DNA glycosylases perform a genome-wide search to locate damaged nucleotides among a great excess of undamaged nucleotides. Many glycosylases are capable of facilitated diffusion, whereby multiple sites along the DNA are sampled during a single binding encounter. Electrostatic interactions between positively charged amino acids and the negatively charged phosphate backbone are crucial for facilitated diffusion, but the extent to which diffusing proteins rely on the double-helical structure DNA is not known. Kinetic assays were used to probe the DNA searching mechanism of human alkyladenine DNA glycosylase (AAG) and to test the extent to which diffusion requires B-form duplex DNA. Although AAG excises εA lesions from single-stranded DNA, it is not processive on single-stranded DNA because dissociation is faster than N-glycosidic bond cleavage. However, the AAG complex with single-stranded DNA is sufficiently stable to allow for DNA annealing when a complementary strand is added. This observation provides evidence of nonspecific association of AAG with single-stranded DNA. Single-strand gaps, bubbles, and bent structures do not impede the search by AAG. Instead, these flexible or bent structures lead to the capture of a nearby site of damage that is more efficient than that of a continuous B-form duplex. The ability of AAG to negotiate these helix discontinuities is inconsistent with a sliding mode of diffusion but can be readily explained by a hopping mode that involves microscopic dissociation and reassociation. These experiments provide evidence of relatively long-range hops that allow a searching protein to navigate around DNA binding proteins that would serve as obstacles to a sliding protein.

Spontaneous deamination of DNA is mutagenic, if it is not repaired by the base excision repair (BER) pathway. Crystallographic data suggest that each BER enzyme has a compact DNA binding site. However, these structures lack information about poorly ordered termini, and the energetic contributions of specific protein-DNA contacts cannot be inferred. Furthermore, these structures do not reveal how DNA repair intermediates are passed between enzyme active sites. We used a functional footprinting approach to define the binding sites of the first two enzymes of the human BER pathway for the repair of deaminated purines, alkyladenine DNA glycosylase (AAG) and AP endonuclease (APE1). Although the functional footprint for full-length AAG is explained by crystal structures of truncated AAG, the footprint for full-length APE1 indicates a much larger binding site than is observed in crystal structures. AAG turnover is stimulated in the presence of APE1, indicating rapid exchange of AAG and APE1 at the abasic site produced by the AAG reaction. The coordinated reaction does not require an extended footprint, suggesting that each enzyme engages the site independently. Functional footprinting provides unique information relative to traditional footprinting approaches and is generally applicable to any DNA modifying enzyme or system of enzymes.

DNA ligases catalyze the joining of DNA strands to complete DNA replication, recombination and repair transactions. To protect the integrity of the genome, DNA ligase 1 (LIG1) discriminates against DNA junctions harboring mutagenic 3'-DNA mismatches or oxidative DNA damage, but how such high-fidelity ligation is enforced is unknown. Here, X-ray structures and kinetic analyses of LIG1 complexes with undamaged and oxidatively damaged DNA unveil that LIG1 employs Mg2+-reinforced DNA binding to validate DNA base pairing during the adenylyl transfer and nick-sealing ligation reaction steps. Our results support a model whereby LIG1 fidelity is governed by a high-fidelity (HiFi) interface between LIG1, Mg2+, and the DNA substrate that tunes the enzyme to release pro-mutagenic DNA nicks. In a second tier of protection, LIG1 activity is surveilled by Aprataxin (APTX), which suppresses mutagenic and abortive ligation at sites of oxidative DNA damage.

The Mag1 and Tpa1 proteins from budding yeast (Saccharomyces cerevisiae) have both been reported to repair alkylation damage in DNA. Mag1 initiates the base excision repair pathway by removing alkylated bases from DNA, and Tpa1 has been proposed to directly repair alkylated bases as does the prototypical oxidative dealkylase AlkB from Escherichia coli However, we found that in vivo repair of methyl methanesulfonate (MMS)-induced alkylation damage in DNA involves Mag1 but not Tpa1. We observed that yeast strains without tpa1 are no more sensitive to MMS than WT yeast, whereas mag1-deficient yeast are ∼500-fold more sensitive to MMS. We therefore investigated the substrate specificity of Mag1 and found that it excises alkylated bases that are known AlkB substrates. In contrast, purified recombinant Tpa1 did not repair these alkylated DNA substrates, but it did exhibit the prolyl hydroxylase activity that has also been ascribed to it. A comparison of several of the kinetic parameters of Mag1 and its E. coli homolog AlkA revealed that Mag1 catalyzes base excision from known AlkB substrates with greater efficiency than does AlkA, consistent with an expanded role of yeast Mag1 in repair of alkylation damage. Our results challenge the proposal that Tpa1 directly functions in DNA repair and suggest that Mag1-initiated base excision repair compensates for the absence of oxidative dealkylation of alkylated nucleobases in budding yeast. This expanded role of Mag1, as compared with alkylation repair glycosylases in other organisms, could explain the extreme sensitivity of Mag1-deficient S. cerevisiae toward alkylation damage.

Double-strand DNA breaks (DSBs) are toxic to cells, and improper repair can cause chromosomal abnormalities that initiate and drive cancer progression. DNA ligases III and IV (LIG3, LIG4) have long been credited for repair of DSBs in mammals, but recent evidence suggests that DNA ligase I (LIG1) has intrinsic end-joining (EJ) activity that can compensate for their loss. To test this model, we employed in vitro biochemical assays to compare EJ by LIG1 and LIG3. The ligases join blunt-end and 3'-overhang-containing DNA substrates with similar catalytic efficiency, but LIG1 joins 5'-overhang-containing DNA substrates ∼20-fold less efficiently than LIG3 under optimal conditions. LIG1-catalyzed EJ is compromised at a physiological concentration of Mg2+, but its activity is restored by increased molecular crowding. In contrast to LIG1, LIG3 efficiently catalyzes EJ reactions at a physiological concentration of Mg2+ with or without molecular crowding. Under all tested conditions, LIG3 has greater affinity than LIG1 for DNA ends. Remarkably, LIG3 can ligate both strands of a DSB during a single binding encounter. The weaker DNA binding affinity of LIG1 causes significant abortive ligation that is sensitive to molecular crowding and DNA terminal structure. These results provide new insights into mechanisms of alternative nonhomologous EJ.

Human alkyladenine DNA glycosylase (AAG) initiates base excision repair (BER) to guard against mutations by excising alkylated and deaminated purines. Counterintuitively, increased expression of AAG has been implicated in increased rates of spontaneous mutation in microsatellite repeats. This microsatellite mutator phenotype is consistent with a model in which AAG excises bulged (unpaired) bases, altering repeat length. To directly test the role of base excision in AAG-induced mutagenesis, we conducted mutation accumulation experiments in yeast overexpressing different variants of AAG and detected mutations via high-depth genome resequencing. We also developed a new software tool, hp_caller, to perform accurate genotyping at homopolymeric repeat loci. Overexpression of wild-type AAG elevated indel mutations in homopolymeric sequences distributed throughout the genome. However, catalytically inactive variants (E125Q/E125A) caused equal or greater increases in frameshift mutations. These results disprove the hypothesis that base excision is the key step in mutagenesis by overexpressed wild-type AAG. Instead, our results provide additional support for the previously published model wherein overexpressed AAG interferes with the mismatch repair (MMR) pathway. In addition to the above results, we observed a dramatic mutator phenotype for N169S AAG, which has increased rates of excision of undamaged purines. This mutant caused a 10-fold increase in point mutations at G:C base pairs and a 50-fold increase in frameshifts in A:T homopolymers. These results demonstrate that it is necessary to consider the relative activities and abundance of many DNA replication and repair proteins when considering mutator phenotypes, as they are relevant to the development of cancer and its resistance to treatment.

Nucleotide flipping is a common feature of DNA-modifying enzymes that allows access to target sites within duplex DNA. Structural studies have identified many intercalating amino acid side chains in a wide variety of enzymes, but the functional contribution of these intercalating residues is poorly understood. We used site-directed mutagenesis and transient kinetic approaches to dissect the energetic contribution of intercalation for human alkyladenine DNA glycosylase, an enzyme that initiates repair of alkylation damage. When AAG flips out a damaged nucleotide, the void in the duplex is filled by a conserved tyrosine (Y162). We find that tyrosine intercalation confers 140-fold stabilization of the extrahelical specific recognition complex, and that Y162 functions as a plug to slow the rate of unflipping by 6000-fold relative to the Y162A mutant. Surprisingly, mutation to the smaller alanine side chain increases the rate of nucleotide flipping by 50-fold relative to the wild-type enzyme. This provides evidence against the popular model that DNA intercalation accelerates nucleotide flipping. In the case of AAG, DNA intercalation contributes to the specific binding of a damaged nucleotide, but this enhanced specificity comes at the cost of reduced speed of nucleotide flipping.

TP53 mutations underlie Barrett's esophagus (BE) progression to dysplasia and cancer. During BE progression, the ubiquitin ligase (E3) RNF128/GRAIL switches expression from isoform 2 (Iso2) to Iso1, stabilizing mutant p53. However, the ubiquitin-conjugating enzyme (E2) that partners with Iso1 to stabilize mutant p53 is unknown.