRAP domain of E. coli yedK

2019 ◽  
Vol 47 (19) ◽  
pp. 10388-10399 ◽  
Author(s):  
Na Wang ◽  
Hongyu Bao ◽  
Liu Chen ◽  
Yanhong Liu ◽  
Yue Li ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Substrate Binding ◽  
Specific Substrate ◽  
Abasic Site ◽  
E Coli ◽  
Abasic Sites ◽  
Ap Sites ◽  
Ap Site

Abstract HMCES and yedK were recently identified as sensors of abasic sites in ssDNA. In this study, we present multiple crystal structures captured in the apo-, nonspecific-substrate-binding, specific-substrate-binding, and product-binding states of yedK. In combination with biochemical data, we unveil the molecular basis of AP site sensing in ssDNA by yedK. Our results indicate that yedK has a strong preference for AP site-containing ssDNA over native ssDNA and that the conserved Glu105 residue is important for identifying AP sites in ssDNA. Moreover, our results reveal that a thiazolidine linkage is formed between yedK and AP sites in ssDNA, with the residues that stabilize the thiazolidine linkage important for the formation of DNA-protein crosslinks between yedK and the AP sites. We propose that our findings offer a unique platform to develop yedK and other SRAP domain-containing proteins as tools for detecting abasic sites in vitro and in vivo.

scholarly journals Bacteriophage-T4 and Micrococcus luteus UV endonucleases are not endonucleases but β-elimination and sometimes βδ-elimination catalysts

1989 ◽  
Vol 259 (3) ◽  
pp. 751-759 ◽  
Author(s):  
V Bailly ◽  
B Sente ◽  
W G Verly
Keyword(s):  
Enzyme Preparation ◽  
Micrococcus Luteus ◽  
Bacteriophage T4 ◽  
Pyrimidine Dimer ◽  
Elimination Reaction ◽  
Ap Sites ◽  
Beta Elimination ◽  
Ap Site ◽  
Polynucleotide Chain

Bacteriophage-T4 UV endonuclease nicks the C(3′)-O-P bond 3′ to AP (apurinic or apyrimidinic) sites by a beta-elimination reaction. The breakage of this bond is sometimes followed by the nicking of the C(5′)-O-P bond 5′ to the AP site, leaving a 3′-phosphate end; delta-elimination is proposed as a mechanism to explain this second reaction. The AP site formed when this enzyme acts on a pyrimidine dimer in a polynucleotide chain undergoes the same nicking reactions. Micrococcus luteus UV endonuclease also nicks the C(3′)-O-P bond 3′ to AP sites by a beta-elimination reaction. No subsequent delta-elimination was observed, but this might be due to the presence of 2-mercaptoethanol in the enzyme preparation.

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.

Proliferating cell nuclear antigen-dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision DNA repair

1994 ◽  
Vol 14 (9) ◽  
pp. 6187-6197
Author(s):  
Y Matsumoto ◽  
K Kim ◽  
D F Bogenhagen
Keyword(s):  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Dna Polymerase Delta ◽  
Polymerase Beta ◽  
Ap Sites ◽  
Base Excision ◽  
Proliferating Cell ◽  
Dependent Pathway ◽  
Ap Site

DNA damage frequently leads to the production of apurinic/apyrimidinic (AP) sites, which are presumed to be repaired through the base excision pathway. For detailed analyses of this repair mechanism, a synthetic analog of an AP site, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran), has been employed in a model system. Tetrahydrofuran residues are efficiently repaired in a Xenopus laevis oocyte extract in which most repair events involve ATP-dependent incorporation of no more than four nucleotides (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989; Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 11:4441-4447, 1991). Using a series of column chromatography procedures to fractionate X. laevis ovarian extracts, we developed a reconstituted system of tetrahydrofuran repair with five fractions, three of which were purified to near homogeneity: proliferating cell nuclear antigen (PCNA), AP endonuclease, and DNA polymerase delta. This PCNA-dependent system repaired natural AP sites as well as tetrahydrofuran residues. DNA polymerase beta was able to replace DNA polymerase delta only for repair of natural AP sites in a reaction that did not require PCNA. DNA polymerase alpha did not support repair of either type of AP site. This result indicates that AP sites can be repaired by two distinct pathways, the PCNA-dependent pathway and the DNA polymerase beta-dependent pathway.

scholarly journals Proliferating cell nuclear antigen-dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision DNA repair.

1994 ◽  
Vol 14 (9) ◽  
pp. 6187-6197 ◽  
Author(s):  
Y Matsumoto ◽  
K Kim ◽  
D F Bogenhagen
Keyword(s):  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Dna Polymerase Delta ◽  
Polymerase Beta ◽  
Ap Sites ◽  
Base Excision ◽  
Proliferating Cell ◽  
Dependent Pathway ◽  
Ap Site

DNA damage frequently leads to the production of apurinic/apyrimidinic (AP) sites, which are presumed to be repaired through the base excision pathway. For detailed analyses of this repair mechanism, a synthetic analog of an AP site, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran), has been employed in a model system. Tetrahydrofuran residues are efficiently repaired in a Xenopus laevis oocyte extract in which most repair events involve ATP-dependent incorporation of no more than four nucleotides (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989; Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 11:4441-4447, 1991). Using a series of column chromatography procedures to fractionate X. laevis ovarian extracts, we developed a reconstituted system of tetrahydrofuran repair with five fractions, three of which were purified to near homogeneity: proliferating cell nuclear antigen (PCNA), AP endonuclease, and DNA polymerase delta. This PCNA-dependent system repaired natural AP sites as well as tetrahydrofuran residues. DNA polymerase beta was able to replace DNA polymerase delta only for repair of natural AP sites in a reaction that did not require PCNA. DNA polymerase alpha did not support repair of either type of AP site. This result indicates that AP sites can be repaired by two distinct pathways, the PCNA-dependent pathway and the DNA polymerase beta-dependent pathway.

scholarly journals YqfS from Bacillus subtilis Is a Spore Protein and a New Functional Member of the Type IV Apurinic/Apyrimidinic-Endonuclease Family

2003 ◽  
Vol 185 (18) ◽  
pp. 5380-5390 ◽  
Author(s):  
José M. Salas-Pacheco ◽  
Norma Urtiz-Estrada ◽  
Guadalupe Martínez-Cadena ◽  
Ronald E. Yasbin ◽  
Mario Pedraza-Reyes
Keyword(s):  
Dna Repair ◽  
Bacillus Subtilis ◽  
Alkylating Agents ◽  
Enzymatic Properties ◽  
Mobility Shift ◽  
Exonuclease Iii ◽  
E Coli ◽  
Type Iv ◽  
Cell Extracts ◽  
Ap Sites

ABSTRACT The enzymatic properties and the physiological function of the type IV apurinic/apyrimidinic (AP)-endonuclease homolog of Bacillus subtilis, encoded by yqfS, a gene specifically expressed in spores, were studied here. To this end, a recombinant YqfS protein containing an N-terminal His6 tag was synthesized in Escherichia coli and purified to homogeneity. An anti-His6-YqfS polyclonal antibody exclusively localized YqfS in cell extracts prepared from B. subtilis spores. The His6-YqfS protein demonstrated enzymatic properties characteristic of the type IV family of DNA repair enzymes, such as AP-endonucleases and 3′-phosphatases. However, the purified protein lacked both 5′-phosphatase and exonuclease III activities. YqfS showed not only a high level of amino acid identity with E. coli Nfo but also a high resistance to inactivation by EDTA, in the presence of DNA containing AP sites (AP-DNA). These results suggest that YqfS possesses a trinuclear Zn center in which the three metal atoms are intimately coordinated by nine conserved basic residues and two water molecules. Electrophoretic mobility shift assays demonstrated that YqfS possesses structural properties that permit it to bind and scan undamaged DNA as well as to strongly interact with AP-DNA. The ability of yqfS to genetically complement the DNA repair deficiency of an E. coli mutant lacking the major AP-endonucleases Nfo and exonuclease III strongly suggests that its product confers protection to cells against the deleterious effects of oxidative promoters and alkylating agents. Thus, we conclude that YqfS of B. subtilis is a spore-specific protein that has structural and enzymatic properties required to participate in the repair of AP sites and 3′ blocking groups of DNA generated during both spore dormancy and germination.

scholarly journals Properties and efficient scrap-and-build repairing of mechanically sheared 3’ DNA ends

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Yoshiyuki Ohtsubo ◽  
Keiichiro Sakai ◽  
Yuji Nagata ◽  
Masataka Tsuda
Keyword(s):  
Dna Polymerase ◽  
Polymerase Activity ◽  
Phosphate Group ◽  
Hydroxyl Group ◽  
Exonuclease Activity ◽  
Exonuclease Iii ◽  
Crucial Step ◽  
Repair Method ◽  
Dna Ends

Abstract Repairing of DNA termini is a crucial step in a variety of DNA handling techniques. In this study, we investigated mechanically-sheared DNA 3’-ends (MSD3Es) to establish an efficient repair method. As opposed to the canonical view of DNA terminus generated by sonication, we showed that approximately 47% and 20% of MSD3Es carried a phosphate group and a hydroxyl group, respectively. The others had unidentified abnormal terminal structures. Notably, a fraction of the abnormal 3’ termini (about 20% of the total) was not repaired after the removal of 3’ phosphates and T4 DNA polymerase (T4DP) treatment. To overcome this limitation, we devised a reaction, in which the 3’− > 5’ exonuclease activity of exonuclease III (3’− > 5’ exonuclease, insensitive to the 3’ phosphate group) was counterbalanced by the 5’− > 3’ polymerase activity of T4DP. This combined reaction, termed “SB-repairing” (for scrap-and-build repairing), will serve as a useful tool for the efficient repair of MSD3Es.

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