16 research outputs found
Genetic dissection of the biochemical activities of human DNA repair protein, APE1
Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme
involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER
pathway, APE1 cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases. In the
NIR pathway, APE1 incises DNA 5' to a number of oxidatively damaged bases. Here we propose to identify
and characterize critical amino acids of APE1 involved in either BER and/or NIR functions by using
the alignment of the known three-dimensional (or tertiary) structures of Xth family AP endonucleases
including the Methanothermobacter thermautotrophicus Mth212, Bacillus subtilis ExoA (1), E. coli Xth
and human APE1 proteins (2)
Genetic dissection of the biochemical activities of human DNA repair protein, APE1
Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme
involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER
pathway, APE1 cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases. In the
NIR pathway, APE1 incises DNA 5' to a number of oxidatively damaged bases. Here we propose to identify
and characterize critical amino acids of APE1 involved in either BER and/or NIR functions by using
the alignment of the known three-dimensional (or tertiary) structures of Xth family AP endonucleases
including the Methanothermobacter thermautotrophicus Mth212, Bacillus subtilis ExoA (1), E. coli Xth
and human APE1 proteins (2)
Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase
Involvement of the Reparative DNA Polymerase Pol X of African Swine Fever Virus in the Maintenance of Viral Genome Stability In Vivo
Functional eukaryotic nuclear localization signals are widespread in terminal proteins of bacteriophages
Uracil in duplex DNA is a substrate for the nucleotide incision repair pathway in human cells
Highly mutagenic exocyclic DNA adducts are substrates for the human nucleotide incision repair pathway
Background: Oxygen free radicals induce lipid peroxidation (LPO) that damages and breaks polyunsaturated fatty acids in cell membranes. LPO-derived aldehydes and hydroxyalkenals react with DNA leading to formation of etheno(ε)-bases including 1,N6-ethenoadenine (εA) and 3,N4-ethenocytosine (εC). The εA and εC residues are highly mutagenic in mammalian cells and eliminated in the base excision repair (BER) pathway and/or by AlkB family proteins in the direct damage reversal process. BER initiated by DNA glycosylases is thought to be the major pathway for the removal of non-bulky endogenous base damage. Alternatively, in the nucleotide incision repair (NIR) pathway, the apurinic/apyrimidinic (AP) endonucleases can directly incise DNA duplex 5’ to a damaged base in a DNA glycosylase-independent manner.
Methodology/Principal Findings: Here, we characterized the substrate specificity of human major AP endonuclease 1, APE1, towards εA, εC, thymine glycol (Tg) and 7,8-dihydro-8-oxoguanine (8oxoG) residues when present in duplex DNA. APE1 cleaves oligonucleotide duplexes containing εA, εC and Tg, but not those containing 8oxoG. The activity depends strongly on sequence context. The apparent kinetic parameters of the reactions suggest that APE1 has high affinity to DNA containing ε-bases but cleaves DNA duplex at an extremely slow rate. Consistent with this observation, the oligonucleotide duplexes containing an ε-base strongly inhibit AP site nicking activity of APE1 with IC50 values in the range of 5-10 nM. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of εA•T and εC•G duplexes generates as expected DNA fragments containing 5’-terminal ε-base residue.
Conclusions/Significance: The fact that ε-bases and Tg in duplex DNA are recognized and cleaved by APE1 in vitro, suggest that NIR may act as a backup pathway to BER one to remove a large variety of genotoxic base lesions in human cells