Lesion Bypass and the Reactivation of Stalled Replication Forks.

Accurate transmission of the genetic information requires complete duplication of the chromosomal DNA each cell division cycle. However, the idea that replication forks would form at origins of DNA replication and proceed without impairment to copy the chromosomes has proven naive. It is now clear that replication forks stall frequently as a result of encounters between the replication machinery and template damage, slow-moving or paused transcription complexes, unrelieved positive superhelical tension, covalent protein-DNA complexes, and as a result of cellular stress responses. These stalled forks are a major source of genome instability. The cell has developed many strategies for ensuring that these obstructions to DNA replication do not result in loss of genetic information, including DNA damage tolerance mechanisms such as lesion skipping, whereby the replisome jumps the lesion and continues downstream; template switching both behind template damage and at the stalled fork; and the error-prone pathway of translesion synthesis.

Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses.

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.

Mitotic CDK Promotes Replisome Disassembly, Fork Breakage, and Complex DNA Rearrangements.

DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C. elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements.

Tinker, tailor, antiviral: RNA virus inhibition by induced recombination.

Nucleotide analogs can help to combat RNA virus growth by stalling the viral RNA polymerase or by introducing lethal mutations into the viral genome. Janissen and Woodman et al. have used single-molecule, sequencing, and virological methods to reveal that antiviral T-1106 provides a third mechanism of counterattack: inducing recombination.

Generation of de novo miRNAs from template switching during DNA replication.

The mechanisms generating novel genes and genetic information are poorly known, even for microRNA (miRNA) genes with an extremely constrained design. All miRNA primary transcripts need to fold into a stem-loop structure to yield short gene products ([Formula: see text]22 nt) that bind and repress their mRNA targets. While a substantial number of miRNA genes are ancient and highly conserved, short secondary structures coding for entirely novel miRNA genes have been shown to emerge in a lineage-specific manner. Template switching is a DNA-replication-related mutation mechanism that can introduce complex changes and generate perfect base pairing for entire hairpin structures in a single event. Here, we show that the template-switching mutations (TSMs) have participated in the emergence of over 6,000 suitable hairpin structures in the primate lineage to yield at least 18 new human miRNA genes, that is 26% of the miRNAs inferred to have arisen since the origin of primates. While the mechanism appears random, the TSM-generated miRNAs are enriched in introns where they can be expressed with their host genes. The high frequency of TSM events provides raw material for evolution. Being orders of magnitude faster than other mechanisms proposed for de novo creation of genes, TSM-generated miRNAs enable near-instant rewiring of genetic information and rapid adaptation to changing environments.

Genotype imputation accuracy and the quality metrics of the minor ancestry in multi-ancestry reference panels.

Large-scale imputation reference panels are currently available and have contributed to efficient genome-wide association studies through genotype imputation. However, whether large-size multi-ancestry or small-size population-specific reference panels are the optimal choices for under-represented populations continues to be debated. We imputed genotypes of East Asian (180k Japanese) subjects using the Trans-Omics for Precision Medicine reference panel and found that the standard imputation quality metric (Rsq) overestimated dosage r2 (squared correlation between imputed dosage and true genotype) particularly in marginal-quality bins. Variance component analysis of Rsq revealed that the increased imputed-genotype certainty (dosages closer to 0, 1 or 2) caused upward bias, indicating some systemic bias in the imputation. Through systematic simulations using different template switching rates (θ value) in the hidden Markov model, we revealed that the lower θ value increased the imputed-genotype certainty and Rsq; however, dosage r2 was insensitive to the θ value, thereby causing a deviation. In simulated reference panels with different sizes and ancestral diversities, the θ value estimates from Minimac decreased with the size of a single ancestry and increased with the ancestral diversity. Thus, Rsq could be deviated from dosage r2 for a subpopulation in the multi-ancestry panel, and the deviation represents different imputed-dosage distributions. Finally, despite the impact of the θ value, distant ancestries in the reference panel contributed only a few additional variants passing a predefined Rsq threshold. We conclude that the θ value substantially impacts the imputed dosage and the imputation quality metric value.

Eukaryotic DNA Polymerases in Homologous Recombination.

Homologous recombination (HR) is a central process to ensure genomic stability in somatic cells and during meiosis. HR-associated DNA synthesis determines in large part the fidelity of the process. A number of recent studies have demonstrated that DNA synthesis during HR is conservative, less processive, and more mutagenic than replicative DNA synthesis. In this review, we describe mechanistic features of DNA synthesis during different types of HR-mediated DNA repair, including synthesis-dependent strand annealing, break-induced replication, and meiotic recombination. We highlight recent findings from diverse eukaryotic organisms, including humans, that suggest both replicative and translesion DNA polymerases are involved in HR-associated DNA synthesis. Our focus is to integrate the emerging literature about DNA polymerase involvement during HR with the unique aspects of these repair mechanisms, including mutagenesis and template switching.

Making Choices: DNA Replication Fork Recovery Mechanisms.

DNA replication is laden with obstacles that slow, stall, collapse, and break DNA replication forks. At each obstacle, there is a decision to be made whether to bypass the lesion, repair or restart the damaged fork, or to protect stalled forks from further demise. Each "decision" draws upon multitude of proteins participating in various mechanisms that allow repair and restart of replication forks. Specific functions for many of these proteins have been described and an understanding of how they come together in supporting replication forks is starting to emerge. Many questions, however, remain regarding selection of the mechanisms that enable faithful genome duplication and how "normal" intermediates in these mechanisms are sometimes funneled into "rogue" processes that destabilize the genome and lead to cancer, cell death, and emergence of chemotherapeutic resistance. In this review we will discuss molecular mechanisms of DNA damage bypass and replication fork protection and repair. We will specifically focus on the key players that define which mechanism is employed including: PCNA and its control by posttranslational modifications, translesion synthesis DNA polymerases, molecular motors that catalyze reversal of stalled replication forks, proteins that antagonize fork reversal and protect reversed forks from nucleolytic degradation, and the machinery of homologous recombination that helps to reestablish broken forks. We will also discuss risks to genome integrity inherent in each of these mechanisms.

Simple and accurate transcriptional start site identification using Smar2C2 and examination of conserved promoter features.

The precise and accurate identification and quantification of transcriptional start sites (TSSs) is key to understanding the control of transcription. The core promoter consists of the TSS and proximal non-coding sequences, which are critical in transcriptional regulation. Therefore, the accurate identification of TSSs is important for understanding the molecular regulation of transcription. Existing protocols for TSS identification are challenging and expensive, leaving high-quality data available for a small subset of organisms. This sparsity of data impairs study of TSS usage across tissues or in an evolutionary context. To address these shortcomings, we developed Smart-Seq2 Rolling Circle to Concatemeric Consensus (Smar2C2), which identifies and quantifies TSSs and transcription termination sites. Smar2C2 incorporates unique molecular identifiers that allowed for the identification of as many as 70 million sites, with no known upper limit. We have also generated TSS data sets from as little as 40 pg of total RNA, which was the smallest input tested. In this study, we used Smar2C2 to identify TSSs in Glycine max (soybean), Oryza sativa (rice), Sorghum bicolor (sorghum), Triticum aestivum (wheat) and Zea mays (maize) across multiple tissues. This wide panel of plant TSSs facilitated the identification of evolutionarily conserved features, such as novel patterns in the dinucleotides that compose the initiator element (Inr), that correlated with promoter expression levels across all species examined. We also discovered sequence variations in known promoter motifs that are positioned reliably close to the TSS, such as differences in the TATA box and in the Inr that may prove significant to our understanding and control of transcription initiation. Smar2C2 allows for the easy study of these critical sequences, providing a tool to facilitate discovery.

Non-recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance.

DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error-prone, HR uses the sister chromatid and is mostly error-free. We report that the HR protein Rad52-but not Rad51 and Rad57-acts in concert with the TLS machinery (Rad6/Rad18-mediated PCNA ubiquitylation and polymerases Rev1/Pol ζ) to repair MMS and UV light-induced ssDNA gaps through a non-recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS- and UV-induced HR); accordingly, Rad52 is required for efficient DNA damage-induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage-induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57-dependent and -independent processes, providing a novel role for the recombination proteins in maintaining genome integrity.

Error-free DNA damage tolerance pathway is facilitated by the Irc5 translocase through cohesin.

DNA damage tolerance (DDT) mechanisms facilitate replication resumption and completion when DNA replication is blocked by bulky DNA lesions. In budding yeast, template switching (TS) via the Rad18/Rad5 pathway is a favored DDT pathway that involves usage of the sister chromatid as a template to bypass DNA lesions in an error-free recombination-like process. Here, we establish that the Snf2 family translocase Irc5 is a novel factor that promotes TS and averts single-stranded DNA persistence during replication. We demonstrate that, during replication stress, Irc5 enables replication progression by assisting enrichment of cohesin complexes, recruited in an Scc2/Scc4-dependent fashion, near blocked replication forks. This allows efficient formation of sister chromatid junctions that are crucial for error-free DNA lesion bypass. Our results support the notion of a key role of cohesin in the completion of DNA synthesis under replication stress and reveal that the Rad18/Rad5-mediated DDT pathway is linked to cohesin enrichment at sites of perturbed replication via the Snf2 family translocase Irc5.

Cellular Protein HuR Regulates the Switching of Genomic RNA Templates for Differential Functions during the Coxsackievirus B3 Life Cycle.

Coxsackievirus B3 (CVB3) is an enterovirus belonging to the family Picornaviridae. Its 5' untranslated region (UTR) contains a cloverleaf structure followed by an internal ribosome entry site (IRES). The cloverleaf forms an RNA-protein complex known to regulate virus replication, translation, and stability of the genome, and the IRES regulates virus RNA translation. For positive-strand RNA-containing viruses, such as members of the flaviviruses or enteroviruses, the genomic RNA is used for translation, replication, and encapsidation. Only a few regulatory mechanisms which govern the accessibility of genomic RNA templates for translation or replication have been reported. Here, we report the role of human antigen R (HuR) in regulating the fate of CVB3 positive-strand RNA into the replication cycle or translation cycle. We have observed that synthesis of HuR is induced during CVB3 infection, and it suppresses viral replication by displacing PCBP-2 (a positive regulator of virus replication) at the cloverleaf RNA. Silencing of HuR increases viral RNA replication and consequently reduces viral RNA translation in a replication-dependent manner. Furthermore, we have shown that HuR level is upregulated upon CVB3 infection. Moreover, HuR limits virus replication and can coordinate the availability of genomic RNA templates for translation, replication, or encapsidation. Our study highlights the fact that the relative abundance of translation factors and replication factors in the cell decides the outcome of viral infection. IMPORTANCE A positive-strand RNA virus must balance the availability of its genomic template for different viral processes at different stages of its life cycle. A few host proteins are shown to be important to help the virus in switching the usage of a template between these processes. These proteins inhibit translation either by displacing a stimulator of translation or by binding to an alternative site. Both mechanisms lead to ribosome clearance and availability of the genomic strand for replication. We have shown that HuR also helps in maintaining this balance by inhibiting replication and subsequently promoting translation and packaging.

Chromosome rearrangements via template switching between diverged repeated sequences.

Recent high-resolution genome analyses of cancer and other diseases have revealed the occurrence of microhomology-mediated chromosome rearrangements and copy number changes. Although some of these rearrangements appear to involve nonhomologous end-joining, many must have involved mechanisms requiring new DNA synthesis. Models such as microhomology-mediated break-induced replication (MM-BIR) have been invoked to explain these rearrangements. We examined BIR and template switching between highly diverged sequences in Saccharomyces cerevisiae, induced during repair of a site-specific double-strand break (DSB). Our data show that such template switches are robust mechanisms that give rise to complex rearrangements. Template switches between highly divergent sequences appear to be mechanistically distinct from the initial strand invasions that establish BIR. In particular, such jumps are less constrained by sequence divergence and exhibit a different pattern of microhomology junctions. BIR traversing repeated DNA sequences frequently results in complex translocations analogous to those seen in mammalian cells. These results suggest that template switching among repeated genes is a potent driver of genome instability and evolution.

Post-replication repair: Rad5/HLTF regulation, activity on undamaged templates, and relationship to cancer.

The eukaryotic post-replication repair (PRR) pathway allows completion of DNA replication when replication forks encounter lesions on the DNA template and are mediated by post-translational ubiquitination of the DNA sliding clamp proliferating cell nuclear antigen (PCNA). Monoubiquitinated PCNA recruits translesion synthesis (TLS) polymerases to replicate past DNA lesions in an error-prone manner while addition of K63-linked polyubiquitin chains signals for error-free template switching to the sister chromatid. Central to both branches is the E3 ubiquitin ligase and DNA helicase Rad5/helicase-like transcription factor (HLTF). Mutations in PRR pathway components lead to genomic rearrangements, cancer predisposition, and cancer progression. Recent studies have challenged the notion that the PRR pathway is involved only in DNA lesion tolerance and have shed new light on its roles in cancer progression. Molecular details of Rad5/HLTF recruitment and function at replication forks have emerged. Mounting evidence indicates that PRR is required during lesion-less replication stress, leading to TLS polymerase activity on undamaged templates. Analysis of PRR mutation status in human cancers and PRR function in cancer models indicates that down regulation of PRR activity is a viable strategy to inhibit cancer cell growth and reduce chemoresistance. Here, we review these findings, discuss how they change our views of current PRR models, and look forward to targeting the PRR pathway in the clinic.

Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) provides new insights to address biological and medical questions, and it will benefit more from the ultralow input RNA or subcellular sequencing. RESULTS: Here, we present a highly sensitive library construction protocol for ultralow input RNA sequencing (ulRNA-seq). We systematically evaluate experimental conditions of this protocol, such as reverse transcriptase, template-switching oligos (TSO), and template RNA structure. It was found that Maxima H Minus reverse transcriptase and rN modified TSO, as well as all RNA templates capped with m7G improved the sequencing sensitivity and low abundance gene detection ability. RNA-seq libraries were successfully prepared from total RNA samples as low as 0.5 pg, and more than 2000 genes have been identified. CONCLUSIONS: The ability of low abundance gene detection and sensitivity were largely enhanced with this optimized protocol. It was also confirmed in single-cell sequencing, that more genes and cell markers were identified compared to conventional sequencing method. We expect that ulRNA-seq will sequence and transcriptome characterization for the subcellular of disease tissue, to find the corresponding treatment plan.

Duplex formation between the template and the nascent strand in the transcription-regulating sequences is associated with the site of template switching in SARS - CoV-2.

Recently published transcriptomic data of the SARS-CoV-2 coronavirus show that there is a large variation in the frequency and steady state levels of subgenomic mRNA sequences. This variation is derived from discontinuous subgenomic RNA synthesis, where the polymerase switches template from a 3' proximal genome body sequence to a 5' untranslated leader sequence. This leads to a fusion between the common 5' leader sequence and a 3' proximal body sequence in the RNA product. This process revolves around a common core sequence (CS) that is present at both the template sites that make up the fusion junction. Base-pairing between the leader CS and the nascent complementary minus strand body CS, and flanking regions (together called the transcription regulating sequence, TRS) is vital for this template switching event. However, various factors can influence the site of template switching within the same TRS duplex. Here, we model the duplexes formed between the leader and complementary body TRS regions, hypothesizing the role of the stability of the TRS duplex in determining the major sites of template switching for the most abundant mRNAs. We indicate that the stability of secondary structures and the speed of transcription play key roles in determining the probability of template switching in the production of subgenomic RNAs. We speculate on the effect of reported variant nucleotide substitutions on our models.

SUMOylation of PCNA by PIAS1 and PIAS4 promotes template switch in the chicken and human B cell lines.

DNA damage tolerance (DDT) releases replication blockage caused by damaged nucleotides on template strands employing two alternative pathways, error-prone translesion DNA synthesis (TLS) and error-free template switch (TS). Lys164 of proliferating cell nuclear antigen (PCNA) is SUMOylated during the physiological cell cycle. To explore the role for SUMOylation of PCNA in DDT, we characterized chicken DT40 and human TK6 B cells deficient in the PIAS1 and PIAS4 small ubiquitin-like modifier (SUMO) E3 ligases. DT40 cells have a unique advantage in the phenotypic analysis of DDT as they continuously diversify their immunoglobulin (Ig) variable genes by TLS and TS [Ig gene conversion (GC)], both relieving replication blocks at abasic sites without accompanying by DNA breakage. Remarkably, PIAS1-/-/PIAS4-/- cells displayed a multifold decrease in SUMOylation of PCNA at Lys164 and over a 90% decrease in the rate of TS. Likewise, PIAS1-/-/PIAS4-/- TK6 cells showed a shift of DDT from TS to TLS at a chemosynthetic UV lesion inserted into the genomic DNA. The PCNAK164R/K164R mutation caused a ∼90% decrease in the rate of Ig GC and no additional impact on PIAS1-/-/PIAS4-/- cells. This epistatic relationship between the PCNAK164R/K164R and the PIAS1-/-/PIAS4-/- mutations suggests that PIAS1 and PIAS4 promote TS mainly through SUMOylation of PCNA at Lys164. This idea is further supported by the data that overexpression of a PCNA-SUMO1 chimeric protein restores defects in TS in PIAS1-/-/PIAS4-/- cells. In conclusion, SUMOylation of PCNA at Lys164 promoted by PIAS1 and PIAS4 ensures the error-free release of replication blockage during physiological DNA replication in metazoan cells.

Non-templated addition and template switching by Moloney murine leukemia virus (MMLV)-based reverse transcriptases co-occur and compete with each other.

Single-cell RNA-Seq (scRNA-Seq) has led to an unprecedented understanding of gene expression and regulation in individual cells. Many scRNA-Seq approaches rely upon the template switching property of Moloney murine leukemia virus (MMLV)-type reverse transcriptases. Template switching is believed to happen in a sequential process involving nontemplated addition of three protruding nucleotides (+CCC) to the 3'-end of the nascent cDNA, which can then anneal to the matching rGrGrG 3'-end of the template-switching oligo (TSO), allowing the reverse transcriptase (RT) to switch templates and continue copying the TSO sequence. In this study, we present a detailed analysis of template switching biases with respect to the RNA template, specifically of the role of the sequence and nature of its 5'-end (capped versus noncapped) in these biases. Our findings confirmed that the presence of a 5'-m7G cap enhances template switching efficiency. We also profiled the composition of the nontemplated addition in the absence of TSO and observed that the 5'-end of RNA template influences the terminal transferase activity of the RT. Furthermore, we found that designing new TSOs that pair with the most common nontemplated additions did little to improve template switching efficiency. Our results provide evidence suggesting that, in contrast to the current understanding of the template switching process, nontemplated addition and template switching are concurrent and competing processes.

Prevention of unwanted recombination at damaged replication forks.

Homologous recombination is essential for the maintenance of genome integrity but must be strictly controlled to avoid dangerous outcomes that produce the opposite effect, genomic instability. During unperturbed chromosome replication, recombination is globally inhibited at ongoing DNA replication forks, which helps to prevent deleterious genomic rearrangements. This inhibition is carried out by Srs2, a helicase that binds to SUMOylated PCNA and has an anti-recombinogenic function at replication forks. However, at damaged stalled forks, Srs2 is counteracted and DNA lesion bypass can be achieved by recombination-mediated template switching. In budding yeast, template switching is dependent on Rad5. In the absence of this protein, replication forks stall in the presence of DNA lesions and cells die. Recently, we showed that in cells lacking Rad5 that are exposed to DNA damage or replicative stress, elimination of the conserved Mgs1/WRNIP1 ATPase allows an alternative mode of DNA damage bypass that is driven by recombination and facilitates completion of chromosome replication and cell viability. We have proposed that Mgs1 is important to prevent a potentially harmful salvage pathway of recombination at damaged stalled forks. In this review, we summarize our current understanding of how unwanted recombination is prevented at damaged stalled replication forks.

DNA damage tolerance in hematopoietic stem and progenitor cells in mice.

DNA damage tolerance (DDT) enables bypassing of DNA lesions during replication, thereby preventing fork stalling, replication stress, and secondary DNA damage related to fork stalling. Three modes of DDT have been documented: translesion synthesis (TLS), template switching (TS), and repriming. TLS and TS depend on site-specific PCNA K164 monoubiquitination and polyubiquitination, respectively. To investigate the role of DDT in maintaining hematopoietic stem cells (HSCs) and progenitors, we used PcnaK164R/K164R mice as a unique DDT-defective mouse model. Analysis of the composition of HSCs and HSC-derived multipotent progenitors (MPPs) revealed a significantly reduced number of HSCs, likely owing to increased differentiation of HSCs toward myeloid/erythroid-associated MPP2s. This skewing came at the expense of the number of lymphoid-primed MPP4s, which appeared to be compensated for by increased MPP4 proliferation. Furthermore, defective DDT decreased the numbers of MPP-derived common lymphoid progenitor (CLP), common myeloid progenitor (CMP), megakaryocyte-erythroid progenitor (MEP), and granulocyte-macrophage progenitor (GMP) cells, accompanied by increased cell cycle arrest in CMPs. The HSC and MPP phenotypes are reminiscent of premature aging and stressed hematopoiesis, and indeed progressed with age and were exacerbated on cisplatin exposure. Bone marrow transplantations revealed a strong cell intrinsic defect of DDT-deficient HSCs in reconstituting lethally irradiated mice and a strong competitive disadvantage when cotransplanted with wild-type HSCs. These findings indicate a critical role of DDT in maintaining HSCs and progenitor cells, and in preventing premature aging.