uracil-DNA Glycosylase Research Articles

Construction of a genome-editing system for the thermophilic actinomycete streptomyces thermodiastaticus K5 strain.

Thermophilic actinomycetes significantly contribute to the terrestrial carbon cycle via the rapid degradation of lignocellulosic polysaccharides in composts. In this study, a genome-editing system was constructed for the thermophilic actinomycetes Streptomyces thermodiastaticus K5 strain, which was isolated from compost. The genome-editing plasmid (pGEK5) harboring niccase Cas9 was derived from the high-copy plasmid pL99 and used for the K5 strain. It was found that pGEK5 could easily be lost from the transformed clone through cultivation on apramycin-free medium and spore formation, enabling its reuse for subsequent genome-editing cycles. With the aid of this plasmid, mutations were sequentially introduced to two uracil-DNA glycosylase genes (Udg1 and Udg2) and one β-glucosidase gene (Bgl1). Thus, the genome-editing system using pGEK5 enables us to start the functional modification of this thermophilic actinomycete, especially for improved conversion of lignocellulosic biomass.

Uracil base PCR implemented for reliable DNA walking

PCR-based DNA walking is of efficacy for capturing unknown flanking genomic sequences. Here, an uracil base PCR (UB-PCR) with satisfying specificity has been devised for DNA walking. Primary UB-PCR replaces thymine base with uracil base, resulting in a primary PCR product composed of U-DNAs. A single-primer (primary nested sequence-specific primer) single-cycle amplification, using the four normal bases (adenine, thymine, cytosine, and guanine) as substrate, is then performed on the primary PCR product. Clearly, only those U-DNAs, ended by the primary nested sequence-specific primer at least at one side, will produce the corresponding normal single strands. Next, the single-cycle product undergoes uracil-DNA glycosylase treatment to destroy the U-DNAs, while the normal single strands are unaffected. Afterward, secondary even tertiary PCR is performed to exclusively enrich the target product. The feasibility of UB-PCR has been checked by obtaining unknown sequences bordering the three selected genetic sites.

Global screening of base excision repair in nucleosome core particles

DNA damage is a fundamental molecular cause of genomic instability. Base excision repair (BER) is one line of defense to minimize the potential mutagenicity and/or toxicity derived from damaged nucleobase lesions. However, BER in the context of chromatin, in which eukaryotic genomic DNA is compacted through a hierarchy of DNA-histone protein interactions, is not fully understood. Here, we investigate the activity of BER enzymes at 27 unique geometric locations in a nucleosome core particle (NCP), which is the minimal unit of packaging in chromatin. The BER enzymes include uracil DNA glycosylase (UDG), AP endonuclease 1 (APE1), DNA polymerase β (Pol β), and DNA ligase IIIα complexed with X-ray repair cross complementing group 1 (LigIIIα/XRCC1). This global analysis of BER reveals that initiation of the repair event by UDG is dictated by the rotational position of the lesion. APE1 has robust activity at locations where repair is initiated whereas the repair event stalls at the Pol β nucleotide incorporation step within the central ∼45 bp of nucleosomal DNA. The final step of the repair, catalyzed by LigIIIα/XRCC1, is achieved only in the entry/exit regions of the NCP when nick sites are transiently exposed by unwrapping from the histones. Kinetic assays further elucidate that the location of the damaged lesion modulates enzymatic activity. Notably, these data indicate that some of the BER enzymes can act at a significant number of locations even in the absence of chromatin remodelers or other cellular factors. These results inform genome wide maps of DNA damage and mutations and contribute to our understanding of mutational hotspots and signatures.

DUF99 family proteins are novel endonucleases that cleave deoxyuridine on DNA substrates

DNA deamination occurs constantly in a cell and causes DNA damage. As this damage can be deleterious, organisms have evolved many systems to eliminate it, such as endonuclease V (Endo V). DUF99 family protein contains a domain of unknown function similar to Endo V, but had not been experimentally characterized to date. Here, we show that DUF99 family proteins cleave the 3'-side of deoxyuridine (dU) on DNA substrates. Based on phylogenetic analysis, we designated this new protein family as Endonuclease dU (Endo_dU). We also observed that Endo_dU coding gene frequently colocalizes with that for uracil-DNA glycosylase (UDG) in halophilic archaea, and we further performed gene knockout of Endo_dU gene on Haloferax volcanii. The transcription level of UDG gene on Endo_dU knockout strain was increased when induced by sodium bisulfite. Thus, we hypothesize that Endo_dU establishes a new endonuclease family with broad phylogenetic distribution and may participate in DNA repair.

Sequentially Activated-Dumbbell DNA Nanodevices for Accurate Detection of Uracil-DNA Glycosylase via PER-Based Orthogonal Signal Outputs.

Accurate and reliable detection of uracil-DNA glycosylase (UDG) activity is crucial for clinical diagnosis and prognosis assessment. However, current techniques for accurately monitoring UDG activity still face significant challenges due to the single input or output signal modes. Here, we develop a sequentially activated-dumbbell DNA nanodevice (SEAD) that enables precise and reliable evaluation of UDG activity through primer exchange reactions (PER)-based orthogonal signal output. The SEAD incorporates a double-hairpin structure with a stem containing two deoxyuridine (dU) sites for target recognition and two preblocked primer binding regions for target amplification and signal output. Upon UDG recognition of dU, the SEAD can be cleaved by apurinic/apyrimidinic endonuclease 1 (APE1), generating two different hairpins with exposed primer binding regions. These hairpins serve as templates to initiate the parallel PER, enabling the extending of two different amplification products: a long single-stranded DNA (ssDNA) with repetitive sequences and a short ferrocene-labeled ssDNA with complementary sequences. These products further self-assemble into DNA nano-strings in an orthogonal manner that act as an electrochemiluminescence signal switch, enabling precise detection of low-abundance UDG. This work develops a sequential input and orthogonal output strategy for accurately monitoring UDG activity, highlighting the significant potential in cancer diagnosis and treatment.

DNA polymerase mediated CRISPR/Cas12a trans-cleavage for dual-mode quantification of uracil DNA glycosylase activity

Since an unusual expression of uracil-DNA glycosylase (UDG) is often associated with the pathogenesis of numerous disorders, the detection of UDG activity is regarded as a promising application in disease diagnosis. Here, we develop a DNA polymerase-mediated CRISPR/Cas12a trans cleavage strategy, which can achieve dual-mode determination of UDG activity. By introducing a hairpin DNA probe containing a single uracil base, the probe undergoes specific cleavage and elongation under the existence of UDG only, thus activating the trans cleavage of ssDNA regardless of its length and sequence. To accommodate different detection modes, the ssDNA was further modified by fluorophore-quencher pairs or designed for connecting magnetic microparticles (MMPs) and polystyrene microparticles (PMPs). Finally, the UDG activity is quantified by fluorescence signal and microparticle accumulation length on a microfluidic chip visible to the naked eye. This strategy provides a detectable minimum UDG concentration of 0.00047 U/mL for fluorescent mode and 0.0048 U/mL for microfluidic mode. Furthermore, we performed the UDG inhibition test and detected UDG activity in cell lysates, suggesting its potential for inhibitor screening and detection of UDG in biological samples.

ATP-driven AP endonuclease-active Exo III@zeolitic imidazolate framework for uracil-DNA glycosylase imaging in living cells

In the currently available approaches for intracellular uracil-DNA glycosylase (UDG) detection, their signal outputs usually rely on the cleavage of the residual AP sites in DNA probes by apurinic/apyrimidinic endonuclease 1 (APE1), whose expression level varies across different types of cells. Therefore, reliable visualizing detection of UDG in living cells remains challenging. Herein, a nanoprobe for imaging of intracellular UDG activity has been designed by taking advantages of both zeolitic imidazolate framework-90 (ZIF-90) and endonuclease III. ZIF-90 is used to deliver exonuclease III (Exo III) and hairpin DNA strands into cells. One of the hairpin strands (HP1) contains a deoxyuridine (dU) base in its stem region, while the other (HP2) was modified with FAM molecule and BHQ1 at its 3′ and 5′ ends, respectively. In the presence of UDG, the dU base in HP1 is excised to form an AP site. Exo III can cleave the AP site in HP1 to generate a stem-loop fragment, which can hybridize with HP2 to initiate the recycling digestion of HP2 by Exo III, resulting in amplification of the fluorescence signal and hence sensitive detection of UDG (limit of detection: 2.0×10−6 U/mL). Experimental results have demonstrated that the nanoprobe has merits of good biocompatibility, high sensitivity and selectivity. It realizes in-situ imaging of intracellular UDG without the involvement of APE1, is a promising alternative tool for UDG detection in fields of clinical diagnosis and biomedical research.

Multienzymatic Orthogonal Activation of DNA Codec Enables Tumor-Specific Imaging of Base Excision Repair Activity.

Accurate monitoring of base excision repair (BER) activity in cancer cells is critical for advancing the comprehension of DNA repair processes, gaining insights into cancer development, and guiding treatment strategies. However, current assay techniques for assessing BER activity in cancer cells face challenges due to the heterogeneous origins and diversity of BER enzymes. In this work, we present a highly reliable triple loop-interlocked DNA codec (GATED) that enables precise assessment of BER activity in cancer cells through signal amplification mediated by multienzyme orthogonal activation. The GATED device features a dumbbell-shaped DNA probe to encode two BER enzymes for BER-related signal conversion as well as two bound circular DNA to decode the apurinic/apyrimidinic sites for apurinic/apyrimidinic endonuclease 1 (APE1)-mediated signal amplification. Importantly, GATED is orthogonally activated by multiple target BER enzymes (i.e., uracil DNA glycosylase, thymine DNA glycosylase, and APE1), resulting in a unified fluorescent signal that significantly improves the detection specificity and sensitivity to BER enzymes. Additionally, we demonstrate that the GATED has exceptional biostability within complex biological systems, where it was successfully employed to monitor BER activity in cancer cells with high specificity and enabled cell-based high-throughput screening for BER inhibitors. The GATED provides a much-needed tool for the real-time monitoring of BER activity and the screening of BER inhibitors in cancer cells, potentially advancing both the investigation and clinical application of BER biology.

Herbicide-resistant plants produced by precision adenine base editing in plastid DNA.

CRISPR-free, protein-only cytosine base editors (CBEs) or adenine base editors, composed of DNA-binding proteins such as zinc finger proteins or transcription activator-like effectors (TALEs) and nucleobase cytosine or adenine deaminases, respectively, enable organellar DNA editing in cultured cells, animals and plants1-4. TALE-linked double-stranded DNA deaminase toxin A (DddAtox)-derived CBEs (DdCBEs) and TALE-linked adenine deaminases (TALEDs) install C-to-T and A-to-G single-nucleotide conversions, respectively, in mitochondria and chloroplasts5-9. Interestingly, whereas TALEDs exclusively induce A-to-G conversions without C-to-T conversions in mammalian mitochondrial DNA10, they often install unwanted C-to-T edits in addition to intended A-to-G edits in plastid DNA7,9,11,12. Here we show that uracil DNA glycosylase (UDG)-fused TALEDs (UDG-TALEDs) minimize C-to-T conversions without reducing the A-to-G editing efficiency and install a mutation in the chloroplast psbA gene that encodes a single-amino-acid substitution (S264G), which confers herbicide resistance in the resulting plants.

Biochemical reconstitution of heat-induced mutational processes.

Non-enzymatic spontaneous deamination of 5-methylcytosine, producing thymine, is the proposed etiology of cancer mutational signature 1, which is the most predominant signature in all cancers. Here, the proposed mutational process was reconstituted using synthetic DNA and purified proteins. First, single-stranded DNA containing 5-methylcytosine at CpG context was incubated at an elevated temperature to accelerate spontaneous DNA damage. Then, the DNA was treated with uracil DNA glycosylase to remove uracil residues that were formed by deamination of cytosine. The resulting DNA was then used as a template for DNA synthesis by yeast DNA polymerase δ. The DNA products were analyzed by next-generation DNA sequencing, and mutation frequencies were quantified. The observed mutations after this process were exclusively C>T mutations at CpG context, which was very similar to signature 1. When 5-methylcytosine modification and uracil DNA glycosylase were both omitted, C>T mutations were produced on C residues in all sequence contexts, but these mutations were diminished by uracil DNA glycosylase-treatment. These results indicate that the CpG>TpG mutations were produced by the deamination of 5-methylcytosine. Additional mutations, mainly C>G, were introduced by yeast DNA polymerase ζ on the heat-damaged DNA, indicating that G residues of the templates were also damaged. However, the damage on G residues was not converted to mutations with DNA polymerase δ or ε.

FAM72A degrades UNG2 through the GID/CTLH complex to promote mutagenic repair during antibody maturation

A diverse antibody repertoire is essential for humoral immunity. Antibody diversification requires the introduction of deoxyuridine (dU) mutations within immunoglobulin genes to initiate somatic hypermutation (SHM) and class switch recombination (CSR). dUs are normally recognized and excised by the base excision repair (BER) protein uracil-DNA glycosylase 2 (UNG2). However, FAM72A downregulates UNG2 permitting dUs to persist and trigger SHM and CSR. How FAM72A promotes UNG2 degradation is unknown. Here, we show that FAM72A recruits a C-terminal to LisH (CTLH) E3 ligase complex to target UNG2 for proteasomal degradation. Deficiency in CTLH complex components result in elevated UNG2 and reduced SHM and CSR. Cryo-EM structural analysis reveals FAM72A directly binds to MKLN1 within the CTLH complex to recruit and ubiquitinate UNG2. Our study further suggests that FAM72A hijacks the CTLH complex to promote mutagenesis in cancer. These findings show that FAM72A is an E3 ligase substrate adaptor critical for humoral immunity and cancer development.

Single G-quadruplex-based fluorescence method for the uracil-DNA glycosylase inhibitor screening

Uracil-DNA glycosylase (UDG) plays a pivotal role in the base repair system. Through bioinformatics, we found that the expression of the UDG enzyme in many cancer cells is increased, and its high expression is not conducive to the prognosis of lung cancer patients. The development of analytical techniques for the quantification of UDG activity and the identification of UDG inhibitors is of paramount importance. We found that when the T base in the G4 loop region mutated to uracil, the G4 structure was not disrupted and still retained the characteristics of a G4 structure (emitting strong fluorescence after binding with ThT (Thioflavin T). Inspired by this phenomenon, we developed a detection method for UDG and its inhibitors utilizing a single DNA strand engineered to form a G-quadruplex structure, containing uracil residues within the loop region, designated as G4-dU. The inclusion of uracil-DNA glycosylase (UDG) in the assay environment induces the removal of uracil, resulting in the formation of apurinic sites (AP) within the G4-dU sequence. Subsequent thermal denaturation leads to strand cleavage at AP sites, precluding the reformation of the G-quadruplex configuration and abrogating fluorescence emission. The detection process in this study can be completed in only 30 min to 1 h, offers a straightforward, expedient, and efficacious modality for assessing UDG activity and UDG inhibitor potency, thereby facilitating the discovery of novel therapeutic agents for cancer treatment.

Distance-based paper device coupled with uracil-rich DNA hydrogel for visual quantification of Uracil-DNA glycosylase

Uracil-DNA glycosylase (UDG), an enzyme for repairing uracil-containing DNA damage, is crucial for maintaining genomic stability. Simple and fast quantification of UDG activity is essential for biological assay and clinical diagnosis, since its aberrant level is associated with DNA damage and various diseases. Herein, we developed a fully integrated “sample in-signal out” distance-based paper analytical device (dPAD) for visual quantification of UDG using a flow-controlled uracil-rich DNA hydrogel (URDH). The uracil base sites contained in the DNA hydrogel are mis-incorporated with dUTP by rolling circle amplification (RCA), which simplifies the preparation process of the functionalized hydrogel. In the presence of UDG, the uracil in URDH can be recognized and removed to induce the permeability change of URDH, resulting in the visible distance signal along the paper channel. Using dPAD, as low as 6.4 × 10−4 U/mL of UDG (within 80 min) is visually identified without any instruments and complicated operations. This integrated dPAD is advantageous for its simplicity, cost effectiveness, and ease of use. We envision that it has the great potential for point-of-care testing (POCT) in DNA damage testing, personalized healthcare assessment, and biomedical applications.

Enzyme-Powered, Label-Free DNA Walker for Uracil-DNA Glycosylase Detection at Single-Cell Level.

Uracil-DNA glycosylase (UDG) plays a crucial role in the removal of damaged uracil bases, thereby upholding genetic stability and integrity. An enzyme-powered, label-free DNA walker was devised for UDG activity detection. Initially, a label-free DNA track, incorporating a gold nanoparticle (AuNP), multiple hairpin structures, and various swing arms, was engineered for walking mechanism. The hairpin structure was meticulously crafted to include a G-quadruplex sequence, enabling the generation of a label-free fluorescence signal. The swing arm remained inert in the absence of UDG, but became activated upon the introduction of UDG, thereby initiating the enzyme-powered walking process and generating significant dissociative G-quadruplex sequences. By integrating a selective fluorescent dye into the design, an enhanced label-free fluorescence response was achieved. The proposed DNA walker presented a direct and label-free approach for UDG detection, demonstrating exceptional sensitivity with a detection limit of 0.00004 U/mL. Using the uracil glycosylase inhibitor (UGI) as an inhibitory model, inhibitor assay was conducted with satisfactory precision. Furthermore, successful analysis of cellular UDG at the single-cell level was accomplished. Consequently, the developed DNA walker serves as a label-free, selective, and sensitive tool for UDG activity assessment, showing great potential for applications in disease diagnosis, inhibitor screening, and biomedical investigations.

Hypomorphic mutation in the large subunit of replication protein A affects mutagenesis by human APOBEC cytidine deaminases in yeast.

Human APOBEC single-strand (ss) specific DNA and RNA cytidine deaminases change cytosines to uracils (U's) and function in antiviral innate immunity and RNA editing and can cause hypermutation in chromosomes. The resulting U's can be directly replicated, resulting in C to T mutations, or U-DNA glycosylase can convert the U's to abasic (AP) sites which are then fixed as C to T or C to G mutations by translesion DNA polymerases. We noticed that in yeast and in human cancers, contributions of C to T and C to G mutations depend on the origin of ssDNA mutagenized by APOBECs. Since ssDNA in eukaryotic genomes readily binds to replication protein A (RPA) we asked if RPA could affect APOBEC-induced mutation spectrum in yeast. For that purpose, we expressed human APOBECs in the wild-type (WT) yeast and in strains carrying a hypomorph mutation rfa1-t33 in the large RPA subunit. We confirmed that the rfa1-t33 allele can facilitate mutagenesis by APOBECs. We also found that the rfa1-t33 mutation changed the ratio of APOBEC3A-induced T to C and T to G mutations in replicating yeast to resemble a ratio observed in long persistent ssDNA in yeast and in cancers. We present the data suggesting that RPA may shield APOBEC formed U's in ssDNA from Ung1, thereby facilitating C to T mutagenesis through the accurate copying of U's by replicative DNA polymerases. Unexpectedly, we also found that for U's shielded from Ung1 by WT RPA, the mutagenic outcome is reduced in the presence of translesion DNA polymerase zeta.

Programmable DNA pyrimidine base editing via engineered uracil-DNA glycosylase

DNA base editing technologies predominantly utilize engineered deaminases, limiting their ability to edit thymine and guanine directly. In this study, we successfully achieve base editing of both cytidine and thymine by leveraging the translesion DNA synthesis pathway through the engineering of uracil-DNA glycosylase (UNG). Employing structure-based rational design, exploration of homologous proteins, and mutation screening, we identify a Deinococcus radiodurans UNG mutant capable of effectively editing thymine. When fused with the nickase Cas9, the engineered DrUNG protein facilitates efficient thymine base editing at endogenous sites, achieving editing efficiencies up to 55% without enrichment and exhibiting minimal cellular toxicity. This thymine base editor (TBE) exhibits high editing specificity and significantly restores IDUA enzyme activity in cells derived from patients with Hurler syndrome. TBEs represent efficient, specific, and low-toxicity approaches to base editing with potential applications in treating relevant diseases.

A Lateral Flow Biosensor Based on Isothermal Amplification for Visual Identification of Species and Sex from Bloodstain.

The prompt species identification from biological samples at a crime scene can rapidly filter out truly valuable biometric information for subsequent personal identification. Meanwhile, early sex determination can assist in narrowing the pool of suspects. However, the current methods for forensic DNA analysis, particularly in point-of-care scenarios, are often limited by the intricate equipment for signal generation and the laborious procedure for DNA purification. The present study introduces a novel portable lateral flow biosensor that possesses extraction-free and anti-aerosol characteristics for on-site determination of species and sex. The bloodstain can be directly submitted to loop-mediated isothermal amplification (LAMP) for the analysis of both mitochondrial and nuclear DNA. The incorporation of a lateral flow device with gold magnetic nanoparticle probes allows for visual interpretation of results through colorimetric signals while also preventing interference on result judgment from pigments such as hemoglobin. Carryover contamination, which is a disharmonious factor in LAMP, especially as the inherent contradiction derived from uncapping in the lateral flow strategy, has been effectively addressed through the integration of uracil DNA glycosylase without compromising the isothermy throughout the process. As a proof-of-concept experiment, species and sex can be accurately identified within 40 min from trace bloodstains amidst significant background interference by targeting cytochrome b and Y-chromosomal amelogenin. Furthermore, the single-blind study revealed a concordance rate of up to 100% in both simulative degraded and true dated bloodstains. This suggests that this biosensor has the potential to be utilized in forensic DNA analysis at crime scenes.

Sequencing of Kaposi's Sarcoma Herpesvirus (KSHV) genomes from persons of diverse ethnicities and provenances with KSHV-associated diseases demonstrate multiple infections, novel polymorphisms, and low intra-host variance.

Recently published near full-length KSHV genomes from a Cameroon Kaposi sarcoma case-control study showed strong evidence of viral recombination and mixed infections, but no sequence variations associated with disease. Using the same methodology, an additional 102 KSHV genomes from 76 individuals with KSHV-associated diseases have been sequenced. Diagnoses comprise all KSHV-associated diseases (KAD): Kaposi sarcoma (KS), primary effusion lymphoma (PEL), KSHV-associated large cell lymphoma (KSHV-LCL), a type of multicentric Castleman disease (KSHV-MCD), and KSHV inflammatory cytokine syndrome (KICS). Participants originated from 22 different countries, providing the opportunity to obtain new near full-length sequences of a wide diversity of KSHV genomes. These include near full-length sequence of genomes with KSHV K1 subtypes A, B, C, and F as well as subtype E, for which no full sequence was previously available. High levels of recombination were observed. Fourteen individuals (18%) showed evidence of infection with multiple KSHV variants (from two to four unique genomes). Twenty-six comparisons of sequences, obtained from various sampling sites including PBMC, tissue biopsies, oral fluids, and effusions in the same participants, identified near complete genome conservation between different biological compartments. Polymorphisms were identified in coding and non-coding regions, including indels in the K3 and K15 genes and sequence inversions here reported for the first time. One such polymorphism in KSHV ORF46, specific to the KSHV K1 subtype E2, encoded a mutation in the leucine loop extension of the uracil DNA glycosylase that results in alteration of biochemical functions of this protein. This confirms that KSHV sequence variations can have functional consequences warranting further investigation. This study represents the largest and most diverse analysis of KSHV genome sequences to date among individuals with KAD and provides important new information on global KSHV genomics.

Inhibition of Uracil DNA Glycosylase Alters Frequency and Spectrum of Action-at-a-Distance Mutations Induced by 8-Oxo-7,8-dihydroguanine.

The generation of DNA damage causes mutations and consequently cancer. Reactive oxygen species are important sources of DNA damage and some mutation signatures found in human cancers. 8-Oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) is one of the most abundant oxidized bases and induces a G→T transversion mutation at the modified site. The damaged G base also causes untargeted base substitution mutations at the G bases of 5'-GpA-3' dinucleotides (action-at-a-distance mutations) in human cells, and the cytosine deaminase apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) is involved in the mutation process. The deaminated cytosine, i.e., uracil, bases are expected to be removed by uracil DNA glycosylase. Most of the substitution mutations at the G bases of 5'-GpA-3' might be caused by abasic sites formed by the glycosylase. In this study, we expressed the uracil DNA glycosylase inhibitor from Bacillus subtilis bacteriophage PBS2 in human U2OS cells and examined the effects on the GO-induced action-at-a-distance mutations. The inhibition of uracil DNA glycosylase increased the mutation frequency, and in particular, the frequency of G→A transitions. These results indicated that uracil DNA glycosylase, in addition to APOBEC3, is involved in the untargeted mutation process induced by GO.

Hypomorphic mutation in the large subunit of replication protein A affects mutagenesis by human APOBEC cytidine deaminases in yeast.

Human APOBEC single-strand (ss) specific DNA and RNA cytidine deaminases change cytosines to uracils and function in antiviral innate immunity, RNA editing, and can cause hypermutation in chromosomes. The resulting uracils can be directly replicated, resulting in C to T mutations, or uracil-DNA glycosylase can convert the uracils to abasic (AP) sites which are then fixed as C to T or C to G mutations by translesion DNA polymerases. We noticed that in yeast and in human cancers, contributions of C to T and C to G mutations depends on the origin of ssDNA mutagenized by APOBECs. Since ssDNA in eukaryotic genomes readily binds to replication protein A (RPA) we asked if RPA could affect APOBEC-induced mutation spectrum in yeast. For that purpose, we expressed human APOBECs in the wild-type yeast and in strains carrying a hypomorph mutation rfa1-t33 in the large RPA subunit. We confirmed that the rfa1-t33 allele can facilitate mutagenesis by APOBECs. We also found that the rfa1-t33 mutation changed the ratio of APOBEC3A-induced T to C and T to G mutations in replicating yeast to resemble a ratio observed in long-persistent ssDNA in yeast and in cancers. We present the data suggesting that RPA may shield APOBEC formed uracils in ssDNA from Ung1, thereby facilitating C to T mutagenesis through the accurate copying of uracils by replicative DNA polymerases. Unexpectedly, we also found that for uracils shielded from Ung1 by wild-type RPA the mutagenic outcome is reduced in the presence of translesion DNA polymerase zeta.