Human AP endonuclease (APE1) demonstrates endonucleolytic activity against AP sites in single-stranded DNA

Marenstein, Dina R.; Wilson, David M.; Teebor, George W.

doi:10.1016/j.dnarep.2004.01.010

Cited by 74 publications

(72 citation statements)

References 56 publications

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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).…”

Section: Resultsmentioning

confidence: 64%

“…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. Based on the findings described here, a role for PolX Bs to repair these lesions on ssDNA could be postulated, likely acting in concert with additional protein factors to prevent separation of the two ssDNA regions originated after the action of the AP-endonuclease activity, as it has been proposed to occur in other systems (37).…”

Section: Resultsmentioning

confidence: 74%

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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: 64%

Section: Resultsmentioning

confidence: 74%

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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“…Dependence of Ape1 ss Incision Activity on DNA Secondary StructureApe1 has been shown to effectively incise at AP sites in ssDNA (15), with its endonuclease efficiency seemingly influenced by the potential secondary structure of the oligonucleotide (16). To determine more explicitly the role of DNA secondary structure on Ape1 activity in vitro, incision assays were performed using DNAs unable to form secondary conformations, namely 19 poly(A)-F and 19 poly(T)-F ( Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…For many years, this activity had been characterized on double-stranded (ds) AP-DNA substrates (12)(13)(14), as it was presumed that a successful BER event would take place exclusively on a template-containing (instructional) duplex DNA molecule. Recently, however, it was shown that Ape1 exhibits a robust endonuclease activity at AP sites in single-stranded (ss) oligonucleotides, in some instances greater than in dsDNA, as well as in several complex and biologically relevant ss structures, such as primertemplate duplexes, bubble conformations, and fork-like arrangements (15,16). Presently, little is known about how these more "exotic" activities of Ape1 are modulated or how the resulting incision products are handled by the cell.…”

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confidence: 99%

Nucleotide Sequence and DNA Secondary Structure, as Well as Replication Protein A, Modulate the Single-stranded Abasic Endonuclease Activity of APE1

Fan

Matsumoto

Wilson

2006

Journal of Biological Chemistry

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A major role of the multifunctional human Ape1 protein is to incise at apurinic/apyrimidinic (AP) sites in DNA via site-specific endonuclease activity. This nuclease function has been well characterized on double-stranded (ds) DNA substrates, where the complementary strand provides a template for subsequent base excision repair events. Recently, Ape1 was found to incise efficiently at AP sites positioned within the single-stranded (ss) regions of various biologically relevant DNA configurations. The studies within indicated that the ss endonuclease activity of Ape1 is poorly active on ss AP site-containing polyadenine or polythymine oligonucleotides, suggesting a requirement for some form of DNA secondary structure for efficient cleavage. Apurinic/apyrimidinic (AP)2 endonuclease 1 (Ape1) is the major mammalian repair protein for abasic sites in DNA (1). This enzyme catalyzes incision of the phosphodiester backbone immediately 5Ј to an AP lesion, initiating a cascade of events that involves components of the base excision repair (BER) pathway and aims to remove the abasic residue and re-establish genetic integrity (2). Abasic sites are common products in DNA, arising either spontaneously (at a rate of 10,000 events per mammalian genome per day), via accelerated base release because of chemical modification, or through enzyme (glycosylase)-catalyzed removal of a damaged base (3, 4). If unrepaired, AP lesions present mutagenic and cytotoxic challenges to the cell (5). Deficiencies in Ape1 activity in mice have been associated with increased spontaneous mutation frequencies in both somatic and germ line cells (6), as well as increased cancer susceptibility and reduced survival in the face of exogenous oxidizing agents (7). It is noteworthy that, although uncommon, human Ape1 protein variants with reduced function have been identified (8).It is generally accepted that Ape1 is the predominant AP site incision enzyme in mammals, accounting for Ͼ95% (if not all) of the total cellular AP endonuclease activity (9). In fact, recent evidence argues that its AP endonuclease function is essential for cell viability (10, 11). For many years, this activity had been characterized on double-stranded (ds) AP-DNA substrates (12-14), as it was presumed that a successful BER event would take place exclusively on a template-containing (instructional) duplex DNA molecule. Recently, however, it was shown that Ape1 exhibits a robust endonuclease activity at AP sites in single-stranded (ss) oligonucleotides, in some instances greater than in dsDNA, as well as in several complex and biologically relevant ss structures, such as primertemplate duplexes, bubble conformations, and fork-like arrangements (15, 16). Presently, little is known about how these more "exotic" activities of Ape1 are modulated or how the resulting incision products are handled by the cell.Replication protein A (RPA) is the most abundant ssDNA-binding protein in mammalian cells, present at roughly 100,000 molecules per cell (17). This heterotrimeric complex was origina...

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“…We recently discovered that human APE1 (hAPE1) is acetylated in vivo at Lys 6 / Lys 7 which modulates its regulatory but not endonuclease activity [113]. The endonuclease and 3′ end cleaning phosphodiesterase activity of APE1 is weaker than that of Xth, but more importantly, APE1 has barely detectable DNA 3′ phosphatase activity and has a very limited 3′ exonuclease activity [114,115]. However, 3′ mismatch-specific exonuclease activity has been observed in APE1 although its in vivo significance has not been established [116].…”

Section: Mammalian Ape1 and E Coli Xth Have Distinct Propertiesmentioning

confidence: 99%

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

2008

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Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3′ OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APE1, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3′ phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.

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Human AP endonuclease (APE1) demonstrates endonucleolytic activity against AP sites in single-stranded DNA

Cited by 74 publications

References 56 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

Nucleotide Sequence and DNA Secondary Structure, as Well as Replication Protein A, Modulate the Single-stranded Abasic Endonuclease Activity of APE1

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

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