Tadahide Izumi

DNA-bound structures and mutants reveal abasic DNA binding by APE1 DNA repair and coordination

Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontaneously and by damage-specific DNA glycosylases. The biologically critical human base excision repair enzyme APE1 cleaves the DNA sugar-phosphate backbone at a position 5′ of AP sites to prime DNA repair synthesis. Here we report three co-crystal structures of human APE1 bound to abasic DNA which show that APE1 uses a rigid, pre-formed, positively charged surface to kink the DNA helix and engulf the AP-DNA strand. APE1 inserts loops into both the DNA major and minor grooves and binds a flipped-out AP site in a pocket that excludes DNA bases and racemized β-anomer AP sites. Both the APE1 active-site geometry and a complex with cleaved AP-DNA and Mn2+ support a testable structure-based catalytic mechanism. Alanine substitutions of the residues that penetrate the DNA helix unexpectedly show that human APE1 is structurally optimized to retain the cleaved DNA product. These structural and mutational results show how APE1 probably displaces bound glycosylases and retains the nicked DNA product, suggesting that APE1 acts in vivo to coordinate the orderly transfer of unstable DNA damage intermediates between the excision and synthesis steps of DNA repair.

Identification and characterization of a human DNA glycosylase for repair of modified bases in oxidatively damaged DNA

8-oxoguanine (8-oxoG), ring-opened purines (formamidopyrimidines or Fapys), and other oxidized DNA base lesions generated by reactive oxygen species are often mutagenic and toxic, and have been implicated in the etiology of many diseases, including cancer, and in aging. Repair of these lesions in all organisms occurs primarily via the DNA base excision repair pathway, initiated with their excision by DNA glycosylase/AP lyases, which are of two classes. One class utilizes an internal Lys residue as the active site nucleophile, and includes Escherichia coli Nth and both known mammalian DNA glycosylase/AP lyases, namely, OGG1 and NTH1. E. coli MutM and its paralog Nei, which comprise the second class, use N-terminal Pro as the active site. Here, we report the presence of two human orthologs of E. coli mutM nei genes in the human genome database, and characterize one of their products. Based on the substrate preference, we have named it NEH1 (Nei homolog). The 44-kDa, wild-type recombinant NEH1, purified to homogeneity from E. coli, excises Fapys from damaged DNA, and oxidized pyrimidines and 8-oxoG from oligodeoxynucleotides. Inactivation of the enzyme because of either deletion of N-terminal Pro or Histag fusion at the N terminus supports the role of N-terminal Pro as its active site. The tissue-specific levels of NEH1 and OGG1 mRNAs are distinct, and S phase-specific increase in NEH1 at both RNA and protein levels suggests that NEH1 is involved in replication-associated repair of oxidized bases.

Purification and Characterization of Human NTH1, a Homolog of Escherichia coli Endonuclease III

The human endonuclease III (hNTH1), a homolog of the Escherichia coli enzyme (Nth), is a DNA glycosylase with abasic (apurinic/apyrimidinic (AP)) lyase activity and specifically cleaves oxidatively damaged pyrimidines in DNA. Its cDNA was cloned, and the full-length enzyme (304 amino acid residues) was expressed as a glutathione S-transferase fusion polypeptide in E. coli. Purified wild-type protein with two additional amino acid residues and a truncated protein with deletion of 22 residues at the NH2 terminus were equally active and had absorbance maxima at 280 and 410 nm, the latter due to the presence of a [4Fe-4S]cluster, as in E. coli Nth. The enzyme cleaved thymine glycol-containing form I plasmid DNA and a dihydrouracil (DHU)-containing oligonucleotide duplex. The protein had a molar extinction coefficient of 5.0 × 104 and a pI of 10. With the DHU-containing oligonucleotide duplex as substrate, theK m was 47 nm, andk cat was ∼0.6/min, independent of whether DHU paired with G or A. The enzyme carries out β-elimination and forms a Schiff base between the active site residue and the deoxyribose generated after base removal. The prediction of Lys-212 being the active site was confirmed by sequence analysis of the peptide-oligonucleotide adduct. Furthermore, replacing Lys-212 with Gln inactivated the enzyme. However, replacement with Arg-212 yielded an active enzyme with about 85-fold lower catalytic specificity than the wild-type protein. DNase I footprinting with hNTH1 showed protection of 10 nucleotides centered around the base lesion in the damaged strand and a stretch of 15 nucleotides (with the G opposite the lesion at the 5′-boundary) in the complementary strand. Immunological studies showed that HeLa cells contain a single hNTH species of the predicted size, localized in both the nucleus and the cytoplasm.

Mammalian DNA base excision repair proteins: their interactions and role in repair of oxidative DNA damage

The DNA base excision repair (BER) is a ubiquitous mechanism for removing damage from the genome induced by spontaneous chemical reaction, reactive oxygen species (ROS) and also DNA damage induced by a variety of environmental genotoxicants. DNA repair is essential for maintaining genomic integrity. As we learn more about BER, a more complex mechanism emerges which supersedes the classical, simple pathway requiring only four enzymatic reactions. The key to understand the complete BER process is to elucidate how multiple proteins interact with one another in a coordinated process under specific physiological conditions.

Erratum: DNA-bound structures and mutants reveal abasic DNA binding by APE1 and DNA repair coordination (Nature (2000) 403 (451-456))

This corrects the article DOI: 10.1038/35000249

Role of acetylated human AP-endonuclease (APE1/Ref-1) in regulation of the parathyroid hormone gene

The human AP‐endonuclease (APE1/Ref‐1), a multifunctional protein central to repairing abasic sites and single‐strand breaks in DNA, also plays a role in transcriptional regulation. Besides activating some transcription factors, APE1 is directly involved in Ca2+‐dependent downregulation of parathyroid hormone (PTH) expression by binding to negative calcium response elements (nCaREs) present in the PTH promoter. Here we show that APE1 is acetylated both in vivo and in vitro by the transcriptional co‐activator p300 which is activated by Ca2+. Acetylation at Lys6 or Lys7 enhances binding of APE1 to nCaRE. APE1 stably interacts with class I histone deacetylases (HDACs) in vivo. An increase in extracellular calcium enhances the level of acetylated APE1 which acts as a repressor for the PTH promoter. Moreover, chromatin immunoprecipitation (ChIP) assay revealed that acetylation of APE1 enhanced binding of the APE1–HDACs complex to the PTH promoter. These results indicate that acetylation of APE1 plays an important role in this key repair proteins action in transcriptional regulation.

Identification and characterization of mitochondrial abasic (AP)-endonuclease in mammalian cells

Abasic (AP)-endonuclease (APE) is responsible for repair of AP sites, and single-strand DNA breaks with 3′ blocking groups that are generated either spontaneously or during repair of damaged or abnormal bases via the DNA base excision repair (BER) pathway in both nucleus and mitochondria. Mammalian cells express only one nuclear APE, 36 kDa APE1, which is essential for survival. Mammalian mitochondrial (mt) BER enzymes other than mtAPE have been characterized. In order to identify and characterize mtAPE, we purified the APE activity from beef liver mitochondria to near homogeneity, and showed that the mtAPE which has 3-fold higher specific activity relative to APE1 is derived from the latter with deletion of 33 N-terminal residues which contain the nuclear localization signal. The mtAPE-sized product could be generated by incubating 35S-labeled APE1 with crude mitochondrial extract, but not with cytosolic or nuclear extract, suggesting that cleavage of APE1 by a specific mitochondria-associated N-terminal peptidase is a prerequisite for mitochondrial import. The low abundance of mtAPE, particularly in cultured cells might be the reason for its earlier lack of detection by western analysis.

Choreography of oxidative damage repair in mammalian genomes

The lesions induced by reactive oxygen species in both nuclear and mitochondrial genomes include altered bases, abasic (AP) sites, and single-strand breaks, all repaired primarily via the base excision repair (BER) pathway. Although the basic BER process (consisting of five sequential steps) could be reconstituted in vitro with only four enzymes, it is now evident that repair of oxidative damage, at least in mammalian cell nuclei, is more complex, and involves a number of additional proteins, including transcription- and replication-associated factors. These proteins may be required in sequential repair steps in concert with other cellular changes, starting with nuclear targeting of the early repair enzymes in response to oxidative stress, facilitation of lesion recognition, and access by chromatin unfolding via histone acetylation, and formation of metastable complexes of repair enzymes and other accessory proteins. Distinct, specific subclasses of protein complexes may be formed for repair of oxidative lesions in the nucleus in transcribed vs. nontranscribed sequences in chromatin, in quiescent vs. cycling cells, and in nascent vs. parental DNA strands in replicating cells. Characterizing the proteins for each repair subpathway, their signaling-dependent modifications and interactions in the nuclear as well as mitochondrial repair complexes, will be a major focus of future research in oxidative damage repair.

Identification of Apurinic/apyrimidinic endonuclease 1 (APE1) as the endoribonuclease that cleaves c-myc mRNA

Endonucleolytic cleavage of the coding region determinant (CRD) of c-myc mRNA appears to play a critical role in regulating c-myc mRNA turnover. Using 32P-labeled c-myc CRD RNA as substrate, we have purified and identified two endoribonucleases from rat liver polysomes that are capable of cleaving the transcript in vitro. A 17-kDa enzyme was identified as RNase1. Apurinic/apyrimidinic (AP) DNA endonuclease 1 (APE1) was identified as the 35-kDa endoribonuclease that preferentially cleaves in between UA and CA dinucleotides of c-myc CRD RNA. APE1 was further confirmed to be the 35-kDa endoribonuclease because: (i) the endoribonuclease activity of the purified 35-kDa native enzyme was specifically immuno-depleted with APE1 monoclonal antibody, and (ii) recombinant human APE1 generated identical RNA cleavage patterns as the native liver enzyme. Studies using E96A and H309N mutants of APE1 suggest that the endoribonuclease activity for c-myc CRD RNA shares the same active center with the AP-DNA endonuclease activity. Transient knockdown of APE1 in HeLa cells led to increased steady-state level of c-myc mRNA and its half-life. We conclude that the ability to cleave RNA dinucleotides is a previously unidentified function of APE1 and it can regulate c-myc mRNA level possibly via its endoribonuclease activity.

Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage

The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein, plays a central role in the repair of oxidative base damage via the DNA base excision repair (BER) pathway. The mammalian AP-endonuclease (APE1) overexpression is often observed in tumor cells, and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to those agents via induction of apoptosis. Here we show that wild type (WT) but not mutant p53 negatively regulates APE1 expression. Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53(+/+), but not in the isogenic p53 null mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor. Furthermore, ectopic expression of WTp53 in the p53 null cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion. Chromatin immunoprecipitation assays revealed that endogenous p53 is bound to the APE1 promoter region that includes a Sp1 site. We show here that WTp53 interferes with Sp1 binding to the APE1 promoter, which provides a mechanism for the downregulation of APE1. Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.