A Chemical and Kinetic Perspective on Base Excision Repair of DNA

Abstract

ConspectusOur cellular genome is continuously exposed to a wide spectrum of exogenous and endogenous DNA damaging agents. These agents can lead to formation of an extensive array of DNA lesions including single- and double-stranded breaks, inter- and intrastrand cross-links, abasic sites, and modification of DNA nucleobases. Persistence of these DNA lesions can be both mutagenic and cytotoxic, and can cause altered gene expression and cellular apoptosis leading to aging, cancer, and various neurological disorders. To combat the deleterious effects of DNA lesions, cells have a variety of DNA repair pathways responsible for restoring damaged DNA to its canonical form. Here we examine one of those repair pathways, the base excision repair (BER) pathway, a highly regulated network of enzymes responsible for repair of modified nucleobase and abasic site lesions.The enzymes required to reconstitute BER in vitro have been identified, and the repair event can be considered to occur in two parts: (1) excision of the modified nucleobase by a DNA glycosylase, and (2) filling the resulting “hole” with an undamaged nucleobase by a series of downstream enzymes. DNA glycosylases, which initiate a BER event, recognize and remove specific modified nucleobases and yield an abasic site as the product. The abasic site, a highly reactive BER intermediate, is further processed by AP endonuclease 1 (APE1), which cleaves the DNA backbone 5′ to the abasic site, generating a nick in the DNA backbone. After action of APE1, BER can follow one of two subpathways, the short-patch (SP) or long-patch (LP) version, which differ based on the number of nucleotides a polymerase incorporates at the nick site. DNA ligase is responsible for sealing the nick in the backbone and regenerating undamaged duplex.Not surprisingly, and consistent with the idea that BER maintains genetic stability, deficiency and/or inactivity of BER enzymes can be detrimental and result in cancer. Intriguingly, this DNA repair pathway has also been implicated in causing genetic instability by contributing to the trinucleotide repeat expansion associated with several neurological disorders.Within this Account, we outline the chemistry of the human BER pathway with a mechanistic focus on the DNA glycosylases that initiate the repair event. Furthermore, we describe kinetic studies of many BER enzymes as a means to understand the complex coordination that occurs during this highly regulated event. Finally, we examine the pitfalls associated with deficiency in BER activity, as well as instances when BER goes awry.

Synergistic Effects of an Irreversible DNA Polymerase Inhibitor and DNA Damaging Agents on HeLa Cells

“…1,2 The base excision repair (BER) pathway mitigates the cytotoxicity of these agents by removing the damage and restoring the DNA to its original structure. 3,4 While BER is critical for maintaining the genome integrity of healthy cells, it is a hindrance for anti-cancer modalities that target DNA. 5 Consequently, the enzymes involved in BER are inhibition targets.…”

A role for the base excision repair enzyme NEIL3 in replication-dependent repair of interstrand DNA cross-links derived from psoralen and abasic sites

“…Importantly, substrate recognition and catalysis by glycosylases typically involve “flipping out” (extrusion of) the target base from the double helix – a process that is presumably precluded for bases involved in an interstrand cross-link (Figure 2B) [81–83]. Monofunctional BER enzymes catalyze hydrolytic removal of a nucleobase leaving an intact abasic (Ap) site in the DNA (Figure 2A), while bifunctional DNA glycosylases remove the base and also catalyze a subsequent elimination (lyase) reaction that generates a strand break with a 5′-phosphoryl end group and 3′-deoxyribose phosphate sugar remnant [74, 76, 78, 79, 82, 84–86]. …”

Landscape ofπ–πand sugar–πcontacts in DNA–protein interactions

“…This is perhaps not surprising since these contacts are known to play some important roles in nature, including stabilizing the DNA helix during processing (Mahadevi & Sastry, 2012), contributing to DNA (Wintjens et al, 2000), drug (Zacharias & Dougherty, 2002) or other ligand (Pellequer et al, 2000) binding, and even enhancing protein catalysis (Brooks, Adhikary, Rubinson, & Eichman, 2013;Schermerhorn & Delaney, 2014). In fact, DNA nucleobase-aromatic amino acid interactions assist the repair of DNA damage in cells.…”

“…Unlike other DNA glycosylases, which bind to substrates with high specificity using hydrogen bonding, these nonspecific π-π contacts allow AAG to bind and excise a wide variety of substrates, including both alkylated (i.e., ethenoadenine) and oxidized (i.e. hypoxanthine) nucleobases (Brooks et al, 2013;Schermerhorn & Delaney, 2014). Third, the substrate-enzyme π-π interactions contribute to the catalytic excision of neutral lesions due to stronger π-π interactions following glycosidic bond cleavage (Rutledge & Wetmore, 2011).…”