Mechanisms of glycosylase induced genomic instability

<div><p>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.</p></div

Fluctuation analysis reveals similar mutator phenotypes for E125Q and E125A AAG and preservation of mutator phenotypes during passaging.

<p>Frameshift mutation rates were measured using the <i>LYS2</i> poly-A reporter allele for -1 frameshift events in E134-derived lines (A) and +1 frameshift events in E133-derived lines (B). Mutation rates were measured by fluctuation analysis in unpassaged strains and for a subset of the lines in each strain after passaging, specifically, the passaged lines with the fewest number of mutations. Error bars indicate the standard error of the mean. Fluctuation analysis could not be performed in ending lines marked with “NA” (not applicable), because these lines did not contain the appropriate <i>LYS2</i> reporter allele (see Table B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174041#pone.0174041.s001" target="_blank">S1 File</a>). Fluctuation analysis was not performed (“ND”) in the passaged Y162A lines since this construct did not induce a mutator phenotype in the mutation accumulation experiments.</p

Point mutations in AAG overexpression lines.

<p>(A) The number of point mutations in each mutation accumulation line. Lines with additional mutator phenotypes are indicated with closed squares. Lines with inactivating mutations in the AAG gene are labeled “null” and marked with an ×. The pseudodiploid line is marked with a closed triangle. (B) The mean number of mutations at A:T and G:C base pairs per strain. (C) The mean number of point mutations in each category for the empty vector strain and the N169S strain. Error bars are 95% confidence intervals.</p

High-confidence genotyping at homopolymer loci with hp_caller.

<p>A) The hp_caller program discriminates between a mutant sample (red) and the other seven non-mutant samples in the same strain. Histograms plotting read depth at each observed homopolymer length are shown for the wild-type AAG strain for the T₁₂ homopolymer at chromosome VIII:50981–50993. The mutation is in line 22556 (see Table B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174041#pone.0174041.s001" target="_blank">S1 File</a> for line identification). B) Genotype calls at the <i>LYS2</i> polyA reporter locus are consistent with the lysine requirements of the three strains shown (A₁₄ = 22537, A₁₃ = 22538, A₁₂ = 22539). C) Read depth distributions at all loci with mutations were comparable for strains called as reference (gray) and non-reference (red).</p

Glycosylase-induced indels in A:T homopolymers are predominantly -1 frameshifts.

<p>The fraction of indels of a given size is plotted for the (A) wild-type, (B) E125Q, and (C) N169S AAG strains. Error bars are 95% confidence intervals. The empty vector and Y162A strains are not shown because they have no indel mutations.</p

Models for glycosylase-induced mutagenesis.

<p>For simplicity, only the most relevant pathways and intermediates are shown. Replication (not shown) occasionally gives rise to bulged species in models A and B (see Fig O in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174041#pone.0174041.s001" target="_blank">S1 File</a> for more detailed models). (A) The MMR interference model proposes that glycosylase binding to bulged, undamaged bases prevents MMR from repairing the bulged base. The pathway shown is for -1 frameshift events, but +1 frameshifts can be stabilized by the same mechanism. (B) The bulge-excision model proposes that bulged bases are deleted by the BER pathway and could be elevated by base damage. This pathway produces only -1 frameshifts, because nascent +1 frameshifts (not shown) would be faithfully repaired. (C) The gratuitous excision model postulates that removal of undamaged bases is mutagenic. In the case of the N169S mutant, most of the gratuitous excision occurs at undamaged guanines. In yeast, abasic sites are paired with A and C during replication. Pairing with C is non-mutagenic in this case, and is not shown. (D) Gratuitous excision in homopolymers effectively increases the number of times that a homopolymer is replicated, thus increasing frameshift rates. Both -1 and +1 frameshift events would occur by this mechanism, but only a -1 frameshift is shown.</p

Strains expressing mutator glycosylase alleles have decreased pYES2 expression plasmid copy numbers.

<p>(A) pYES2 copy numbers for the parental (unpassaged) strains, as well as each of the ending strains. The two lines with inactivating mutations in the AAG gene are shown as the strain “N169S null”. (B) Endogenous 2-micron circle copy numbers for the parental strains and ending strains. (C) U-test comparing the distribution of plasmid copy numbers between the mutator strains (E125Q + N169S) and the nonmutator strains (empty + WT + Y162A) for the pYES2 expression vector. (D) U-test comparing the copy numbers of the endogenous 2-micron circle for mutator and nonmutator strains. Horizontal lines in all panels indicate the median of each distribution.</p

Frameshift mutations in A:T homopolymers are increased in the wild-type, E125Q, and N169S strains.

<p>(A) The number of frameshift mutations in A:T homopolymers in each line. (B) The average rate of frameshift mutations at all A:T homopolymers with lengths between 7 and 16 nt. Error bars are 95% confidence intervals calculated by the Clopper-Pearson method. Asterisks indicate p-values: *, p < 0.05; **, p < 0.005; ***, p < 0.0005; Fisher’s exact test for indicated pairwise comparisons. (C) Mutation rates in A:T homopolymers as a function of homopolymer length.</p

The wild-type and mutant AAG enzymes exhibit different affinities for bulged and duplex DNA.

<p>(A) Sample EMSA gel image for E125A. Singly and multiply bound complexes were combined in calculating the fraction of DNA bound, because these species are not cleanly separated. (B) Example binding curves for the E125A mutant. (C) Apparent <i>K</i>_D values were determined by EMSA for the indicated substrates, containing a centrally located, bulged undamaged adenosine, or fully duplex DNA (undamaged). n = 3; error bars are SEM. E125Q and E125A both exhibit tighter binding than wild-type to a bulged site, whereas the Y162A exhibits much weaker binding and does not distinguish between bulged and intact duplexes. Binding data for all constructs is shown in Fig M in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174041#pone.0174041.s001" target="_blank">S1 File</a>.</p