Single‐Stranded Oligodeoxyribonucleotides Are Substrates of Fpg Protein from Escherichia Coli

Ищенко, А. А.; Bulychev, N. V.; Maksakova, G. A.; Johnson, Francis; Nevinsky, Georgy A.

doi:10.1080/713803570

Cited by 15 publications

(6 citation statements)

References 10 publications

(10 reference statements)

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“…This property has been reported in AP endonucleases as hApeI (37) and Chlamydia pneumoniae AP endonuclease IV (38). In addition, other enzymes involved in BER, as several phylogenetically diverse DNA N-glycosylases, have shown activity against damaged bases in ssDNA (39)(40)(41)(42)(43)(44)(45). The presence of abasic sites in ssDNA regions is highly deleterious because, in addition to block replication and transcription, the nicking action of a canonical AP endonuclease could induce lethal dsDNA breaks.…”

Section: Resultsmentioning

confidence: 80%

Intrinsic apurinic/apyrimidinic (AP) endonuclease activity enables Bacillus subtilis DNA polymerase X to recognize, incise, and further repair abasic sites

Baños

Villar

Salas

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The N-glycosidic bond can be hydrolyzed spontaneously or by glycosylases during removal of damaged bases by the base excision repair pathway, leading to the formation of highly mutagenic apurinic/apyrimidinic (AP) sites. Organisms encode for evolutionarily conserved repair machinery, including specific AP endonucleases that cleave the DNA backbone 5′ to the AP site to prime further DNA repair synthesis. We report on the DNA polymerase X from the bacterium Bacillus subtilis (PolX Bs ) that, along with polymerization and 3′-5′-exonuclease activities, possesses an intrinsic AP-endonuclease activity. Both, AP-endonuclease and 3′-5′-exonuclease activities are genetically linked and governed by the same metal ligands located at the C-terminal polymerase and histidinol phosphatase domain of the polymerase. The different catalytic functions of PolX Bs enable it to perform recognition and incision at an AP site and further restoration (repair) of the original nucleotide in a standalone AP-endonuclease-independent way. apurinic/apyrimidinic-lyase | site-directed mutagenesis G enomes are continuously insulted by exogenous and endogenous genotoxic agents, as ionizing radiation, drugs, and (by)products of normal cellular metabolism that generate reactive oxygen species (ROS) leading to mainly nonbulky DNA lesions (1). Base excision repair (BER) is the major pathway involved in the removal of this type of damage, and its importance for cell survival is reflected by its conservation from bacteria to eukaryotes (2). During the first steps of BER, highly mutagenic apurinic/apyrimidinic (AP) intermediates are produced as a result of hydrolytic cleavage of the altered base-sugar bond by mono-(class II) and/or bifunctional (class I) DNA N-glycosylases (ref. 3 and references therein), or from spontaneous DNA base loss, causing replication and transcription inhibition if left unrepaired (4, 5). AP endonucleases play a crucial role in BER because they recognize the abasic residue and hydrolyze the phosphodiester bond 5′ to the AP site, leaving a gapped DNA intermediate with an extendable 3′-OH end (ref. 2 and references therein).Members of the family X of DNA polymerases (hereafter, PolX) are widely spread in nature from virus to humans, being involved in the DNA synthesis step during BER and DNA double-strand break repair by virtue of a common Polβ-like core adapted to fill the gapped DNA intermediates very proficiently (6-9).PolX Bs (570-aa long) is a prototypic bacterial/archaeal PolX member from Bacillus subtilis with a N-terminal Polβ-like core (residues 1-317) responsible for catalysis of DNA polymerization (10), and a C-terminal polymerase and histidinol phosphatase (PHP) domain (residues 333-570) containing highly conserved residues that catalyze a Mn 2þ -dependent 3′-5′-exonuclease activity (11-14), which shows a preferential processing of unannealed 3′ termini (12). Due to this fact and to its adaptation to perform filling of small gaps (10), PolX Bs was proposed to play a potential role in the DNA synthesis step of repair p...

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Section: Resultsmentioning

confidence: 80%

Intrinsic apurinic/apyrimidinic (AP) endonuclease activity enables Bacillus subtilis DNA polymerase X to recognize, incise, and further repair abasic sites

Baños

Villar

Salas

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Although E. coli Fpg processes both ss and ds 8-oxodG oligomers, it has been shown that E. coli Fpg processes ds DNA more efficiently than ss DNA as reported from in vitro experiments by Ishchenko et al The K m and k cat /K m for ds 8-oxodG DNA are 6.0 nM and 500 μM –1 min –1 whereas K m and k cat /K m for ss 8-oxodG DNA are 1.0 μM and 0.135 μM –1 min –1 , respectively . The binding preference of Fpg to 8-oxodG base-paired opposite the four nucleotides is in the following order of affinity: dC (8.9 ± 2.1 nM) > dG (21 ± 6 nM) > dT (29 ± 6 nM) > dA (340 ± 29 nM) …”

Section: Resultsmentioning

confidence: 88%

“…The presence of a second carbonyl group at C8 position in 8-oxoguanosine leads to reorganization of the electron density in the purine ring, resulting in an absorption spectrum markedly different from that of guanosine spectrum as well as a Raman intensity pattern distinct from that of the guanosine Raman spectrum . Since E. coli Fpg has been shown to process single and double-stranded 8-oxodG containing oligonucleotides, we have studied complexes of E3Q Fpg with both ss and ds stranded 8-oxodG oligomers.…”

Section: Resultsmentioning

confidence: 99%

Mechanism of Discrimination of 8-Oxoguanosine versus Guanosine by Escherichia coli Fpg

Jayanth

Puranik

2017

J. Phys. Chem. B

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The mutagenic 8-oxoguanosine monophosphate, the predominant product of DNA oxidation, is excised by formamidopyrimidine glycosylase (Fpg) in bacteria. The mechanism of recognition of 8-oxodG, which differs subtly from its normal counterpart, guanosine monophosphate (dG), by Escherichia coli Fpg remains elusive due to the lack of structural data of E. coli Fpg bound to 8-oxodG. Here, we present solution-state structure of 8-oxodG oligomer bound to E. coli E3Q Fpg using UV resonance Raman (UVRR) spectroscopy. The vibrational spectra report on the π-stacking and hydrogen bonding interactions established by 8-oxodG with E. coli E3Q Fpg. Furthermore, we report on the interactions of E. coli E3Q Fpg with the normal, undamaged nucleotide, dG. We show that E. coli Fpg recognizes 8-oxodG and dG through their C2-amino group but only 8-oxodG forms extensive contacts with E. coli Fpg. Our findings provide a basis for mechanism of lesion recognition by E. coli Fpg.

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“…Notably MtuFpg1 and MtuNth showed no activity against any lesion in single-stranded DNA, unlike EcoFpg which recognizes 8-oxoG in single-stranded DNA (Fig. 4A and [62,63]); essentially complete cleavage was observed for all enzymes tested on the single-stranded oligodeoxynucleotide containing an AP site (Fig. 4A).…”

Section: Resultsmentioning

confidence: 99%

“…4A and [48,76]) exhibit lyase activity on single-stranded substrates and EcoFpg recognizes a single-stranded substrate containing 8-oxoG (Fig. 4A and [62,63]). Taken together with the recent observations on Nei orthologs from Mimivirus [64] as well as Fpg homologs derived from Candida albicans and Arabidopsis thaliana [34], the data suggest that recognizing lesions in single-stranded DNA as well as other DNA structures containing single-stranded regions is not exclusively a function of the human/mammalian NEILs [26,29,61,75,77], but rather a characteristic of the Fpg/Nei family members in general.…”

Section: Discussionmentioning

confidence: 99%

The oxidative DNA glycosylases of Mycobacterium tuberculosis exhibit different substrate preferences from their Escherichia coli counterparts

et al. 2010

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The DNA glycosylases that remove oxidized DNA bases fall into two general families: the Fpg/Nei family and the Nth superfamily. Based on protein sequence alignments, we identified four putative Fpg/Nei family members, as well as a putative Nth protein in Mycobacterium tuberculosis H37Rv. All four Fpg/Nei proteins were successfully overexpressed using a bicistronic vector created in our laboratory. The MtuNth protein was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligodeoxynucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GC/MS) analysis of products released from γ-irradiated DNA. MtuFpg1 has a substrate specificity similar to that of EcoFpg. Both EcoFpg and MtuFpg1 are more efficient at removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG). However, MtuFpg1 shows a substantially increased opposite base discrimination compared to EcoFpg. MtuFpg2 contains only the C-terminal domain of an Fpg protein and has no detectable DNA binding activity or DNA glycosylase/lyase activity and thus appears to be a pseudogene. MtuNei1 recognizes oxidized pyrimidines on both double-stranded and single-stranded DNA and exhibits uracil DNA glycosylase activity. MtuNth recognizes a variety of oxidized bases, including urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5-OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd). Both MtuNei1 and MtuNth excise thymine glycol (Tg); however, MtuNei1 strongly prefers the (5R) isomers, whereas MtuNth recognizes only the (5S) isomers. MtuNei2 did not demonstrate activity in vitro as a recombinant protein, but like MtuNei1 when expressed in Escherichia coli, it decreased the spontaneous mutation frequency of both the fpg mutY nei triple and nei nth double mutants, suggesting that MtuNei2 is functionally active in vivo recognizing both guanine and cytosine oxidation products. The kinetic b Current address: Mylan Pharmaceuticals, 3711 Collins Ferry Rd, Morgantown, WV 26505Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

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Single‐Stranded Oligodeoxyribonucleotides Are Substrates of Fpg Protein from Escherichia Coli

Abstract: SummaryThe interaction of Escherichia coli Fpg protein, which catalyzes excision of several damaged purine bases including 8-oxoguanin e (oxoG) from DNA with a set of single-(ss) and double-stranded (

Cited by 15 publications

References 10 publications

Intrinsic apurinic/apyrimidinic (AP) endonuclease activity enables Bacillus subtilis DNA polymerase X to recognize, incise, and further repair abasic sites

Intrinsic apurinic/apyrimidinic (AP) endonuclease activity enables Bacillus subtilis DNA polymerase X to recognize, incise, and further repair abasic sites

Mechanism of Discrimination of 8-Oxoguanosine versus Guanosine by Escherichia coli Fpg

The oxidative DNA glycosylases of Mycobacterium tuberculosis exhibit different substrate preferences from their Escherichia coli counterparts

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