Genetic inhibitors of APOBEC3B-induced mutagenesis

The cytidine deaminases APOBEC3A (A3A) and APOBEC3B (A3B) are prominent mutators of human cancer genomes. However, tumor-specific genetic modulators of APOBEC-induced mutagenesis are poorly defined. Here, we used a screen to identify 61 gene deletions that increase A3B-induced mutations in yeast. We also determined whether each deletion was epistatic with Ung1 loss, which indicated whether the encoded factors participate in the homologous recombination (HR)–dependent bypass of A3B/Ung1-dependent abasic sites or suppress A3B-catalyzed deamination by protecting against aberrant formation of single-stranded DNA (ssDNA). We found that the mutation spectra of A3B-induced mutations revealed genotype-specific patterns of strand-specific ssDNA formation and nucleotide incorporation across APOBEC-induced lesions. Combining these three metrics, we were able to establish a multifactorial signature of APOBEC-induced mutations specific to (1) failure to remove H3K56 acetylation, (2) defective CTF18–RFC complex function, and (3) defective HR-mediated bypass of APOBEC-induced lesions. We extended these results by analyzing mutation data for human tumors and found BRCA1/2-deficient breast cancers display three- to fourfold more APOBEC-induced mutations. Mirroring our results in yeast, Rev1-mediated C-to-G substitutions are mainly responsible for increased APOBEC-signature mutations in BRCA1/2-deficient tumors, and these mutations associate with lagging strand synthesis during replication. These results identify important factors that influence DNA replication dynamics and likely the abundance of APOBEC-induced mutation during tumor progression. They also highlight a novel role for BRCA1/2 during HR-dependent lesion bypass of APOBEC-induced lesions during cancer cell replication.

UNG1 deletion strains were created by either transforming yeast strains with PCRgenerated NatMX deletion cassettes or by a mating yTM-03 and strains from the deletion library, sporulating, and selecting haploid yeast on SC-Leu+G418.Yeast strains with the rev1-D467A, E468A mutation were generated with CRISPR-Cas9 using methods as in (Vandenberg et al. 2023) and homology directed repair donor oligos SAR616 and SAR617.The rev1-D467A, E468A strains were validated by PCR amplification using oligos oTM-1200 and oTM-1203 and Sanger sequencing with oligo SAR622.For genotypes and additional details about individual strains, (see Supplemental Table S7) and for primer sequences used to construct and verify gene deletions (see Supplemental Table S8).

Plasmid construction
The A3A yeast expression plasmid, pVH-A3A, was constructed by ligating a DraI and PfimI (NEB) digested gBlock containing the A3A CDS with the yeast ACT1 intron inserted 20 nucleotides 3' of the A3A start codon (i.e.SRO235; gBlocks® Gene Fragment technology; IDT) into the PmeI and PflmI restriction sites of a previously constructed yeast A3A expression vector, pSR435 (Chan et al. 2015).Correct cloning was confirmed by Sanger sequencing.
Sequencing of RT-PCR products for the exogenously expressed A3A from pVH-A3A in yeast with primer SRO251(Supplemental Table S8) confirmed accurate splicing of the A3A mRNA.

RT-qPCR to assess changes in A3B expression.
Yeast strains containing pSR-440 (A3B expression) were grown overnight in 20 mL of YPDA+hygromycinB and cell pellets from these cultures were frozen in liquid nitrogen.RNA was extracted from these pellets using TRIzol and glass beads, which was followed by a second purification using the E.Z.N.A.Total RNA Kit I (Omega Biotek) with an on-column DNase I digest.cDNA was generated using MashUp RT, a reverse transcriptase based on FeLV-RT, which was purified as in (Alekseenko et al. 2021).qPCR was performed using Forget-Me-Not EvaGreen qPCR Master Mix (Biotium) and primers oTM-115 and oTM-117 (A3B specific) and oTM-1348 and oTM-1349 (ALD6 specific) (Supplemental Table S8).This large increase in A3B induced mutagenesis is suppressed by deletion of either ASF1 or RTT109, which indicated it is dependent on hyperacetylated H3K56.Interestingly, combining ung1Δ and hst3Δ did not further increase APOBEC-induced mutagenesis, which indicates a lack of HR-meditated bypass in hst3Δ yeast, which is consistent with literature indicating establishment and removal of H3K56ac is important for multiple aspects of homologous recombination.(C) Defects in the CTF18-RFC complex increase in the mutation rate greater than ung1Δ alone and further increase the mutation rate when combined with ung1Δ, which indicates that deletion of CTF8, CTF18, and DCC1 likely increase ssDNA.A significant bias to dG residues in the CAN1 coding sequence points to elevated ssDNA on the leading strand, which likely results from decreased loading/unloading of PCNA on the leading strand and reduced efficiency of DNA replication and/or lesion bypass.

B. H3 K56 acetylation
Results in C-to-T and C-to-G mutations in the CAN1 coding sequence Supplemental FigureS1.Models for increased APOBEC-induced mutagenesis.Data from https://cerevisiae.
of APOBEC-induced mutagenesis in deletion strains with defective HR-directed repair, H3K56ac, and CTF18-RFC.(A) For yeast strains with deletions known to result in defective homology-directed repair, the A3B-induced Can R rates for yeast strains with and without UNG1, rate of mutations at C and G nucleotides (stand-bias of A3B-induced mutations), and spectra of A3B-induced substitutions (indicative Rev1-dependent TLS usage) are compared.(B) For yeast strains with deletions of genes encoding proteins that modulate H3K56ac, the A3B-induced Can R rates for yeast strains with and without UNG1, rate of mutations at C and G nucleotides (stand-bias of A3B-induced mutations), and spectra of A3B-induced substitutions (indicative Rev1-dependent TLS usage) are compared.(C) For yeast strains with deletions of genes encoding members of the CTF18-RFC complex, the A3B-induced Can R rates for yeast strains with and without UNG1, rate of mutations at C and G nucleotides (stand-bias of A3B-induced mutations), and spectra of A3B-induced substitutions (indicative Rev1-dependent TLS usage) are compared.deficient breast cancers have elevated APOBEC signature mutagenesis.Amounts of SBS2 and 13 in breast cancers with germline mutations in (A) BRCA1 or (B) BRCA2 compared to tumors without germline BRCA1/2 mutations.(C) Amounts of SBS2 and 13 in breast cancers with and without SBS3 (indicative of HR-deficiency).(D) Amounts of SBS2 and 13 in breast cancers with SBS3 but no germline BRCA1/2 mutations (i.e.somatically acquired HR-deficiency) compared to those in tumors with germline BRCA1/2 mutations (i.e.germline HR-deficiency).Central dashed line indicates median values.P-values were calculated by Mann-Whitney U test.
oridb.org indicates that the CAN1 gene is replicated most often from ARS507, which places dC residues of the CAN1 coding sequence on the lagging strand template during DNA replication.This observation is consistent with our data showing APOBEC-induced mutations at dC residues are 2.1-fold more frequent than those at dG residues.Blue arrows in the depiction of replication forks indicate DNA synthesis.Black arrows indicate steps that control outcomes of dC residues deaminated by A3B and are weighted more heavily for steps that lead to the predominant outcomes.Maroon arrows indicated how outcomes are changed by defective processes.The dC residues deaminated by A3B are highlighted yellow throughout the figure.(A)Outcomes of APOBEC-induced deamination in wild type yeast and strains lacking Ung1 or HR-dependent lesion bypass.Approximately 80% of APOBEC-generated dU residues are converted to abasic sites by Ung1.In wild type yeast, the vast majority of these abasic sites are channeled into HR-dependent lesion bypass, which avoids mutations that would be generated by TLS.Loss of Ung1 results in all APOBEC-generated dU residues templating during DNA replication resulting in C-to-T mutations.Loss of HR-dependent lesion bypass results in abasic sites being bypassed by TLS with insertion of dC across form the abasic site by Rev1 and extension by Polζ being the predominate mechanism, which results in increased C-to-G mutations.(B) Deletion of HST3 led to the largest increase in A3B-induced mutagenesis and a mutation rate approximately twice that of ung1Δ yeast, which would indicate hst3Δ increases ssDNA.