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

Abstract

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...

DNA secondary structure of the released strand stimulates WRN helicase action on forked duplexes without coordinate action of WRN exonuclease

“…4A). We used 34G and 34 poly(T), which have been reported to have hairpin loops previously, as positive controls [20]. The 34G oligonucleotide migrated more quickly than the 34 poly(T) (Fig.…”

APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER

“…Both XPA and RPA preferentially bind to cisplatin-damaged DNA [52, 53] and may also play a role in subsequent steps in NER through interaction with other repair proteins [54]. RPA has been found to interact with APE1 [55], although to date this interaction has not been observed in sensory neurons nor in the context of cisplatin repair in neurons. Given our findings demonstrating APE1 knockdown altering Pt adduct removal and alterations in RPA and XPA, it will be interesting to see whether increasing damage recognition ability by overexpressing damage recognition factors such as XPA and RPA can actually enhance repair of platinum damage and how altering APE1 levels influences this DNA repair response.…”

The Many Functions of APE1/Ref-1: Not Only a DNA Repair Enzyme

“…This implies the coordinated interaction of the various players in the BER process and, when necessary, their further interaction with the DNA replication machinery, as demonstrated by the co-immunoprecipitation of the BER repair proteins with cyclin A and DNA replication proteins (103). In this context, it is noteworthy that RPA proteins are able to suppress the APE1 endonuclease activity in ssDNA of a replicative fork but not in a transcription bubble or in dsDNA (27) and that Cockayne syndrome B protein potentiates the APE1 activity on fully paired AP-DNA but much more on bubble AP-DNA, suggesting a role for this protein in the transcription-repair pathway (143).…”