APOBEC3B cytidine deaminase targets the non-transcribed strand of tRNA genes in yeast

Abstract

Variations in mutation rates across the genome have been demonstrated both in model organisms and in cancers. This phenomenon is largely driven by the damage specificity of diverse mutagens and the differences in DNA repair efficiency in given genomic contexts. Here, we demonstrate that the single-strand DNA-specific cytidine deaminase APOBEC3B (A3B) damages tRNA genes at a 1000-fold higher efficiency than other non-tRNA genomic regions in budding yeast. We found that A3B-induced lesions in tRNA genes were predominantly located on the non-transcribed strand, while no transcriptional strand bias was observed in protein coding genes. Furthermore, tRNA gene mutations were exacerbated in cells where RNaseH expression was completely abolished (Δrnh1Δrnh35). These data suggest a transcription-dependent mechanism for A3B-induced tRNA gene hypermutation. Interestingly, in strains proficient in DNA repair, only 1% of the abasic sites formed upon excision of A3B-deaminated cytosines were not repaired leading to mutations in tRNA genes, while 18% of these lesions failed to be repaired in the remainder of the genome. A3B-induced mutagenesis in tRNA genes was found to be efficiently suppressed by the redundant activities of both base excision repair (BER) and the error-free DNA damage bypass pathway. On the other hand, deficiencies in BER did not have a profound effect on A3B-induced mutations in CAN1, the reporter for protein coding genes. We hypothesize that differences in the mechanisms underlying ssDNA formation at tRNA genes and other genomic loci are the key determinants of the choice of the repair pathways and consequently the efficiency of DNA damage repair in these regions. Overall, our results indicate that tRNA genes are highly susceptible to ssDNA-specific DNA damaging agents. However, increased DNA repair efficacy in tRNA genes can prevent their hypermutation and maintain both genome and proteome homeostasis.

Fig. 1. A3B-induced mutation load and spread in tRNA genes. (A)

The number of mutations in tRNA genes in each isolate sequenced and the median are depicted. (B) A3B-induced mutations in the Δung1 background are enriched within tRNA genes and spread up to 200 bases within the genes.

Fig. 2. A3B-induced mutation density is higher in tRNA genes than the remaining genome. (A)

Density of A3B-mutations in tC or cC motifs per tc or cc dinucleotide present in tRNA genes and non-tRNA genomic regions in the Δung1 background is calculated (mutated residue is capitalized). (B) The mutation rates per targeted nucleotide in CAN1 leading to Can^R (non-tRNA) and Trp-tRNA(tRNA) genes leading to Trp⁺ phenotype in the Δung1 background are depicted. The target size was calculated as the number of tC and cC motifs multiplied by the total number of strains sequenced.

Fig. 3. Replication and transcription-dependent strand-bias in A3B-induced mutations in tRNA genes and other genomic regions

(A) Relative number of A3B-induced C→T and G→A changes in tRNA genes and non-tRNA genomic regions are plotted against the fractional distance between neighboring origins of replication in the yeast genome. The X-axis denotes the distance between neighboring origins of replication divided into 5 bins. (B) The percentages of A3B-induced mutations in the transcribed and non-transcribed strands of tRNA genes and other genes in the Δung1 strain are depicted. (C) The relative proportion of C→T changes in the transcribed and non-transcribed strands along the distance of tRNA genes in the Δung1 strain is shown. On the X-axis, the relative distances from tRNA gene start position is plotted in increments of 100 bases. The negative values denote regions upstream of tRNA genes, while the positive values represent genomic positions downstream of tRNA start positions.

Fig. 4. Model for A3B-induced mutagenesis and repair in tRNA genes and non-tRNA genomic regions

ssDNA mutated by A3B (yellow circle) in tRNA genes is stabilized by RNA:DNA hybrids formed during transcription (red = RNA) while in other genomic regions ssDNA is formed on the lagging strand during DNA replication (blue arrows). Moreover, the RNA transcript at tRNA genes could directly recruit A3B to the ssDNA formed during transcription. In the absence of Ung1 (blue circle), replication across dU residues leads to a C→T transition (red). Ung1 (blue circle) can act on dU residues and form abasic sites in DNA (empty squares). In tRNA genes, removal of RNA leads to reannealing of the strands, and base excision repair machinery (pink circle) can repair the abasic site in an error-free manner. On the other hand, persistent RNA:DNA hybrids, would prevent repair by BER, and abasic site would later be presented as a block to the replication fork. Abasic sites at the fork, can either be undergo error-free repair by the error-free DNA damage bypass pathway (orange circle) or are bypassed by translesion synthesis polymerases (green circle) which can lead to C→T or C→G substitutions (red).