Her2 amplification, Rel-A, and Bach1 can influence APOBEC3A expression in breast cancer cells.

APOBEC-induced mutations occur in 50% of sequenced human tumors, with APOBEC3A (A3A) being a major contributor to mutagenesis in breast cancer cells. The mechanisms that cause A3A activation and mutagenesis in breast cancers are still unknown. Here, we describe factors that influence basal A3A mRNA transcript levels in breast cancer cells. We found that basal A3A mRNA correlates with A3A protein levels and predicts the amount of APOBEC signature mutations in a panel of breast cancer cell lines, indicating that increased basal transcription may be one mechanism leading to breast cancer mutagenesis. We also show that alteration of ERBB2 expression can drive A3A mRNA levels, suggesting the enrichment of the APOBEC mutation signature in Her2-enriched breast cancer could in part result from elevated A3A transcription. Hierarchical clustering of transcripts in primary breast cancers determined that A3A mRNA was co-expressed with other genes functioning in viral restriction and interferon responses. However, reduction of STAT signaling via inhibitors or shRNA in breast cancer cell lines had only minor impact on A3A abundance. Analysis of single cell RNA-seq from primary tumors indicated that A3A mRNA was highest in infiltrating immune cells within the tumor, indicating that correlations of A3A with STAT signaling in primary tumors may be result from higher immune infiltrates and are not reflective of STAT signaling controlling A3A expression in breast cancer cells. Analysis of ATAC-seq data in multiple breast cancer cell lines identified two transcription factor sites in the APOBEC3A promoter region that could promote A3A transcription. We determined that Rel-A, and Bach1, which have binding sites in these peaks, elevated basal A3A expression. Our findings highlight a complex and variable set of transcriptional activators for A3A in breast cancer cells.

An impaired ubiquitin-proteasome system increases APOBEC3A abundance.

Apolipoprotein B messenger RNA (mRNA) editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases cause genetic instability during cancer development. Elevated APOBEC3A (A3A) levels result in APOBEC signature mutations; however, mechanisms regulating A3A abundance in breast cancer are unknown. Here, we show that dysregulating the ubiquitin-proteasome system with proteasome inhibitors, including Food and Drug Administration-approved anticancer drugs, increased A3A abundance in breast cancer and multiple myeloma cell lines. Unexpectedly, elevated A3A occurs via an ∼100-fold increase in A3A mRNA levels, indicating that proteasome inhibition triggers a transcriptional response as opposed to or in addition to blocking A3A degradation. This transcriptional regulation is mediated in part through FBXO22, a protein that functions in SKP1-cullin-F-box ubiquitin ligase complexes and becomes dysregulated during carcinogenesis. Proteasome inhibitors increased cellular cytidine deaminase activity, decreased cellular proliferation and increased genomic DNA damage in an A3A-dependent manner. Our findings suggest that proteasome dysfunction, either acquired during cancer development or induced therapeutically, could increase A3A-induced genetic heterogeneity and thereby influence therapeutic responses in patients.

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.

Genetic modifiers of APOBEC-induced mutagenesis.

The cytidine deaminases APOBEC3A and APOBEC3B (A3B) are prominent mutators of human cancer genomes. However, tumor-specific genetic modulators of APOBEC-induced mutagenesis are poorly defined. Here, we utilized a screen to identify 61 gene deletions that increase A3B-induced mutations in yeast. Also, we determined whether each deletion was epistatic with UNG1 loss, which indicated whether the encoded factors participate in the error-free bypass of A3B/Ung1-dependent abasic sites or suppress A3B-catalyzed deamination by protecting against aberrant formation of single stranded DNA (ssDNA). Additionally, we determined 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, which results in extremely high A3B-induced mutagenesis, (2) defective CTF18-RFC complex function, which results in high levels of A3B induced mutations specifically on the leading strand template that synergistically increase with loss of UNG1, and (3) defective HR-mediated bypass of APOBEC-induced lesions, which were epistatic with Ung1 loss and result from increased Rev1-mediated C-to-G substitutions. We extended these results by analyzing mutation data for human tumors and found BRCA1/2-deficient breast cancer tumors display 3- to 4-fold more APOBEC-induced mutations. Mirroring our results in yeast, for BRCA1/2 deficient tumors Rev1-mediated C-to-G substitutions are solely responsible for increased APOBEC-signature mutations and these mutations occur on the lagging strand during DNA replication. Together these results identify important factors that influence the dynamics of DNA replication and likely the abundance of APOBEC-induced mutation during tumor progression as well as a novel mechanistic role for BRCA1/2 during HR-dependent lesion bypass of APOBEC-induced lesions during cancer cell replication.

APOBEC-Induced Mutagenesis in Cancer.

The initiation, progression, and relapse of cancers often result from mutations occurring within somatic cells. Consequently, processes that elevate mutation rates accelerate carcinogenesis and hinder the development of long-lasting therapeutics. Recent sequencing of human cancer genomes has identified patterns of mutations, termed mutation signatures, many of which correspond to specific environmentally induced and endogenous mutation processes. Some of the most frequently observed mutation signatures are caused by dysregulated activity of APOBECs, which deaminate cytidines in single-stranded DNA at specific sequence motifs causing C-to-T and C-to-G substitutions. In humans, APOBEC-generated genetic heterogeneity in tumor cells contributes to carcinogenesis, metastasis, and resistance to therapeutics. Here, we review the current understanding of APOBECs' role in cancer mutagenesis and impact on disease and the biological processes that influence APOBEC mutagenic capacity.

The Shu complex prevents mutagenesis and cytotoxicity of single-strand specific alkylation lesions.

Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Thus, our work identifies a novel homologous recombination-based mechanism mediated by the Shu complex for coping with alkylation adducts.

Single-stranded DNA binding proteins influence APOBEC3A substrate preference.

The cytidine deaminase, APOBEC3A (A3A), is a prominent source of mutations in multiple cancer types. These APOBEC-signature mutations are non-uniformly distributed across cancer genomes, associating with single-stranded (ss) DNA formed during DNA replication and hairpin-forming sequences. The biochemical and cellular factors that influence these specificities are unclear. We measured A3A's cytidine deaminase activity in vitro on substrates that model potential sources of ssDNA in the cell and found that A3A is more active on hairpins containing 4 nt ssDNA loops compared to hairpins with larger loops, bubble structures, replication fork mimics, ssDNA gaps, or linear DNA. Despite pre-bent ssDNAs being expected to fit better in the A3A active site, we determined A3A favors a 4 nt hairpin substrate only 2- to fivefold over linear ssDNA substrates. Addition of whole cell lysates or purified RPA to cytidine deaminase assays more severely reduced A3A activity on linear ssDNA (45 nt) compared to hairpin substrates. These results indicate that the large enrichment of A3A-driven mutations in hairpin-forming sequences in tumor genomes is likely driven in part by other proteins that preferentially bind longer ssDNA regions, which limit A3A's access. Furthermore, A3A activity is reduced at ssDNA associated with a stalled T7 RNA polymerase, suggesting that potential protein occlusion by RNA polymerase also limits A3A activity. These results help explain the small transcriptional strand bias for APOBEC mutation signatures in cancer genomes and the general targeting of hairpin-forming sequences in the lagging strand template during DNA replication.

APOBEC3A is a prominent cytidine deaminase in breast cancer.

APOBEC cytidine deaminases are the second-most prominent source of mutagenesis in sequenced tumors. Previous studies have proposed that APOBEC3B (A3B) is the major source of mutagenesis in breast cancer (BRCA). We show that APOBEC3A (A3A) is the only APOBEC whose expression correlates with APOBEC-induced mutation load and that A3A expression is responsible for cytidine deamination in multiple BRCA cell lines. Comparative analysis of A3A and A3B expression by qRT-PCR, RSEM-normalized RNA-seq, and unambiguous RNA-seq validated the use of RNA-seq to measure APOBEC expression, which indicates that A3A is the primary correlate with APOBEC-mutation load in primary BRCA tumors. We also demonstrate that A3A has >100-fold more cytidine deamination activity than A3B in the presence of cellular RNA, likely explaining why higher levels of A3B expression contributes less to mutagenesis in BRCA. Our findings identify A3A as a major source of cytidine deaminase activity in breast cancer cells and possibly a prominent contributor to the APOBEC mutation signature.

The Rad51 paralogs facilitate a novel DNA strand specific damage tolerance pathway.

Accurate DNA replication is essential for genomic stability and cancer prevention. Homologous recombination is important for high-fidelity DNA damage tolerance during replication. How the homologous recombination machinery is recruited to replication intermediates is unknown. Here, we provide evidence that a Rad51 paralog-containing complex, the budding yeast Shu complex, directly recognizes and enables tolerance of predominantly lagging strand abasic sites. We show that the Shu complex becomes chromatin associated when cells accumulate abasic sites during S phase. We also demonstrate that purified recombinant Shu complex recognizes an abasic analog on a double-flap substrate, which prevents AP endonuclease activity and endonuclease-induced double-strand break formation. Shu complex DNA binding mutants are sensitive to methyl methanesulfonate, are not chromatin enriched, and exhibit increased mutation rates. We propose a role for the Shu complex in recognizing abasic sites at replication intermediates, where it recruits the homologous recombination machinery to mediate strand specific damage tolerance.

Risks at the DNA Replication Fork: Effects upon Carcinogenesis and Tumor Heterogeneity.

The ability of all organisms to copy their genetic information via DNA replication is a prerequisite for cell division and a biological imperative of life. In multicellular organisms, however, mutations arising from DNA replication errors in the germline and somatic cells are the basis of genetic diseases and cancer, respectively. Within human tumors, replication errors additionally contribute to mutator phenotypes and tumor heterogeneity, which are major confounding factors for cancer therapeutics. Successful DNA replication involves the coordination of many large-scale, complex cellular processes. In this review, we focus on the roles that defects in enzymes that normally act at the replication fork and dysregulation of enzymes that inappropriately damage single-stranded DNA at the fork play in causing mutations that contribute to carcinogenesis. We focus on tumor data and experimental evidence that error-prone variants of replicative polymerases promote carcinogenesis and on research indicating that the primary target mutated by APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like) cytidine deaminases is ssDNA present at the replication fork. Furthermore, we discuss evidence from model systems that indicate replication stress and other cancer-associated metabolic changes may modulate mutagenic enzymatic activities at the replication fork.

APOBEC3A and APOBEC3B Preferentially Deaminate the Lagging Strand Template during DNA Replication.

APOBEC family cytidine deaminases have recently been implicated as powerful mutators of cancer genomes. How APOBECs, which are ssDNA-specific enzymes, gain access to chromosomal DNA is unclear. To ascertain the chromosomal ssDNA substrates of the APOBECs, we expressed APOBEC3A and APOBEC3B, the two most probable APOBECs mediating cancer mutagenesis, in a yeast model system. We demonstrate, using mutation reporters and whole genome sequencing, that APOBEC3A- and APOBEC3B-induced mutagenesis primarily results from the deamination of the lagging strand template during DNA replication. Moreover, our results indicate that both genetic deficiencies in replication fork-stabilizing proteins and chemical induction of replication stress greatly augment the mutagenesis of APOBEC3A and APOBEC3B. Taken together, these results strongly indicate that ssDNA formed during DNA lagging strand synthesis is a major substrate for APOBECs and may be the principal substrate in human cancers experiencing replication stress.

DNA polymerase ζ-dependent lesion bypass in Saccharomyces cerevisiae is accompanied by error-prone copying of long stretches of adjacent DNA.

Translesion synthesis (TLS) helps cells to accomplish chromosomal replication in the presence of unrepaired DNA lesions. In eukaryotes, the bypass of most lesions involves a nucleotide insertion opposite the lesion by either a replicative or a specialized DNA polymerase, followed by extension of the resulting distorted primer terminus by DNA polymerase ζ (Polζ). The subsequent events leading to disengagement of the error-prone Polζ from the primer terminus and its replacement with an accurate replicative DNA polymerase remain largely unknown. As a first step toward understanding these events, we aimed to determine the length of DNA stretches synthesized in an error-prone manner during the Polζ-dependent lesion bypass. We developed new in vivo assays to identify the products of mutagenic TLS through a plasmid-borne tetrahydrofuran lesion and a UV-induced chromosomal lesion. We then surveyed the region downstream of the lesion site (in respect to the direction of TLS) for the presence of mutations indicative of an error-prone polymerase activity. The bypass of both lesions was associated with an approximately 300,000-fold increase in the mutation rate in the adjacent DNA segment, in comparison to the mutation rate during normal replication. The hypermutated tract extended 200 bp from the lesion in the plasmid-based assay and as far as 1 kb from the lesion in the chromosome-based assay. The mutation rate in this region was similar to the rate of errors produced by purified Polζ during copying of undamaged DNA in vitro. Further, no mutations downstream of the lesion were observed in rare TLS products recovered from Polζ-deficient cells. This led us to conclude that error-prone Polζ synthesis continues for several hundred nucleotides after the lesion bypass is completed. These results provide insight into the late steps of TLS and show that error-prone TLS tracts span a substantially larger region than previously appreciated.

Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity.

Defects in DNA polymerases δ (Polδ) and ε (Polε) cause hereditary colorectal cancer and have been implicated in the etiology of some sporadic colorectal and endometrial tumors. We previously reported that the yeast pol3-R696W allele mimicking a human cancer-associated variant, POLD1-R689W, causes a catastrophic increase in spontaneous mutagenesis. Here, we describe the mechanism of this extraordinary mutator effect. We found that the mutation rate increased synergistically when the R696W mutation was combined with defects in Polδ proofreading or mismatch repair, indicating that pathways correcting DNA replication errors are not compromised in pol3-R696W mutants. DNA synthesis by purified Polδ-R696W was error-prone, but not to the extent that could account for the unprecedented mutator phenotype of pol3-R696W strains. In a search for cellular factors that augment the mutagenic potential of Polδ-R696W, we discovered that pol3-R696W causes S-phase checkpoint-dependent elevation of dNTP pools. Abrogating this elevation by strategic mutations in dNTP metabolism genes eliminated the mutator effect of pol3-R696W, whereas restoration of high intracellular dNTP levels restored the mutator phenotype. Further, the use of dNTP concentrations present in pol3-R696W cells for in vitro DNA synthesis greatly decreased the fidelity of Polδ-R696W and produced a mutation spectrum strikingly similar to the spectrum observed in vivo. The results support a model in which (i) faulty synthesis by Polδ-R696W leads to a checkpoint-dependent increase in dNTP levels and (ii) this increase mediates the hypermutator effect of Polδ-R696W by facilitating the extension of mismatched primer termini it creates and by promoting further errors that continue to fuel the mutagenic pathway.

DNA polymerases ζ and Rev1 mediate error-prone bypass of non-B DNA structures.

DNA polymerase ζ (Pol ζ) and Rev1 are key players in translesion DNA synthesis. The error-prone Pol ζ can also participate in replication of undamaged DNA when the normal replisome is impaired. Here we define the nature of the replication disturbances that trigger the recruitment of error-prone polymerases in the absence of DNA damage and describe the specific roles of Rev1 and Pol ζ in handling these disturbances. We show that Pol ζ/Rev1-dependent mutations occur at sites of replication stalling at short repeated sequences capable of forming hairpin structures. The Rev1 deoxycytidyl transferase can take over the stalled replicative polymerase and incorporate an additional 'C' at the hairpin base. Full hairpin bypass often involves template-switching DNA synthesis, subsequent realignment generating multiply mismatched primer termini and extension of these termini by Pol ζ. The postreplicative pathway dependent on polyubiquitylation of proliferating cell nuclear antigen provides a backup mechanism for accurate bypass of these sequences that is primarily used when the Pol ζ/Rev1-dependent pathway is inactive. The results emphasize the pivotal role of noncanonical DNA structures in mutagenesis and reveal the long-sought-after mechanism of complex mutations that represent a unique signature of Pol ζ.

A reversible histone H3 acetylation cooperates with mismatch repair and replicative polymerases in maintaining genome stability.

Mutations are a major driving force of evolution and genetic disease. In eukaryotes, mutations are produced in the chromatin environment, but the impact of chromatin on mutagenesis is poorly understood. Previous studies have determined that in yeast Saccharomyces cerevisiae, Rtt109-dependent acetylation of histone H3 on K56 is an abundant modification that is introduced in chromatin in S phase and removed by Hst3 and Hst4 in G2/M. We show here that the chromatin deacetylation on histone H3 K56 by Hst3 and Hst4 is required for the suppression of spontaneous gross chromosomal rearrangements, base substitutions, 1-bp insertions/deletions, and complex mutations. The rate of base substitutions in hst3Δ hst4Δ is similar to that in isogenic mismatch repair-deficient msh2Δ mutant. We also provide evidence that H3 K56 acetylation by Rtt109 is important for safeguarding DNA from small insertions/deletions and complex mutations. Furthermore, we reveal that both the deacetylation and acetylation on histone H3 K56 are involved in mutation avoidance mechanisms that cooperate with mismatch repair and the proofreading activities of replicative DNA polymerases in suppressing spontaneous mutagenesis. Our results suggest that cyclic acetylation and deacetylation of chromatin contribute to replication fidelity and play important roles in the protection of nuclear DNA from diverse spontaneous mutations.

Histone deacetylase complexes promote trinucleotide repeat expansions.

Expansions of DNA trinucleotide repeats cause at least 17 inherited neurodegenerative diseases, such as Huntington's disease. Expansions can occur at frequencies approaching 100% in affected families and in transgenic mice, suggesting that specific cellular proteins actively promote (favor) expansions. The inference is that expansions arise due to the presence of these promoting proteins, not their absence, and that interfering with these proteins can suppress expansions. The goal of this study was to identify novel factors that promote expansions. We discovered that specific histone deacetylase complexes (HDACs) promote CTG•CAG repeat expansions in budding yeast and human cells. Mutation or inhibition of yeast Rpd3L or Hda1 suppressed up to 90% of expansions. In cultured human astrocytes, expansions were suppressed by 75% upon inhibition or knockdown of HDAC3, whereas siRNA against the histone acetyltransferases CBP/p300 stimulated expansions. Genetic and molecular analysis both indicated that HDACs act at a distance from the triplet repeat to promote expansions. Expansion assays with nuclease mutants indicated that Sae2 is one of the relevant factors regulated by Rpd3L and Hda1. The causal relationship between HDACs and expansions indicates that HDACs can promote mutagenesis at some DNA sequences. This relationship further implies that HDAC3 inhibitors being tested for relief of expansion-associated gene silencing may also suppress somatic expansions that contribute to disease progression.

A cancer-associated DNA polymerase delta variant modeled in yeast causes a catastrophic increase in genomic instability.

Accurate DNA synthesis by the replicative DNA polymerases alpha, delta, and epsilon is critical for genome stability in eukaryotes. In humans, over 20 SNPs were reported that result in amino-acid changes in Poldelta or Polepsilon. In addition, Poldelta variants were found in colon-cancer cell lines and in sporadic colorectal carcinomas. Using the yeast-model system, we examined the functional consequences of two cancer-associated Poldelta mutations and four polymorphisms affecting well-conserved regions of Poldelta or Polepsilon. We show that the R696W substitution in Poldelta (analog of the R689W change in the human cancer-cell line DLD-1) is lethal in haploid and homozygous diploid yeast. The cell death results from a catastrophic increase in spontaneous mutagenesis attributed to low-fidelity DNA synthesis by Poldelta-R696W. Heterozygotes survive, and the mutation rate depends on the relative expression level of wild-type versus mutant alleles. Based on these observations, we propose that the mutation rate in heterozygous human cells could be regulated by transient changes in gene expression leading to a temporary excess of Poldelta-R689W. The similarities between the mutational spectra of the yeast strains producing Poldelta-R696W and DLD-1 cells suggest that the altered Poldelta could be responsible for a significant proportion of spontaneous mutations in this cancer cell line. These results suggest that the highly error-prone Poldelta-R689W could contribute to cancer initiation and/or progression in humans.

Postreplication repair inhibits CAG.CTG repeat expansions in Saccharomyces cerevisiae.

Trinucleotide repeats (TNRs) are unique DNA microsatellites that can expand to cause human disease. Recently, Srs2 was identified as a protein that inhibits TNR expansions in Saccharomyces cerevisiae. Here, we demonstrate that Srs2 inhibits CAG . CTG expansions in conjunction with the error-free branch of postreplication repair (PRR). Like srs2 mutants, expansions are elevated in rad18 and rad5 mutants, as well as the PRR-specific PCNA alleles pol30-K164R and pol30-K127/164R. Epistasis analysis indicates that Srs2 acts upstream of these PRR proteins. Also, like srs2 mutants, the pol30-K127/164R phenotype is specific for expansions, as this allele does not alter mutation rates at dinucleotide repeats, at nonrepeating sequences, or for CAG . CTG repeat contractions. Our results suggest that Srs2 action and PRR processing inhibit TNR expansions. We also investigated the relationship between PRR and Rad27 (Fen1), a well-established inhibitor of TNR expansions that acts at 5' flaps. Our results indicate that PRR protects against expansions arising from the 3' terminus, presumably replication slippage events. This work provides the first evidence that CAG . CTG expansions can occur by 3' slippage, and our results help define PRR as a key cellular mechanism that protects against expansions.