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.

Strand-biased cytosine deamination at the replication fork causes cytosine to thymine mutations in Escherichia coli

“…It is possible that a larger mutational harvest would reveal a significant transcription strand bias, especially in highly transcribed genes, but in our data, any such bias was overwhelmed by the observed replication strand bias. In contrast, Lada et al found that most mutations caused by the lamprey cytosine deaminase, PmCDA1, in nondividing yeast were correlated with transcription (40). Together, these observations suggest that in dividing cells, the greatest source of C:G to T:A mutations is the deamination of cytosines in the template for the lagging strand synthesis, but transcription may play a larger role in nondividing cells.…”

“…One characteristic of cancer genome mutations is that they are found in clusters, suggesting that they occur in genomic regions that contain stretches of ssDNA (34). Although a number of cellular processes, including replication, transcription, and recombination, have been proposed as sources of ssDNA targets for APOBEC3s (13,34,(40)(41)(42), experimental evidence for these ideas is limited. The data presented here suggest that the LGST at the replication forks of rapidly dividing cancer cells would be accessible to these enzymes, and hence the cytosines mutated in cancer genomes should be found preferentially in the LGST compared with the LDST.…”

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

“…Cytidine deamination by AID on ssDNA during transcription of variable regions in the immunoglobulin locus is required for the initiation of both class-switch recombination and somatic hypermutation [18,19]. Interestingly, the activity of AID is regulated at the transcription bubble through the interaction with transcriptional co factors that assist with AID catalytic activity [20,21]. The expression of AID in B cells is essential as AID deficiency leads to Hyper-IgM syndrome and the inability to produce antibodies other than IgM M2 [22].…”

“…For a related family member AID, which normally deaminates the immunoglobulin genes to enable antibody maturation and class switching [11], targeting of AID to the correct genomic region and maximizing its access to both strands of the genomic DNA requires many interacting partner [186]. Although AID can access other regions of the genome, it is thought that interactions with transcription machinery occur to facilitate its specific role in deaminating cytosine in immunoglobulin genes [20,21]. These protein-protein interactions may not only assist in targeting immunoglobulin genes during B-cell activation, but may be required for AID to catalyze deaminations since its catalytic rate is slow [187].…”

“…The generation of increased amounts of ssDNA promotes the activity of A3 and increases the mutational frequency Thus, it is notable that A3B is not problematic until a cellular alteration occurs that increases A3B expression and increases replication stress [177,192,203]. In contrast, the ssDNA generated on the nontranscribed strand during transcription is accessible at multiple times during the cell cycle of a normal or transformed cell, especially during RNA polymerase stalling [20,21]. Although A3 enzymes would still need to access transiently available nontranscribed ssDNA that interacts with the RNA polymerase, the ssDNA is solvent accessible [204].…”