Avoidance of APOBEC3B-induced mutation by error-free lesion bypass

Abstract

APOBEC cytidine deaminases mutate cancer genomes by converting cytidines into uridines within ssDNA during replication. Although uracil DNA glycosylases limit APOBEC-induced mutation, it is unknown if subsequent base excision repair (BER) steps function on replication-associated ssDNA. Hence, we measured APOBEC3B-induced CAN1 mutation frequencies in yeast deficient in BER endonucleases or DNA damage tolerance proteins. Strains lacking Apn1, Apn2, Ntg1, Ntg2 or Rev3 displayed wild-type frequencies of APOBEC3B-induced canavanine resistance (CanR). However, strains without error-free lesion bypass proteins Ubc13, Mms2 and Mph1 displayed respective 4.9-, 2.8- and 7.8-fold higher frequency of APOBEC3B-induced CanR. These results indicate that mutations resulting from APOBEC activity are avoided by deoxyuridine conversion to abasic sites ahead of nascent lagging strand DNA synthesis and subsequent bypass by error-free template switching. We found this mechanism also functions during telomere re-synthesis, but with a diminished requirement for Ubc13. Interestingly, reduction of G to C substitutions in Ubc13-deficient strains uncovered a previously unknown role of Ubc13 in controlling the activity of the translesion synthesis polymerase, Rev1. Our results highlight a novel mechanism for error-free bypass of deoxyuridines generated within ssDNA and suggest that the APOBEC mutation signature observed in cancer genomes may under-represent the genomic damage these enzymes induce.

Figure 1.

Reporters used in this study. (A) The CAN1 forward mutation reporter was placed 16 kb centromere proximal to the replication origin ARS216 on Chr II. This system has been previously shown to accumulate mutations predominantly through A3B-induced deamination during replication. During DNA replication the lagging strand template is exposed to A3B, resulting in targeted dC to dU deamination. This confers a mutation in CAN1 which allows the yeast to survive on selective canavanine media. Based upon the location of CAN1 to the centromere proximal side of ARS216, the bottom DNA strand of the gene is the lagging strand template. Thus, cytidine deamination occurs on the bottom strand, resulting in mutations at G bases (sequencing reports mutations relative to the top DNA strand). (B) The CAN1 forward mutation reporter was placed 7.5 kb from the end of the telomere of Chr V. This system contains the cdc13-1 mutant allele which uncaps telomeres at nonpermissive temperatures and halts the growth of Saccharomyces cerevisiae at G₂ phase following DNA replication. Following telomere uncapping, 5΄ to 3΄ resection initiates, resulting in the exposure of dC in ssDNA to A3B. When sequenced, the mutations in the re-synthesized strand are reported as mutations in dG.

Figure 2.

BER is not a predominant method of removing A3B-induced dU formed during replication. (A) The frequencies of canavanine-resistance (Can^R) induced in WT, ung1Δ, apn1Δ, apn2Δ, apn1Δ apn2Δ, ntg1Δ, ntg2Δ and ntg1Δ ntg2Δ yeast following transformation with vector control plasmid, or A3B expression plasmid were assessed. Can^R frequency was determined in isogenic strains harboring the CAN1 reporter on Chr II. Horizontal bars and numeric values indicate the median frequency of six independent replicates. Statistical significance was determined by a two-tailed non-parametric Mann–Whitney rank sum test. (B) The strand bias of CAN1 mutations from A3B-expressing wild-type, apn1Δ apn2Δ and ung1Δ yeast strains were assessed by Sanger sequencing. The mutation spectra frequency represents the proportion of the Can^R frequency in (A) that can be attributed to the individual mutation types. The numerical values above the bars indicate the number of mutations in each strand for each strain. Statistical significance of strand bias in each genotype was determined by a two-tailed G-test of goodness-of-fit. **:P < 0.02.

Figure 3.

Error-free bypass is the DDT pathway used to avoid A3B-induced lesions incurred ahead of polymerase δ lagging strand DNA synthesis. (A) Possible mechanisms for processing dU and their mutagenic outcomes. If left within DNA, the A3B-induced dU will confer a G to A mutation due to templating during replication, as indicated in black. Glycolytic removal of the uracil base by Ung1, converts dU to an abasic site, which can then serve to induce DNA damage tolerance (DDT) pathways to bypass the lesion. Specific modifications on the PCNA clamp determine pathway choice. PCNA SUMOylation by Ubc9-Siz1 controls recombination-mediated fork restart, noted in red. Monoubiquitination of PCNA by Rad18-Rad6 can result in two outcomes: the initiation of TLS and the insertion of untemplated nucleotides across from the abasic site (purple) or the extension of the ubiquitin chain by Rad5-Ubc13-Mms2 (blue). The latter results in the initiation of error-free lesion bypass. (B and C) Frequencies and mutational spectra of Can^R following transformation with A3B expression plasmid assessed in yeast strains harboring the CAN1 reporter on Chr II were assessed. (B) rev3Δ, siz1Δ, ubc13Δ, mms2Δ, mph1Δ and mte1Δ yeast strains were compared to discern the usage of the various DDT pathways to avoid abasic sites generated during replication. Horizontal bars and numerical values indicate the median frequency of six or seven independent replicates. Statistical significance was determined by a two-tailed non-parametric Mann-Whitney rank sum test. WT and ung1Δ values from Figure 2 are included for reference. (C) The mutation spectra of the CAN1 in independent WT, ubc13Δ, mms2Δ or ung1Δ Can^R yeast were determined through PacBio and Sanger Sequencing. The mutation spectra frequency represents the proportion of the Can^R frequency in (A) that can be attributed to the individual mutation types. The numerical values above the bars indicate the number of isolates with the reported mutation. Statistical significance of G to A and G to C mutation ratios in each genetic backgrounds were determined by two-tailed G-test of goodness-of-fit test. *:P < 0.05, **: P < 0.01.

Figure 4.

Mutagenesis in error-free bypass-deficient strains is TLS-dependent. A3B-induced Can^R frequencies WT, rev3Δ, ubc13Δ, ubc13Δ rev3Δ, mph1Δ and mph1Δ rev3Δ yeast. Can^R frequency was determined with CAN1 reporter on Chr II. Values for WT, rev3Δ, ubc13Δ and mph1Δ from Figure 3 are included for reference. Horizontal bars and numeric values indicate the median frequency of six or seven independent replicates. Statistical significance was determined by a two-tailed non-parametric Mann–Whitney rank sum test.

Figure 5.

Ubc13 is dispensable for error-free bypass of abasic sites in non-replicative DNA synthesis but plays a role in Rev1 usage. Frequencies and mutational spectra of Can^R following transformation with vector control plasmid, or A3B expression plasmid assessed in yeast strains harboring the CAN1 reporter on Chr V. (A) cdc13-1, cdc13-1 ung1Δ, cdc13-1 mph1Δ, cdc13-1 rev3Δ and cdc13-1 ubc13Δ yeast strains were assessed to discern the usage of the error-free bypass pathway to avoid abasic sites generated during nonreplicative DNA synthesis. Horizontal bars and numerical values indicate the median frequency of six or seven independent replicates. Statistical significance was determined by a two-tailed non-parametric Mann–Whitney rank sum test. (B) The mutation spectra of the CAN1 in independent cdc13-1, cdc13-1 ung1Δ and cdc13-1 ubc13Δ Can^R yeast were assessed through PacBio sequencing. The mutation spectra frequency represents the proportion of the Can^R frequency in (A) that can be attributed to the individual mutation types. The numerical values above the bars indicate the number of isolates with the reported mutation. Statistical significance of G to A and G to C mutation ratios between genetic backgrounds were determined by two-tailed Fisher's exact test.

Figure 6.

Model for avoidance of APOBEC-induced lesions ahead of lagging strand DNA synthesis. (A) Single stranded DNA in the lagging strand template contain cytidines susceptible for APOBEC-induced deamination. These dU are excised by Ung1 ahead of the replicative polymerase, generating a fork-stalling abasic site (denoted as AP). The majority of these lesions are bypassed in an error-free manner by template switching, which is dependent on Ubc13, Mms2, Rad5 and Mph1. Some abasic sites are bypassed via the error-prone TLS pathway, dependent on Rev1 and polymerase ζ. Here, Ubc13 and Rad5 influence polymerase choice by promoting Rev1-mediated dC insertion, resulting in G to C substitutions. Alternatively, A-rule insertion by a different polymerase results in G to A substitutions. (B) Following end resection, cytidines on the non-resected DNA strand are exposed to APOBEC activity. These dU are excised by Ung1, generating abasic sites. During gap-fill synthesis, the polymerase stalls at the abasic sites. At this point, the lesion is predominantly bypassed through Mph1-dependent error-free template switch, potentially using its sister chromatid as a template or through error-prone TLS, dependent on Rev1 and polymerase ζ. As with (A), Ubc13 promotes Rev1 cytidine transferase activity during TLS, however, Ubc13-mediated polyubiquitination of PCNA is unnecessary for initiating error-free template switching.