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1.
PeerJ ; 11: e16671, 2023.
Article in English | MEDLINE | ID: mdl-38107580

ABSTRACT

Background: Francis Crick's central dogma provides a residue-by-residue mechanistic explanation of the flow of genetic information in living systems. However, this principle may not be sufficient for explaining how random mutations cause continuous variation of quantitative highly polygenic complex traits. Chargaff's second parity rule (CSPR), also referred to as intrastrand DNA symmetry, defined as near-exact equalities G ≈ C and A ≈ T within a single DNA strand, is a statistical property of cellular genomes. The phenomenon of intrastrand DNA symmetry was discovered more than 50 years ago; at present, it remains unclear what its biological role is, what the mechanisms are that force cellular genomes to comply strictly with CSPR, and why genomes of certain noncellular organisms have broken intrastrand DNA symmetry. The present work is aimed at studying a possible link between intrastrand DNA symmetry and the origin of genetic interactions in quantitative traits. Methods: Computational analysis of single-nucleotide polymorphisms in human and mouse populations and of nucleotide composition biases at different codon positions in bacterial and human proteomes. Results: The analysis of mutation spectra inferred from single-nucleotide polymorphisms observed in murine and human populations revealed near-exact equalities of numbers of reverse complementary mutations, indicating that random genetic variations obey CSPR. Furthermore, nucleotide compositions of coding sequences proved to be statistically interwoven via CSPR because pyrimidine bias at the 3rd codon position compensates purine bias at the 1st and 2nd positions. Conclusions: According to Fisher's infinitesimal model, we propose that accumulation of reverse complementary mutations results in a continuous phenotypic variation due to small additive effects of statistically interwoven genetic variations. Therefore, additive genetic interactions can be inferred as a statistical entanglement of nucleotide compositions of separate genetic loci. CSPR challenges the neutral theory of molecular evolution-because all random mutations participate in variation of a trait-and provides an alternative solution to Haldane's dilemma by making a gene function diffuse. We propose that CSPR is symmetry of Fisher's infinitesimal model and that genetic information can be transferred in an implicit contactless manner.


Subject(s)
DNA , Evolution, Molecular , Animals , Humans , Mice , DNA/chemistry , Mutation , Nucleotides/genetics , Codon
2.
Nucleic Acids Res ; 51(12): 6321-6336, 2023 07 07.
Article in English | MEDLINE | ID: mdl-37216593

ABSTRACT

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and as base excision repair (BER) intermediates. AP sites and their derivatives readily trap DNA-bound proteins, resulting in DNA-protein cross-links. Those are subject to proteolysis but the fate of the resulting AP-peptide cross-links (APPXLs) is unclear. Here, we report two in vitro models of APPXLs synthesized by cross-linking of DNA glycosylases Fpg and OGG1 to DNA followed by trypsinolysis. The reaction with Fpg produces a 10-mer peptide cross-linked through its N-terminus, while OGG1 yields a 23-mer peptide attached through an internal lysine. Both adducts strongly blocked Klenow fragment, phage RB69 polymerase, Saccharolobus solfataricus Dpo4, and African swine fever virus PolX. In the residual lesion bypass, mostly dAMP and dGMP were incorporated by Klenow and RB69 polymerases, while Dpo4 and PolX used primer/template misalignment. Of AP endonucleases involved in BER, Escherichia coli endonuclease IV and its yeast homolog Apn1p efficiently hydrolyzed both adducts. In contrast, E. coli exonuclease III and human APE1 showed little activity on APPXL substrates. Our data suggest that APPXLs produced by proteolysis of AP site-trapped proteins may be removed by the BER pathway, at least in bacterial and yeast cells.


Subject(s)
DNA Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase , Animals , Humans , African Swine Fever Virus/metabolism , DNA Damage , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Endonucleases/metabolism , Escherichia coli/metabolism , Peptides , Saccharomyces cerevisiae/metabolism , Swine , DNA Polymerase beta/metabolism
3.
Adv Exp Med Biol ; 1241: 77-100, 2020.
Article in English | MEDLINE | ID: mdl-32383117

ABSTRACT

A variety of endogenous and exogenous factors induce chemical and structural alterations in cellular DNA in addition to the errors occurring throughout DNA synthesis. These types of DNA damage are cytotoxic, miscoding or both and are believed to be at the origin of cancer and other age-related diseases. A human cell, aside from nuclear DNA, contains thousands of copies of mitochondrial DNA (mtDNA), a double-stranded, circular molecule of 16,569 bp. It has been proposed that mtDNA is a critical target of reactive oxygen species: by-products of oxidative phosphorylation that are generated in the organelle during aerobic respiration. Indeed, oxidative damage to mtDNA is more extensive and persistent as compared to that to nuclear DNA. Although transversions are the hallmark of mutations induced by reactive oxygen species, paradoxically, the majority of mtDNA mutations that occur during ageing and cancer are transitions. Furthermore, these mutations show a striking strand orientation bias: T→C/G→A transitions preferentially occur on the light strand, whereas C→T/A→G on the heavy strand of mtDNA. Here, we propose that the majority of mtDNA progenies, created after multiple rounds of DNA replication, are derived from the heavy strand only, owing to asymmetric replication of the DNA strand anchored to the inner membrane via the D-loop structure.


Subject(s)
DNA Repair , DNA, Mitochondrial/genetics , Mitochondria/genetics , Mutagenesis , Vertebrates , Animals , Humans , Vertebrates/genetics
4.
Mutagenesis ; 35(1): 107-118, 2020 02 13.
Article in English | MEDLINE | ID: mdl-31782485

ABSTRACT

Genotoxic stress generates single- and double-strand DNA breaks either through direct damage by reactive oxygen species or as intermediates of DNA repair. Failure to detect and repair DNA strand breaks leads to deleterious consequences such as chromosomal aberrations, genomic instability and cell death. DNA strand breaks disrupt the superhelical state of cellular DNA, which further disturbs the chromatin architecture and gene activity regulation. Proteins from the poly(ADP-ribose) polymerase (PARP) family, such as PARP1 and PARP2, use NAD+ as a substrate to catalyse the synthesis of polymeric chains consisting of ADP-ribose units covalently attached to an acceptor molecule. PARP1 and PARP2 are regarded as DNA damage sensors that, upon activation by strand breaks, poly(ADP-ribosyl)ate themselves and nuclear acceptor proteins. Noteworthy, the regularly branched structure of poly(ADP-ribose) polymer suggests that the mechanism of its synthesis may involve circular movement of PARP1 around the DNA helix, with a branching point in PAR corresponding to one complete 360° turn. We propose that PARP1 stays bound to a DNA strand break end, but rotates around the helix displaced by the growing poly(ADP-ribose) chain, and that this rotation could introduce positive supercoils into damaged chromosomal DNA. This topology modulation would enable nucleosome displacement and chromatin decondensation around the lesion site, facilitating the access of DNA repair proteins or transcription factors. PARP1-mediated DNA supercoiling can be transmitted over long distances, resulting in changes in the high-order chromatin structures. The available structures of PARP1 are consistent with the strand break-induced PAR synthesis as a driving force for PARP1 rotation around the DNA axis.


Subject(s)
DNA Repair , DNA/chemistry , DNA/metabolism , Poly (ADP-Ribose) Polymerase-1/chemistry , Poly (ADP-Ribose) Polymerase-1/metabolism , Animals , DNA Breaks, Double-Stranded , DNA Damage , DNA, Superhelical , Drosophila/genetics , Fungi/genetics , Humans , Mice , Nucleic Acid Conformation , Poly (ADP-Ribose) Polymerase-1/genetics , Poly ADP Ribosylation , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism
5.
Sci Rep ; 7(1): 17438, 2017 12 12.
Article in English | MEDLINE | ID: mdl-29234069

ABSTRACT

Interstrand cross-links (ICLs) are highly cytotoxic DNA lesions that block DNA replication and transcription by preventing strand separation. Previously, we demonstrated that the bacterial and human DNA glycosylases Nei and NEIL1 excise unhooked psoralen-derived ICLs in three-stranded DNA via hydrolysis of the glycosidic bond between the crosslinked base and deoxyribose sugar. Furthermore, NEIL3 from Xenopus laevis has been shown to cleave psoralen- and abasic site-induced ICLs in Xenopus egg extracts. Here we report that human NEIL3 cleaves psoralen-induced DNA-DNA cross-links in three-stranded and four-stranded DNA substrates to generate unhooked DNA fragments containing either an abasic site or a psoralen-thymine monoadduct. Furthermore, while Nei and NEIL1 also cleave a psoralen-induced four-stranded DNA substrate to generate two unhooked DNA duplexes with a nick, NEIL3 targets both DNA strands in the ICL without generating single-strand breaks. The DNA substrate specificities of these Nei-like enzymes imply the occurrence of long uninterrupted three- and four-stranded crosslinked DNA-DNA structures that may originate in vivo from DNA replication fork bypass of an ICL. In conclusion, the Nei-like DNA glycosylases unhook psoralen-derived ICLs in various DNA structures via a genuine repair mechanism in which complex DNA lesions can be removed without generation of highly toxic double-strand breaks.


Subject(s)
Cross-Linking Reagents/pharmacology , DNA Glycosylases/metabolism , DNA/drug effects , DNA/metabolism , Ficusin/pharmacology , N-Glycosyl Hydrolases/metabolism , DNA Breaks , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Escherichia coli , Escherichia coli Proteins/metabolism , Humans , Hydrolysis , Nucleic Acid Conformation/drug effects
6.
Mol Biosyst ; 13(12): 2638-2649, 2017 Nov 21.
Article in English | MEDLINE | ID: mdl-29051947

ABSTRACT

In all organisms, DNA glycosylases initiate base excision repair pathways resulting in removal of aberrant bases from DNA. Human SMUG1 belongs to the superfamily of uracil-DNA glycosylases catalyzing the hydrolysis of the N-glycosidic bond of uridine and uridine lesions bearing oxidized groups at C5: 5-hydroxymethyluridine (5hmU), 5-formyluridine (5fU), and 5-hydroxyuridine (5hoU). An apurinic/apyrimidinic (AP) site formed as the product of an N-glycosylase reaction is tightly bound to hSMUG1, thus inhibiting the downstream action of AP-endonuclease APE1. The steady-state kinetic parameters (kcat and KM; obtained from the literature) correspond to the enzyme turnover process limited by the release of hSMUG1 from the complex with the AP-site. In the present study, our objective was to carry out a stopped-flow fluorescence analysis of the interaction of hSMUG1 with a DNA substrate containing a dU:dG base pair to follow the pre-steady-state kinetics of conformational changes in both molecules. A comparison of kinetic data obtained by means of Trp and 2-aminopurine fluorescence and Förster resonance energy transfer (FRET) detection allowed us to elucidate the stages of specific and nonspecific DNA binding, to propose the mechanism of damaged base recognition by hSMUG1, and to determine the true rate of the catalytic step. Our results shed light on the kinetic mechanism underlying the initiation of base excision repair by hSMUG1 using the "wedge" strategy for DNA lesion search.


Subject(s)
Uracil-DNA Glycosidase/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Fluorescence Resonance Energy Transfer , Humans , Kinetics , Uridine/analogs & derivatives , Uridine/chemistry
7.
Free Radic Biol Med ; 107: 266-277, 2017 06.
Article in English | MEDLINE | ID: mdl-27890638

ABSTRACT

In cellular organisms composition of DNA is constrained to only four nucleobases A, G, T and C, except for minor DNA base modifications such as methylation which serves for defence against foreign DNA or gene expression regulation. Interestingly, this severe evolutionary constraint among other things demands DNA repair systems to discriminate between regular and modified bases. DNA glycosylases specifically recognize and excise damaged bases among vast majority of regular bases in the base excision repair (BER) pathway. However, the mismatched base pairs in DNA can occur from a spontaneous conversion of 5-methylcytosine to thymine and DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved special DNA repair systems that target the non-damaged DNA strand in a duplex to remove mismatched regular DNA bases. Mismatch-specific adenine- and thymine-DNA glycosylases (MutY/MUTYH and TDG/MBD4, respectively) initiated BER and mismatch repair (MMR) pathways can recognize and remove normal DNA bases in mismatched DNA duplexes. Importantly, in DNA repair deficient cells bacterial MutY, human TDG and mammalian MMR can act in the aberrant manner: MutY and TDG removes adenine and thymine opposite misincorporated 8-oxoguanine and damaged adenine, respectively, whereas MMR removes thymine opposite to O6-methylguanine. These unusual activities lead either to mutations or futile DNA repair, thus indicating that the DNA repair pathways which target non-damaged DNA strand can act in aberrant manner and introduce genome instability in the presence of unrepaired DNA lesions. Evidences accumulated showing that in addition to the accumulation of oxidatively damaged DNA in cells, the aberrant DNA repair can also contribute to cancer, brain disorders and premature senescence. For example, the aberrant BER and MMR pathways for oxidized guanine residues can lead to trinucleotide expansion that underlies Huntington's disease, a severe hereditary neurodegenerative syndrome. This review summarises the present knowledge about the aberrant DNA repair pathways for oxidized base modifications and their possible role in age-related diseases.


Subject(s)
DNA Damage , DNA Repair/genetics , DNA/metabolism , Neoplasms/genetics , Neurodegenerative Diseases/genetics , Animals , Cellular Senescence , DNA/chemistry , Humans , Oxidation-Reduction , Oxidative Stress
8.
Nucleic Acids Res ; 44(19): 9279-9295, 2016 Nov 02.
Article in English | MEDLINE | ID: mdl-27471034

ABSTRACT

Poly(ADP-ribose) polymerases (PARPs/ARTDs) use nicotinamide adenine dinucleotide (NAD+) to catalyse the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARPs act on single- and double-stranded DNA breaks by recruiting DNA repair factors. Here, in in vitro biochemical experiments, we found that the mammalian PARP1 and PARP2 proteins can directly ADP-ribosylate the termini of DNA oligonucleotides. PARP1 preferentially catalysed covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 3'-cordycepin, 5'- and 3'-phosphate and also to 5'-phosphate of a single-stranded oligonucleotide. PARP2 preferentially ADP-ribosylated the nicked/gapped DNA duplexes containing 5'-phosphate at the double-stranded termini. PAR glycohydrolase (PARG) restored native DNA structure by hydrolysing PAR-DNA adducts generated by PARP1 and PARP2. Biochemical and mass spectrometry analyses of the adducts suggested that PARPs utilise DNA termini as an alternative to 2'-hydroxyl of ADP-ribose and protein acceptor residues to catalyse PAR chain initiation either via the 2',1″-O-glycosidic ribose-ribose bond or via phosphodiester bond formation between C1' of ADP-ribose and the phosphate of a terminal deoxyribonucleotide. This new type of post-replicative modification of DNA provides novel insights into the molecular mechanisms underlying biological phenomena of ADP-ribosylation mediated by PARPs.


Subject(s)
DNA Breaks, Double-Stranded , DNA/genetics , DNA/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Animals , Catalysis , DNA Adducts , Humans , Hydrolysis , Mice , NAD/metabolism , Poly (ADP-Ribose) Polymerase-1/metabolism , Protein Binding , Substrate Specificity
9.
J Biol Chem ; 290(23): 14338-49, 2015 Jun 05.
Article in English | MEDLINE | ID: mdl-25869130

ABSTRACT

Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3'-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln(41) and Leu(81) as DNA lesion sensors.


Subject(s)
DNA, Bacterial/metabolism , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Base Sequence , DNA Repair , DNA, Bacterial/chemistry , Deoxyribonuclease (Pyrimidine Dimer)/chemistry , Escherichia coli/chemistry , Escherichia coli Proteins/chemistry , Kinetics , Models, Molecular , Nucleic Acid Conformation , Protein Conformation
10.
Biochim Biophys Acta ; 1840(10): 3042-51, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25086253

ABSTRACT

BACKGROUND: DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through ß- or ß,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases. METHODS: Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4(cat) and UNG from different structural superfamilies were used. RESULTS: We found that all DNA glycosylases may utilise direct protein-protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. CONCLUSIONS: We hypothesize a fast "flip-flop" exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4(cat), AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. GENERAL SIGNIFICANCE: Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway.


Subject(s)
DNA Damage , DNA Glycosylases/chemistry , DNA Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry , Fluorescence , Humans
11.
Nucleic Acids Res ; 42(10): 6300-13, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24692658

ABSTRACT

The human thymine-DNA glycosylase (TDG) initiates the base excision repair (BER) pathway to remove spontaneous and induced DNA base damage. It was first biochemically characterized for its ability to remove T mispaired with G in CpG context. TDG is involved in the epigenetic regulation of gene expressions by protecting CpG-rich promoters from de novo DNA methylation. Here we demonstrate that TDG initiates aberrant repair by excising T when it is paired with a damaged adenine residue in DNA duplex. TDG targets the non-damaged DNA strand and efficiently excises T opposite of hypoxanthine (Hx), 1,N(6)-ethenoadenine, 7,8-dihydro-8-oxoadenine and abasic site in TpG/CpX context, where X is a modified residue. In vitro reconstitution of BER with duplex DNA containing Hx•T pair and TDG results in incorporation of cytosine across Hx. Furthermore, analysis of the mutation spectra inferred from single nucleotide polymorphisms in human population revealed a highly biased mutation pattern within CpG islands (CGIs), with enhanced mutation rate at CpA and TpG sites. These findings demonstrate that under experimental conditions used TDG catalyzes sequence context-dependent aberrant removal of thymine, which results in TpG, CpA→CpG mutations, thus providing a plausible mechanism for the putative evolutionary origin of the CGIs in mammalian genomes.


Subject(s)
CpG Islands , DNA Repair , Mutation , Thymine DNA Glycosylase/metabolism , Adenine/chemistry , Animals , Base Pair Mismatch , Cells, Cultured , DNA/metabolism , DNA Damage , Humans , Mice , Oligonucleotides/chemistry , Polymorphism, Single Nucleotide , Thymine/chemistry , Thymine/metabolism
12.
Biochim Biophys Acta ; 1840(1): 387-95, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24096108

ABSTRACT

BACKGROUND: Extensive structural studies of human DNA glycosylase hOGG1 have revealed essential conformational changes of the enzyme. However, at present there is little information about the time scale of the rearrangements of the protein structure as well as the dynamic behavior of individual amino acids. METHODS: Using pre-steady-state kinetic analysis with Trp and 2-aminopurine fluorescence detection the conformational dynamics of hOGG1 wild-type (WT) and mutants Y203W, Y203A, H270W, F45W, F319W and K249Q as well as DNA-substrates was examined. RESULTS: The roles of catalytically important amino acids F45, Y203, K249, H270, and F319 in the hOGG1 enzymatic pathway and their involvement in the step-by-step mechanism of oxidative DNA lesion recognition and catalysis were elucidated. CONCLUSIONS: The results show that Tyr-203 participates in the initial steps of the lesion site recognition. The interaction of the His-270 residue with the oxoG base plays a key role in the insertion of the damaged base into the active site. Lys-249 participates not only in the catalytic stages but also in the processes of local duplex distortion and flipping out of the oxoG residue. Non-damaged DNA does not form a stable complex with hOGG1, although a complex with a flipped out guanine base can be formed transiently. GENERAL SIGNIFICANCE: The kinetic data obtained in this study significantly improves our understanding of the molecular mechanism of lesion recognition by hOGG1.


Subject(s)
DNA Damage/genetics , DNA Glycosylases/metabolism , Guanine/analogs & derivatives , Mutant Proteins/metabolism , Binding Sites , Catalysis , DNA Glycosylases/genetics , DNA Repair , Guanine/metabolism , Humans , Kinetics , Molecular Conformation , Mutant Proteins/genetics , Mutation/genetics , Spectrometry, Fluorescence
13.
Proc Natl Acad Sci U S A ; 110(39): E3695-703, 2013 Sep 24.
Article in English | MEDLINE | ID: mdl-24023064

ABSTRACT

Spontaneous hydrolytic deamination of cytosine to uracil (U) in DNA is a constant source of genome instability in cells. This mutagenic process is greatly enhanced at high temperatures and in single-stranded DNA. If not repaired, these uracil residues give rise to C → T transitions, which are the most common spontaneous mutations occurring in living organisms and are frequently found in human tumors. In the majority of species, uracil residues are removed from DNA by specific uracil-DNA glycosylases in the base excision repair pathway. Alternatively, in certain archaeal organisms, uracil residues are eliminated by apurinic/apyrimidinic (AP) endonucleases in the nucleotide incision repair pathway. Here, we characterized the substrate specificity of the major human AP endonuclease 1, APE1, toward U in duplex DNA. APE1 cleaves oligonucleotide duplexes containing a single U⋅G base pair; this activity depends strongly on the sequence context and the base opposite to U. The apparent kinetic parameters of the reactions show that APE1 has high affinity for DNA containing U but cleaves the DNA duplex at an extremely low rate. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of a U⋅G duplex generates the expected DNA fragments containing a 5'-terminal deoxyuridine monophosphate. The fact that U in duplex DNA is recognized and cleaved by APE1 in vitro suggests that this property of the exonuclease III family of AP endonucleases is remarkably conserved from Archaea to humans. We propose that nucleotide incision repair may act as a backup pathway to base excision repair to remove uracils arising from cytosine deamination.


Subject(s)
DNA Repair , DNA/metabolism , Nucleotides/metabolism , Signal Transduction , Uracil/metabolism , Base Sequence , Biocatalysis , Cell Line , Cytosine/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Deamination , Humans , Kinetics , Methanosarcina/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate Specificity , Sulfites , Thymine DNA Glycosylase/metabolism
14.
FEBS Lett ; 587(18): 3129-34, 2013 Sep 17.
Article in English | MEDLINE | ID: mdl-23954288

ABSTRACT

CpG dinucleotides are targets for epigenetic methylation, many of them bearing 5-methylcytosine (mCyt) in the human genome. Guanine in this context can be easily oxidized to 8-oxoguanine (oxoGua), which is repaired by 8-oxoguanine-DNA glycosylase (OGG1). We have studied how methylation affects the efficiency of oxoGua excision from damaged CpG dinucleotides. Methylation of the adjacent cytosine moderately decreased the oxoGua excision rate while methylation opposite oxoGua lowered the rate of product release. Cytosine methylation abolished stimulation of OGG1 by repair endonuclease APEX1. The OGG1 S326C polymorphic variant associated with lung cancer showed poorer base excision and lost sensitivity to the opposite-base methylation. The overall repair in the system reconstituted from purified proteins decreased for CpG with mCyt in the damaged strand.


Subject(s)
CpG Islands/genetics , DNA Glycosylases/metabolism , Epigenesis, Genetic , Guanine/analogs & derivatives , Neoplasm Proteins/metabolism , 5-Methylcytosine/metabolism , DNA/metabolism , DNA Damage , DNA Glycosylases/genetics , DNA Methylation , DNA Polymerase II/genetics , DNA Polymerase II/metabolism , DNA Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Guanine/metabolism , Humans , Kinetics , Mutation , Neoplasm Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity
15.
Proc Natl Acad Sci U S A ; 110(33): E3071-80, 2013 Aug 13.
Article in English | MEDLINE | ID: mdl-23898172

ABSTRACT

8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine generated in DNA by both endogenous oxidative stress and ionizing radiation are helix-distorting lesions and strong blocks for DNA replication and transcription. In duplex DNA, these lesions are repaired in the nucleotide excision repair (NER) pathway. However, lesions at DNA strand breaks are most likely poor substrates for NER. Here we report that the apurinic/apyrimidinic (AP) endonucleases--Escherichia coli Xth and human APE1--can remove 5'S cdA (S-cdA) at 3' termini of duplex DNA. In contrast, E. coli Nfo and yeast Apn1 are unable to carry out this reaction. None of these enzymes can remove S-cdA adduct located at 1 or more nt away from the 3' end. To understand the structural basis of 3' repair activity, we determined a high-resolution crystal structure of E. coli Nfo-H69A mutant bound to a duplex DNA containing an α-anomeric 2'-deoxyadenosine:T base pair. Surprisingly, the structure reveals a bound nucleotide incision repair (NIR) product with an abortive 3'-terminal dC close to the scissile position in the enzyme active site, providing insight into the mechanism for Nfo-catalyzed 3'→5' exonuclease function and its inhibition by 3'-terminal S-cdA residue. This structure was used as a template to model 3'-terminal residues in the APE1 active site and to explain biochemical data on APE1-catalyzed 3' repair activities. We propose that Xth and APE1 may act as a complementary repair pathway to NER to remove S-cdA adducts from 3' DNA termini in E. coli and human cells, respectively.


Subject(s)
DNA Adducts/metabolism , DNA Repair/physiology , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Deoxyribonuclease IV (Phage T4-Induced)/chemistry , Escherichia coli Proteins/chemistry , Exonucleases/metabolism , Models, Molecular , Protein Conformation , DNA Adducts/chemistry , DNA Repair/genetics , Denaturing Gradient Gel Electrophoresis , Deoxyadenosines/chemistry , Deoxyadenosines/metabolism , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/chemistry , Deoxyguanosine/metabolism , Escherichia coli , Humans , Molecular Structure , Oligonucleotides/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , X-Ray Diffraction , Yeasts
16.
DNA Repair (Amst) ; 11(10): 811-22, 2012 Oct 01.
Article in English | MEDLINE | ID: mdl-22819077

ABSTRACT

Caenorhabditis elegans possesses two distinct DNA repair enzymes EXO-3 and APN-1 that have been identified by cross-specie complementation analysis of the Saccharomyces cerevisiae apn1Δapn2Δtpp1Δ triple mutant deficient in the ability to repair apurinic/apyrimidinc (AP) sites and DNA strand breaks with blocked 3'-ends. While purified EXO-3 directly incises AP sites and removes 3'-blocking groups, such characterization has not been previously reported for APN-1. We recently documented that C. elegans knockdown for apn-1 is unable to maintain integrity of the genome. Despite the presence of EXO-3, the apn-1 knockdown animals are also defective in the division of the P1 blastomere, an observation consistent with the accumulation of unrepaired DNA lesions suggesting a unique role for APN-1 DNA repair functions. Herein, we show that C. elegans APN-1 is stably expressed as GST-fusion protein in S. cerevisiae only when it carries a nuclear localization signal, and with this requirement rescued the DNA repair defects of the S. cerevisiae apn1Δapn2Δtpp1Δ triple mutant. We purified the APN-1 from the yeast expression system and established that it displays AP endonuclease and 3'-diesterase activities. In addition, we showed that APN-1 also possesses a 3'- to 5'-exonuclease and the nucleotide incision repair activity. This latter activity is capable of directly incising DNA at the 5'-side of various oxidatively damaged bases, as previously observed for Escherichia coli endonuclease IV and S. cerevisiae Apn1, underscoring the importance of this family of enzymes in removing these types of lesions. Glycine substitution of the conserved amino acid residue Glu261 of APN-1, corresponding to Glu145 involved in coordinating Zn(2+) ions in the active site pocket of E. coli endonuclease IV, resulted in an inactive variant that lose the ability to rescue the DNA repair defects of S. cerevisiae apn1Δapn2Δtpp1Δ mutant. Interestingly, the Glu261Gly variant did not sustain purification and yielded a truncated polypeptide. These data suggest that the Glu261 residue of APN-1 may have a broader role in maintaining the structure of the protein.


Subject(s)
Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/metabolism , DNA Repair Enzymes/chemistry , DNA Repair Enzymes/metabolism , DNA Repair , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Amino Acid Sequence , Animals , Caenorhabditis elegans/enzymology , Caenorhabditis elegans Proteins/genetics , DNA Breaks , DNA Repair Enzymes/genetics , DNA, Fungal/genetics , DNA, Fungal/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Endodeoxyribonucleases/genetics , Glycine/genetics , Molecular Sequence Data , Nuclear Localization Signals/chemistry , Nucleotidases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Zinc/chemistry
17.
PLoS One ; 6(9): e24063, 2011.
Article in English | MEDLINE | ID: mdl-21912662

ABSTRACT

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme in the base excision repair (BER) and nucleotide incision repair (NIR) pathways. We recently analyzed the conformational dynamics and kinetic mechanism of wild-type (wt) protein, in a stopped-flow fluorescence study. In this study, we investigated the mutant enzyme APE1K98A using the same approach. Lys98 was known to hydrogen bond to the carboxyl group of Asp70, a residue implicated in binding the divalent metal ion. Our data suggested that the conformational selection and induced fit occur during the enzyme action. We expanded upon the evidence that APE1 can pre-exist in two conformations. The isomerization of an enzyme-product complex in the BER process and the additional isomerization stage of enzyme-substrate complex in the NIR process were established for APE1K98A. These stages had not been registered for the wtAPE1. We found that the K98A substitution resulted in a 12-fold reduction of catalytic constant of 5'-phosphodiester bond hydrolysis in (3-hydroxytetrahydrofuran-2-yl)methyl phosphate (F, tetrahydrofuran) containing substrate, and in 200-fold reduction in 5,6-dihydrouridine (DHU) containing substrate. Thus, the K98A substitution influenced NIR more than BER. We demonstrated that the K98A mutation influenced the formation of primary unspecific enzyme-substrate complex in a complicated manner, depending on the Mg(2+) concentration and pH. This mutation obstructed the induced fit of enzyme in the complex with undamaged DNA and F-containing DNA and appreciably decreased the stability of primary complex upon interaction of enzyme with DNA, containing the natural apurinic/apyrimidinic (AP) site. Furthermore, it significantly delayed the activation of the less active form of enzyme during NIR and slowed down the conformational conversion of the complex of enzyme with the cleavage product of DHU-substrate. Our data revealed that APE1 uses the same active site to catalyze the cleavage of DHU- and AP-substrates.


Subject(s)
Amino Acid Substitution , DNA Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Lysine , Base Sequence , Buffers , Catalytic Domain , DNA/genetics , DNA/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry , Humans , Hydrolysis , Kinetics , Ligands , Mutation , Spectrometry, Fluorescence , Uridine/analogs & derivatives , Uridine/metabolism
18.
Plant Physiol Biochem ; 49(10): 1155-64, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21856164

ABSTRACT

Cereal aleurone cells are specialized endosperm cells that produce enzymes to hydrolyze the starchy endosperm during germination. Aleurone cells can undergo programmed cell death (PCD) when incubated in the presence of gibberellic acid (GA) in contrast to abscisic acid (ABA) which inhibits the process. The progression of PCD in aleurone layer cells of wheat grain is accompanied by an increase in deoxyribonuclease (DNase) activities and the internucleosomal degradation of nuclear DNA. Reactive oxygen species (ROS) are increased during PCD in the aleurone cells owing to the ß-oxidation of triglycerides and inhibition of the antioxidant enzymes possibly leading to extensive oxidative damage to DNA. ROS generate mainly non-bulky DNA base lesions which are removed in the base excision repair (BER) pathway, initiated by the DNA glycosylases. At present, very little is known about oxidative DNA damage repair in cereals. Here, we study DNA repair in the cell-free extracts of wheat aleurone layer incubated or not with phytohormones. We show, for the first time, the presence of 8-oxoguanine-DNA and ethenoadenine-DNA glycosylase activities in wheat aleurone cells. Interestingly, the DNA glycosylase and AP endonuclease activities are strongly induced in the presence of GA. Based on these data we propose that GA in addition to activation of nuclear DNases also induces the DNA repair activities which remove oxidized DNA bases in the BER pathway. Potential roles of the wheat DNA glycosylases in GA-induced oligonucleosomal fragmentation of DNA and metabolic activation of aleurone layer cells via repair of transcribed regions are discussed.


Subject(s)
DNA Repair Enzymes/metabolism , DNA Repair , Deoxyribonucleases/metabolism , Endosperm/enzymology , Triticum/enzymology , Abscisic Acid/pharmacology , Acid Anhydride Hydrolases/metabolism , Cell Death , Cytoplasm/enzymology , DNA Fragmentation , DNA, Plant/genetics , DNA, Plant/isolation & purification , Endosperm/drug effects , Endosperm/genetics , Enzyme Activation , Gibberellins/pharmacology , Glucosephosphate Dehydrogenase/metabolism , Oxidative Stress , Plant Extracts/chemistry , Reactive Oxygen Species/metabolism , Triticum/drug effects , Triticum/genetics
19.
Environ Mol Mutagen ; 51(6): 508-19, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20120016

ABSTRACT

Fanconi anemia (FA) is a recessive cancer prone syndrome featuring bone marrow failure and hypersensitivity to DNA interstrand crosslinks (ICLs) and, to a milder extension, to ionizing radiation and oxidative stress. Recently, we reported that human oxidative DNA glycosylase, NEIL1 excises with high efficiency the unhooked crosslinked oligomer within three-stranded DNA repair intermediate induced by photoactivated psoralen exposure. Complete reconstitution of repair of the ICL within a three-stranded DNA structure shows that it is processed in the short-patch base excision repair (BER) pathway. To examine whether the DNA damage hypersensitivity in FA cells follows impaired BER activities, we measured DNA glycosylase and AP endonuclease activities in cell-free extracts from wild-type, FA, and FA-corrected cells. We showed that immortalized lymphoid cells of FA complementation Groups A, C, and D and from control cells from normal donors contain similar BER activities. Intriguingly, the cellular level of NEIL1 protein strongly depends on the intact FA pathway suggesting that the hypersensitivity of FA cells to ICLs may, at least in part, arise from downregulation or degradation of NEIL1. Consistent with this result, plasmid-based expression of the FLAG-tagged NEIL1 protein partially complements the hypersensitivity FA cells to the crosslinking agents exposures, suggesting that NEIL1 specifically complements impaired capability of FA cells to repair ICLs and oxidative DNA damage. These findings shed light to how the FA pathway may regulate DNA repair proteins and bring explanation for the long-time disputed problem of the oxidative stress sensitive phenotype of FA cells.


Subject(s)
DNA Glycosylases/metabolism , DNA Repair , Fanconi Anemia/metabolism , Cell Line, Tumor , Cross-Linking Reagents/pharmacology , DNA Glycosylases/drug effects , DNA Glycosylases/genetics , Down-Regulation , Fanconi Anemia/genetics , Humans , Signal Transduction
20.
Mutat Res ; 685(1-2): 70-9, 2010 Mar 01.
Article in English | MEDLINE | ID: mdl-19751747

ABSTRACT

Aerobic respiration generates reactive oxygen species (ROS) as a by-product of cellular metabolism which can damage DNA. The complex nature of oxidative DNA damage requires actions of several repair pathways. Oxidized DNA bases are substrates for two overlapping pathways: base excision repair (BER) and nucleotide incision repair (NIR). In the BER pathway a DNA glycosylase cleaves the N-glycosylic bond between the abnormal base and deoxyribose, leaving either an abasic site or single-stranded DNA break. Alternatively, in the NIR pathway, an apurinic/apyrimidinic (AP) endonuclease incises duplex DNA 5' next to oxidatively damaged nucleotide. The multifunctional Escherichia coli endonuclease IV (Nfo) is involved in both BER and NIR pathways. Nfo incises duplex DNA 5' of a damaged residue but also possesses an intrinsic 3'-->5' exonuclease activity. Herein, we demonstrate that Nfo-catalyzed NIR and exonuclease activities can generate a single-strand gap at the 5' side of 5,6-dihydrouracil residue. Furthermore, we show that Nfo mutants carrying amino acid substitutions H69A and G149D are deficient in both NIR and exonuclease activities, suggesting that these two functions are genetically linked and governed by the same amino acid residues. The crystal structure of Nfo-H69A mutant reveals the loss of one of the active site zinc atoms (Zn1) and rearrangements of the catalytic site, but no gross changes in the overall enzyme conformation. We hypothesize that these minor changes strongly affect the DNA binding of Nfo. Decreased affinity may lead to a different kinking angle of the DNA helix and this in turn thwart nucleotide incision and exonuclease activities of Nfo mutants but to lesser extent of their AP endonuclease function. Based on the biochemical and genetic data we propose a model where nucleotide incision coupled to 3'-->5' exonuclease activity prevents formation of lethal double-strand breaks when repairing bi-stranded clustered DNA damage.


Subject(s)
DNA Damage , DNA Repair , Deoxyribonuclease IV (Phage T4-Induced)/chemistry , Escherichia coli Proteins/chemistry , Amino Acid Substitution , Catalytic Domain , DNA Breaks, Single-Stranded , Deoxyribonuclease IV (Phage T4-Induced)/genetics , Deoxyribonuclease IV (Phage T4-Induced)/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Models, Genetic , Models, Molecular , Mutation , Oxidation-Reduction , Protein Conformation
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