Base excision repair (BER) maintains genomic stability through the repair of DNA damage. Within BER, AP-endonuclease 1 (APE1) is a multifunctional enzyme that processes DNA intermediates through its backbone cleavage activity. To accomplish these repair activities, APE1 must recognize and accommodate several diverse DNA substrates. This is hypothesized to occur through a DNA sculpting mechanism where structural adjustments of the DNA substrate are imposed by the protein; however, how APE1 uniquely sculpts each substrate within a single rigid active site remains unclear. Here, we utilize structural and biochemical approaches to probe the DNA sculpting mechanism of APE1, specifically by characterizing a protein loop that intercalates the minor groove of the DNA (termed the intercalating loop). Pre-steady-state kinetics reveal a tyrosine residue within the intercalating loop (Y269) that is critical for AP-endonuclease activity. Using X-ray crystallography and molecular dynamics simulations, we determined the Y269 residue acts to anchor the intercalating loop on abasic DNA. Atomic force microscopy reveals the Y269 residue is required for proper DNA bending by APE1, providing evidence for the importance of this mechanism. We conclude that this previously unappreciated tyrosine residue is key to anchoring the intercalating loop and stabilizing the DNA in the APE1 active site.

DNA polymerases are vital for the synthesis of new DNA strands. Since the discovery of DNA polymerase I in Escherichia coli, a diverse library of mammalian DNA polymerases involved in DNA replication, DNA repair, antibody generation, and cell checkpoint signaling has emerged. While the unique functions of these DNA polymerases are differentiated by their association with accessory factors and/or the presence of distinctive catalytic domains, atomic resolution structures of DNA polymerases in complex with their DNA substrates have revealed mechanistic subtleties that contribute to their specialization. In this review, the structure and function of all 15 mammalian DNA polymerases from families B, Y, X, and A will be reviewed and discussed with special emphasis on the insights gleaned from recently published atomic resolution structures.

Genomic DNA is continually exposed to endogenous and exogenous factors that promote DNA damage. Eukaryotic genomic DNA is packaged into nucleosomes, which present a barrier to accessing and effectively repairing DNA damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the nucleosome and protect genomic stability is unknown. Here, we determine how the base excision repair (BER) endonuclease AP-endonuclease 1 (APE1) recognizes and cleaves DNA damage in the nucleosome. Kinetic assays determine that APE1 cleaves solvent-exposed AP sites in the nucleosome with 3 − 6 orders of magnitude higher efficiency than occluded AP sites. A cryo-electron microscopy structure of APE1 bound to a nucleosome containing a solvent-exposed AP site reveal that APE1 uses a DNA sculpting mechanism for AP site recognition, where APE1 bends the nucleosomal DNA to access the AP site. Notably, additional biochemical and structural characterization of occluded AP sites identify contacts between the nucleosomal DNA and histone octamer that prevent efficient processing of the AP site by APE1. These findings provide a rationale for the position-dependent activity of BER proteins in the nucleosome and suggests the ability of BER proteins to sculpt nucleosomal DNA drives efficient BER in chromatin.

During oxidative stress, inflammation, or environmental exposure, ribo- and deoxyribonucleotides are oxidatively modified. 8-Oxo-7,8-dihydro-2′-guanosine (8-oxo-G) is a common oxidized nucleobase whose deoxyribonucleotide form, 8-oxo-dGTP, has been widely studied and demonstrated to be a mutagenic substrate for DNA polymerases. Guanine ribonucleotides are analogously oxidized to r8-oxo-GTP, which can constitute up to 5% of the rGTP pool. Because ribonucleotides are commonly misinserted into DNA, and 8-oxo-G causes replication errors, we were motivated to investigate how the oxidized ribonucleotide is utilized by DNA polymerases. To do this, here we employed human DNA polymerase β (pol β) and characterized r8-oxo-GTP insertion with DNA substrates containing either a templating cytosine (nonmutagenic) or adenine (mutagenic). Our results show that pol β has a diminished catalytic efficiency for r8-oxo-GTP compared with canonical deoxyribonucleotides but that r8-oxo-GTP is inserted mutagenically at a rate similar to those of other common DNA replication errors (i.e. ribonucleotide and mismatch insertions). Using FRET assays to monitor conformational changes of pol β with r8-oxo-GTP, we demonstrate impaired pol β closure that correlates with a reduced insertion efficiency. X-ray crystallographic analyses revealed that, similar to 8-oxo-dGTP, r8-oxo-GTP adopts an anti conformation opposite a templating cytosine and a syn conformation opposite adenine. However, unlike 8-oxo-dGTP, r8-oxo-GTP did not form a planar base pair with either templating base. These results suggest that r8-oxo-GTP is a potential mutagenic substrate for DNA polymerases and provide structural insights into how r8-oxo-GTP is processed by DNA polymerases.