Disruption of Transcriptional Coactivator Sub1 Leads to Genome-Wide Re-distribution of Clustered Mutations Induced by APOBEC in Active Yeast Genes

Abstract

Mutations in genomes of species are frequently distributed non-randomly, resulting in mutation clusters, including recently discovered kataegis in tumors. DNA editing deaminases play the prominent role in the etiology of these mutations. To gain insight into the enigmatic mechanisms of localized hypermutagenesis that lead to cluster formation, we analyzed the mutational single nucleotide variations (SNV) data obtained by whole-genome sequencing of drug-resistant mutants induced in yeast diploids by AID/APOBEC deaminase and base analog 6-HAP. Deaminase from sea lamprey, PmCDA1, induced robust clusters, while 6-HAP induced a few weak ones. We found that PmCDA1, AID, and APOBEC1 deaminases preferentially mutate the beginning of the actively transcribed genes. Inactivation of transcription initiation factor Sub1 strongly reduced deaminase-induced can1 mutation frequency, but, surprisingly, did not decrease the total SNV load in genomes. However, the SNVs in the genomes of the sub1 clones were re-distributed, and the effect of mutation clustering in the regions of transcription initiation was even more pronounced. At the same time, the mutation density in the protein-coding regions was reduced, resulting in the decrease of phenotypically detected mutants. We propose that the induction of clustered mutations by deaminases involves: a) the exposure of ssDNA strands during transcription and loss of protection of ssDNA due to the depletion of ssDNA-binding proteins, such as Sub1, and b) attainment of conditions favorable for APOBEC action in subpopulation of cells, leading to enzymatic deamination within the currently expressed genes. This model is applicable to both the initial and the later stages of oncogenic transformation and explains variations in the distribution of mutations and kataegis events in different tumor cells.

Author Summary

Genomes of tumors are heavily enriched with mutations. Some of these mutations are distributed non-randomly, forming mutational clusters. Editing cytosine deaminases from APOBEC superfamily are responsible for the formation of many of these clusters. We have expressed APOBEC enzyme in diploid yeast cells and found that most of the mutations occur in the beginning of the active genes, where transcription starts. Clusters of mutations overlapped with promoters/transcription start sites. This is likely due to the weaker protection of ssDNA, an ultimate APOBEC deaminase enzyme target, in the beginning of the genes. This hypothesis was reinforced by the finding that inactivation of Sub1 transcription initiation factor, which is found predominantly in the regions of transcription initiation, leads to further increase in mutagenesis in the beginning of the genes. Interestingly, the total number of mutations in the genomes of Sub1-deficient clones did not change, despite the 100-fold decrease in frequency of mutants in a reporter gene. Thus, the drastic change in genome-wide distribution of mutations can be caused by inactivation of a single gene. We propose that the loss of ssDNA protection factors causes formation of mutation clusters in human cancer.