Structural basis for a Polθ helicase small-molecule inhibitor revealed by cryo-EM.

DNA polymerase theta (Polθ) is a DNA helicase-polymerase protein that facilitates DNA repair and is synthetic lethal with homology-directed repair (HDR) factors. Thus, Polθ is a promising precision oncology drug-target in HDR-deficient cancers. Here, we characterize the binding and mechanism of action of a Polθ helicase (Polθ-hel) small-molecule inhibitor (AB25583) using cryo-EM. AB25583 exhibits 6 nM IC50 against Polθ-hel, selectively kills BRCA1/2-deficient cells, and acts synergistically with olaparib in cancer cells harboring pathogenic BRCA1/2 mutations. Cryo-EM uncovers predominantly dimeric Polθ-hel:AB25583 complex structures at 3.0-3.2 Å. The structures reveal a binding-pocket deep inside the helicase central-channel, which underscores the high specificity and potency of AB25583. The cryo-EM structures in conjunction with biochemical data indicate that AB25583 inhibits the ATPase activity of Polθ-hel helicase via an allosteric mechanism. These detailed structural data and insights about AB25583 inhibition pave the way for accelerating drug development targeting Polθ-hel in HDR-deficient cancers.

Bulk synthesis and beyond: The roles of eukaryotic replicative DNA polymerases.

An organism's genomic DNA must be accurately duplicated during each cell cycle. DNA synthesis is catalysed by DNA polymerase enzymes, which extend nucleotide polymers in a 5' to 3' direction. This inherent directionality necessitates that one strand is synthesised forwards (leading), while the other is synthesised backwards discontinuously (lagging) to couple synthesis to the unwinding of duplex DNA. Eukaryotic cells possess many diverse polymerases that coordinate to replicate DNA, with the three main replicative polymerases being Pol α, Pol δ and Pol ε. Studies conducted in yeasts and human cells utilising mutant polymerases that incorporate molecular signatures into nascent DNA implicate Pol ε in leading strand synthesis and Pol α and Pol δ in lagging strand replication. Recent structural insights have revealed how the spatial organization of these enzymes around the core helicase facilitates their strand-specific roles. However, various challenging situations during replication require flexibility in the usage of these enzymes, such as during replication initiation or encounters with replication-blocking adducts. This review summarises the roles of the replicative polymerases in bulk DNA replication and explores their flexible and dynamic deployment to complete genome replication. We also examine how polymerase usage patterns can inform our understanding of global replication dynamics by revealing replication fork directionality to identify regions of replication initiation and termination.

Analysis of translesion polymerases in colorectal cancer cells following cetuximab treatment: A network perspective.

INTRODUCTION: Adaptive mutagenesis observed in colorectal cancer (CRC) cells upon exposure to EGFR inhibitors contributes to the development of resistance and recurrence. Multiple investigations have indicated a parallel between cancer cells and bacteria in terms of exhibiting adaptive mutagenesis. This phenomenon entails a transient and coordinated escalation of error-prone translesion synthesis polymerases (TLS polymerases), resulting in mutagenesis of a magnitude sufficient to drive the selection of resistant phenotypes. METHODS: In this study, we conducted a comprehensive pan-transcriptome analysis of the regulatory framework within CRC cells, with the objective of identifying potential transcriptome modules encompassing certain translesion polymerases and the associated transcription factors (TFs) that govern them. Our sampling strategy involved the collection of transcriptomic data from tumors treated with cetuximab, an EGFR inhibitor, untreated CRC tumors, and colorectal-derived cell lines, resulting in a diverse dataset. Subsequently, we identified co-regulated modules using weighted correlation network analysis with a minKMEtostay threshold set at 0.5 to minimize false-positive module identifications and mapped the modules to STRING annotations. Furthermore, we explored the putative TFs influencing these modules using KBoost, a kernel PCA regression model. RESULTS: Our analysis did not reveal a distinct transcriptional profile specific to cetuximab treatment. Moreover, we elucidated co-expression modules housing genes, for example, POLK, POLI, POLQ, REV1, POLN, and POLM. Specifically, POLK, POLI, and POLQ were assigned to the "blue" module, which also encompassed critical DNA damage response enzymes, for example. BRCA1, BRCA2, MSH6, and MSH2. To delineate the transcriptional control of this module, we investigated associated TFs, highlighting the roles of prominent cancer-associated TFs, such as CENPA, HNF1A, and E2F7. CONCLUSION: We found that translesion polymerases are co-regulated with DNA mismatch repair and cell cycle-associated factors. We did not, however, identified any networks specific to cetuximab treatment indicating that the response to EGFR inhibitors relates to a general stress response mechanism.

Enhancing efficiency of protein language models with minimal wet-lab data through few-shot learning.

Accurately modeling the protein fitness landscapes holds great importance for protein engineering. Pre-trained protein language models have achieved state-of-the-art performance in predicting protein fitness without wet-lab experimental data, but their accuracy and interpretability remain limited. On the other hand, traditional supervised deep learning models require abundant labeled training examples for performance improvements, posing a practical barrier. In this work, we introduce FSFP, a training strategy that can effectively optimize protein language models under extreme data scarcity for fitness prediction. By combining meta-transfer learning, learning to rank, and parameter-efficient fine-tuning, FSFP can significantly boost the performance of various protein language models using merely tens of labeled single-site mutants from the target protein. In silico benchmarks across 87 deep mutational scanning datasets demonstrate FSFP's superiority over both unsupervised and supervised baselines. Furthermore, we successfully apply FSFP to engineer the Phi29 DNA polymerase through wet-lab experiments, achieving a 25% increase in the positive rate. These results underscore the potential of our approach in aiding AI-guided protein engineering.

REV3 promotes cellular tolerance to 5-fluorodeoxyuridine by activating translesion DNA synthesis and intra-S checkpoint.

The drug floxuridine (5-fluorodeoxyuridine, FUdR) is an active metabolite of 5-Fluorouracil (5-FU). It converts to 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorodeoxyuridine triphosphate (FdUTP), which on incorporation into the genome inhibits DNA replication. Additionally, it inhibits thymidylate synthase, causing dTMP shortage while increasing dUMP availability, which induces uracil incorporation into the genome. However, the mechanisms underlying cellular tolerance to FUdR are yet to be fully elucidated. In this study, we explored the mechanisms underlying cellular resistance to FUdR by screening for FUdR hypersensitive mutants from a collection of DT40 mutants deficient in each genomic maintenance system. We identified REV3, which is involved in translesion DNA synthesis (TLS), to be a critical factor in FUdR tolerance. Replication using a FUdR-damaged template was attenuated in REV3-/- cells, indicating that the TLS function of REV3 is required to maintain replication on the FUdR-damaged template. Notably, FUdR-exposed REV3-/- cells exhibited defective cell cycle arrest in the early S phase, suggesting that REV3 is involved in intra-S checkpoint activation. Furthermore, REV3-/- cells showed defects in Chk1 phosphorylation, which is required for checkpoint activation, but the survival of FUdR-exposed REV3-/- cells was further reduced by the inhibition of Chk1 or ATR. These data indicate that REV3 mediates DNA checkpoint activation at least through Chk1 phosphorylation, but this signal acts in parallel with ATR-Chk1 DNA damage checkpoint pathway. Collectively, we reveal a previously unappreciated role of REV3 in FUdR tolerance.

A universal fluorescence biosensor based on rolling circle amplification and locking probe for DNA detection.

A stable DNA signal amplification sensor was developed on account of rolling circle amplification (RCA). This sensor includes target DNA-controlled rolling circle amplification technology and locking probe DNA replacement technology, which can be used to detect DNA fragments with genetic information, thus constructing a biosensor for universal detection of DNA. This study takes the homologous DNA of human immunodeficiency virus (HIV) and let-7a as examples to describe this biosensor. The padlock probe is first cyclized by T4 DNA ligase in response to the target's reaction with it. Then, rolling cycle amplification is initiated by Phi29 DNA polymerase, resulting in the formation of a lengthy chain with several triggers. These triggers can open the locked probe LP1 with the fluorescence signal turned off, so that it can continue to react with H2 to form a stable H1-H2 double strand. This regulates the distance between B-DNA modified by the quenching group and H1 modified by fluorescent group, and the fluorescence signal is recovered.

Combination of error-prone PCR (epPCR) and Circular Polymerase Extension Cloning (CPEC) for improving the coverage of random mutagenesis libraries.

Random mutagenesis, such as error-prone PCR (epPCR), is a technique capable of generating a wide variety of a single gene. However, epPCR can produce a large number of mutated gene variants, posing a challenge in ligating these mutated PCR products into plasmid vectors. Typically, the primers for mutagenic PCRs incorporate artificial restriction enzyme sites compatible with chosen plasmids. Products are cleaved and ligated to linearized plasmids, then recircularized by DNA ligase. However, this cut-and-paste method known as ligation-dependent process cloning (LDCP), has limited efficiency, as the loss of potential mutants is inevitable leading to a significant reduction in the library's breadth. An alternative to LDCP is the circular polymerase extension cloning (CPEC) method. This technique involves a reaction where a high-fidelity DNA polymerase extends the overlapping regions between the insert and vector, forming a circular molecule. In this study, our objective was to compare the traditional cut-and-paste enzymatic method with CPEC in producing a variant library from the gene encoding the red fluorescent protein (DsRed2) obtained by epPCR. Our findings suggest that CPEC can accelerate the cloning process in gene library generation, enabling the acquisition of a greater number of gene variants compared to methods reliant on restriction enzymes.

PARG is essential for Polθ-mediated DNA end-joining by removing repressive poly-ADP-ribose marks.

DNA polymerase theta (Polθ)-mediated end-joining (TMEJ) repairs DNA double-strand breaks and confers resistance to genotoxic agents. How Polθ is regulated at the molecular level to exert TMEJ remains poorly characterized. We find that Polθ interacts with and is PARylated by PARP1 in a HPF1-independent manner. PARP1 recruits Polθ to the vicinity of DNA damage via PARylation dependent liquid demixing, however, PARylated Polθ cannot perform TMEJ due to its inability to bind DNA. PARG-mediated de-PARylation of Polθ reactivates its DNA binding and end-joining activities. Consistent with this, PARG is essential for TMEJ and the temporal recruitment of PARG to DNA damage corresponds with TMEJ activation and dissipation of PARP1 and PAR. In conclusion, we show a two-step spatiotemporal mechanism of TMEJ regulation. First, PARP1 PARylates Polθ and facilitates its recruitment to DNA damage sites in an inactivated state. PARG subsequently activates TMEJ by removing repressive PAR marks on Polθ.

REV1 coordinates a multi-faceted tolerance response to DNA alkylation damage and prevents chromosome shattering in Drosophila melanogaster.

When replication forks encounter damaged DNA, cells utilize damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, we have investigated damage tolerance responses in Drosophila melanogaster. We report that tolerance of DNA alkylation damage in rapidly dividing larval tissues depends heavily on translesion synthesis. Furthermore, we show that the REV1 protein plays a multi-faceted role in damage tolerance in Drosophila. Larvae lacking REV1 are hypersensitive to methyl methanesulfonate (MMS) and have highly elevated levels of γ-H2Av (Drosophila γ-H2AX) foci and chromosome aberrations in MMS-treated tissues. Loss of the REV1 C-terminal domain (CTD), which recruits multiple translesion polymerases to damage sites, sensitizes flies to MMS. In the absence of the REV1 CTD, DNA polymerases eta and zeta become critical for MMS tolerance. In addition, flies lacking REV3, the catalytic subunit of polymerase zeta, require the deoxycytidyl transferase activity of REV1 to tolerate MMS. Together, our results demonstrate that Drosophila prioritize the use of multiple translesion polymerases to tolerate alkylation damage and highlight the critical role of REV1 in the coordination of this response to prevent genome instability.

Methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases.

Epigenetic cytosine methylation covers most of genomic CpG dinucleotides in human cells. In addition to common deamination-mediated mutagenesis at CpG sites, an alternative deamination-independent pathway associated with DNA polymerase activity was previously described. This mutagenesis is characterized by the TCG→TTG mutational signature and is believed to arise from dAMP misincorporation opposite 5-methylcytosine (mC) or its oxidized derivative 5-hydroxymethylcytosine (hmC) by B-family replicative DNA polymerases with disrupted proofreading 3→5'-exonuclease activity. In addition to being less stable and pro-mutagenic themselves, cytosine modifications also increase the risk of adjacent nucleotides damage, including the formation of 8-oxo-2'-deoxyguanosine (8-oxoG), a well-known mutagenic lesion. The effect of cytosine methylation on error-prone DNA polymerases lacking proofreading activity and involved in repair and DNA translesion synthesis remains unexplored. Here we analyze the efficiency and fidelity of translesion Y-family polymerases (Pol κ, Pol η, Pol ι and REV1) and primase-polymerase PrimPol opposite mC and hmC as well as opposite 8-oxoG adjacent to mC in the TCG context. We demonstrate that epigenetic cytosine modifications suppress Pol ι and REV1 activities and lead to increasing dAMP misincorporation by PrimPol, Pol κ and Pol ι in vitro. Cytosine methylation also increases misincorporation of dAMP opposite the adjacent 8-oxoG by PrimPol, decreases the TLS activity of Pol η opposite the lesion but increases dCMP incorporation opposite 8-oxoG by REV1. Altogether, these data suggest that methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases.

Human translesion DNA polymerases ι and κ mediate tolerance to temozolomide in MGMT-deficient glioblastoma cells.

Glioblastoma (GBM) is a highly aggressive brain tumor associated with poor patient survival. The current standard treatment involves invasive surgery, radiotherapy, and chemotherapy employing temozolomide (TMZ). Resistance to TMZ is, however, a major challenge. Previous work from our group has identified candidate genes linked to TMZ resistance, including genes encoding translesion synthesis (TLS) DNA polymerases iota (PolÉ©) and kappa (Polκ). These specialized enzymes are known for bypassing lesions and tolerating DNA damage. Here, we investigated the roles of PolÉ© and Polκ in TMZ resistance, employing MGMT-deficient U251-MG glioblastoma cells, with knockout of either POLI or POLK genes encoding PolÉ© and Polκ, respectively, and assess their viability and genotoxic stress responses upon subsequent TMZ treatment. Cells lacking either of these polymerases exhibited a significant decrease in viability following TMZ treatment compared to parental counterparts. The restoration of the missing polymerase led to a recovery of cell viability. Furthermore, knockout cells displayed increased cell cycle arrest, mainly in late S-phase, and lower levels of genotoxic stress after TMZ treatment, as assessed by a reduction of γH2AX foci and flow cytometry data. This implies that TMZ treatment does not trigger a significant H2AX phosphorylation response in the absence of these proteins. Interestingly, combining TMZ with Mirin (double-strand break repair pathway inhibitor) further reduced the cell viability and increased DNA damage and γH2AX positive cells in TLS KO cells, but not in parental cells. These findings underscore the crucial roles of PolÉ© and Polκ in conferring TMZ resistance and the potential backup role of homologous recombination in the absence of these TLS polymerases. Targeting these TLS enzymes, along with double-strand break DNA repair inhibition, could, therefore, provide a promising strategy to enhance TMZ's effectiveness in treating GBM.

Current status and prospect of the DNA double-strand break repair pathway in colorectal cancer development and treatment.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Double-strand break (DSB) is the most severe type of DNA damage. However, few reviews have thoroughly examined the involvement of DSB in CRC. Latest researches demonstrated that DSB repair plays an important role in CRC. For example, DSB-related genes such as BRCA1, Ku-70 and DNA polymerase theta (POLQ) are associated with the occurrence of CRC, and POLQ even showed to affect the prognosis and resistance for radiotherapy in CRC. This review comprehensively summarizes the DSB role in CRC, explores the mechanisms and discusses the association with CRC treatment. Four pathways for DSB have been demonstrated. 1. Nonhomologous end joining (NHEJ) is the major pathway. Its core genes including Ku70 and Ku80 bind to broken ends and recruit repair factors to form a complex that mediates the connection of DNA breaks. 2. Homologous recombination (HR) is another important pathway. Its key genes including BRCA1 and BRCA2 are involved in finding, pairing, and joining broken ends, and ensure the restoration of breaks in a normal double-stranded DNA structure. 3. Single-strand annealing (SSA) pathway, and 4. POLθ-mediated end-joining (alt-EJ) is a backup pathway. This paper elucidates roles of the DSB repair pathways in CRC, which could contribute to the development of potential new treatment approaches and provide new opportunities for CRC treatment and more individualized treatment options based on therapeutic strategies targeting these DNA repair pathways.

Engineering the Activity of a Template-Independent DNA Polymerase.

Enzymatic DNA writing technologies based on the template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT) have the potential to advance DNA information storage. TdT is unique in its ability to synthesize single-stranded DNA de novo but has limitations, including catalytic inhibition by ribonucleotide presence and slower incorporation rates compared to replicative polymerases. We anticipate that protein engineering can improve, modulate, and tailor the enzyme's properties, but there is limited information on TdT sequence-structure-function relationships to facilitate rational approaches. Therefore, we developed an easily modifiable screening assay that can measure the TdT activity in high-throughput to evaluate large TdT mutant libraries. We demonstrated the assay's capabilities by engineering TdT mutants that exhibit both improved catalytic efficiency and improved activity in the presence of an inhibitor. We screened for and identified TdT variants with greater catalytic efficiency in both selectively incorporating deoxyribonucleotides and in the presence of deoxyribonucleotide/ribonucleotide mixes. Using this information from the screening assay, we rationally engineered other TdT homologues with the same properties. The emulsion-based assay we developed is, to the best of our knowledge, the first high-throughput screening assay that can measure TdT activity quantitatively and without the need for protein purification.

Protein Assemblies in Translesion Synthesis.

Translesion synthesis (TLS) is a mechanism of DNA damage tolerance utilized by eukaryotic cells to replicate DNA across lesions that impede the high-fidelity replication machinery. In TLS, a series of specialized DNA polymerases are employed, which recognize specific DNA lesions, insert nucleotides across the damage, and extend the distorted primer-template. This allows cells to preserve genetic integrity at the cost of mutations. In humans, TLS enzymes include the Y-family, inserter polymerases, Polη, Polι, Polκ, Rev1, and the B-family extender polymerase Polζ, while in S. cerevisiae only Polη, Rev1, and Polζ are present. To bypass DNA lesions, TLS polymerases cooperate, assembling into a complex on the eukaryotic sliding clamp, PCNA, termed the TLS mutasome. The mutasome assembly is contingent on protein-protein interactions (PPIs) between the modular domains and subunits of TLS enzymes, and their interactions with PCNA and DNA. While the structural mechanisms of DNA lesion bypass by the TLS polymerases and PPIs of their individual modules are well understood, the mechanisms by which they cooperate in the context of TLS complexes have remained elusive. This review focuses on structural studies of TLS polymerases and describes the case of TLS holoenzyme assemblies in action emerging from recent high-resolution Cryo-EM studies.

DNA polymerase κ participates in early S-phase DNA replication in human cells.

Cycling cells replicate their DNA during the S phase through a defined temporal program known as replication timing. Mutation frequencies, epigenetic chromatin states, and transcriptional activities are different for genomic regions that are replicated early and late in the S phase. Here, we found from ChIP-Seq analysis that DNA polymerase (Pol) κ is enriched in early-replicating genomic regions in HEK293T cells. In addition, by feeding cells with N 2-heptynyl-2'-deoxyguanosine followed by click chemistry-based enrichment and high-throughput sequencing, we observed elevated Pol κ activities in genomic regions that are replicated early in the S phase. On the basis of the established functions of Pol κ in accurate and efficient nucleotide insertion opposite endogenously induced N 2-modified dG lesions, our work suggests that active engagement of Pol κ may contribute to diminished mutation rates observed in early-replicating regions of the human genome, including cancer genomes. Together, our work expands the functions of Pol κ and offered a plausible mechanism underlying replication timing-dependent mutation accrual in the human genome.

Activity of DNA polymerase κ across the genome in human fibroblasts.

DNA polymerase κ (Polκ) is a specialized polymerase that has multiple cellular roles such as translesion DNA synthesis, replication of repetitive sequences, and nucleotide excision repair. We have developed a method for capturing DNA synthesized by Polκ utilizing a Polκ-specific substrate, N2-(4-ethynylbenzyl)-2'-deoxyguanosine (EBndG). After shearing of the DNA into 200 to 500 bp lengths, the EBndG-containing DNA was covalently bound to biotin using the Cu(I)-catalyzed alkyne-azide cycloaddition reaction and isolated with streptavidin beads. Isolated DNA was then ligated to adaptors, followed by PCR amplification and next-generation sequencing to generate genome-wide repair maps. We have termed this method polymerase κ sequencing. Here, we present the human genome maps for Polκ activity in an undamaged cell line. We found that Polκ activity was enhanced in GC-rich regions, euchromatin regions, the promoter of genes, and in DNA that is replicated early in the S phase.

Probing the Conformational Restraints of DNA Damage Recognition with ß-L-Nucleotides.

The DNA building blocks 2'-deoxynucleotides are enantiomeric, with their natural ß-D-configuration dictated by the sugar moiety. Their synthetic ß-L-enantiomers (ßLdNs) can be used to obtain L-DNA, which, when fully substituted, is resistant to nucleases and is finding use in many biosensing and nanotechnology applications. However, much less is known about the enzymatic recognition and processing of individual ßLdNs embedded in D-DNA. Here, we address the template properties of ßLdNs for several DNA polymerases and the ability of base excision repair enzymes to remove these modifications from DNA. The Klenow fragment was fully blocked by ßLdNs, whereas DNA polymerase κ bypassed them in an error-free manner. Phage RB69 DNA polymerase and DNA polymerase ß treated ßLdNs as non-instructive but the latter enzyme shifted towards error-free incorporation on a gapped DNA substrate. DNA glycosylases and AP endonucleases did not process ßLdNs. DNA glycosylases sensitive to the base opposite their cognate lesions also did not recognize ßLdNs as a correct pairing partner. Nevertheless, when placed in a reporter plasmid, pyrimidine ßLdNs were resistant to repair in human cells, whereas purine ßLdNs appear to be partly repaired. Overall, ßLdNs are unique modifications that are mostly non-instructive but have dual non-instructive/instructive properties in special cases.

Activation of Neurotoxic Astrocytes Due to Mitochondrial Dysfunction Triggered by POLG Mutation.

Mitochondrial diseases are associated with neuronal death and mtDNA depletion. Astrocytes respond to injury or stimuli and damage to the central nervous system. Neurodegeneration can cause astrocytes to activate and acquire toxic functions that induce neuronal death. However, astrocyte activation and its impact on neuronal homeostasis in mitochondrial disease remain to be explored. Using patient cells carrying POLG mutations, we generated iPSCs and then differentiated these into astrocytes. POLG astrocytes exhibited mitochondrial dysfunction including loss of mitochondrial membrane potential, energy failure, loss of complex I and IV, disturbed NAD+/NADH metabolism, and mtDNA depletion. Further, POLG derived astrocytes presented an A1-like reactive phenotype with increased proliferation, invasion, upregulation of pathways involved in response to stimulus, immune system process, cell proliferation and cell killing. Under direct and indirect co-culture with neurons, POLG astrocytes manifested a toxic effect leading to the death of neurons. We demonstrate that mitochondrial dysfunction caused by POLG mutations leads not only to intrinsic defects in energy metabolism affecting both neurons and astrocytes, but also to neurotoxic damage driven by astrocytes. These findings reveal a novel role for dysfunctional astrocytes that contribute to the pathogenesis of POLG diseases.

N6-methyladenosine modification of a parvovirus-encoded small noncoding RNA facilitates viral DNA replication through recruiting Y-family DNA polymerases.

Human bocavirus 1 (HBoV1) is a human parvovirus that causes lower respiratory tract infections in young children. It contains a single-stranded (ss) DNA genome of ~5.5 kb that encodes a small noncoding RNA of 140 nucleotides known as bocavirus-encoded small RNA (BocaSR), in addition to viral proteins. Here, we determined the secondary structure of BocaSR in vivo by using DMS-MaPseq. Our findings reveal that BocaSR undergoes N6-methyladenosine (m6A) modification at multiple sites, which is critical for viral DNA replication in both dividing HEK293 cells and nondividing cells of the human airway epithelium. Mechanistically, we found that m6A-modified BocaSR serves as a mediator for recruiting Y-family DNA repair DNA polymerase (Pol) η and Pol κ likely through a direct interaction between BocaSR and the viral DNA replication origin at the right terminus of the viral genome. Thus, this report represents direct involvement of a viral small noncoding RNA in viral DNA replication through m6A modification.

Mapping fast DNA polymerase exchange during replication.

Despite extensive studies on DNA replication, the exchange mechanisms of DNA polymerase during replication remain unclear. Existing models propose that this exchange is facilitated by protein partners like helicase. Here we present data, employing a combination of mechanical DNA manipulation and single fluorescent protein observation, that reveal DNA polymerase undergoing rapid and autonomous exchange during replication not coordinated by other proteins. The DNA polymerase shows fast unbinding and rebinding dynamics, displaying a preference for either exonuclease or polymerase activity, or pausing events, during each brief binding event. We also observed a 'memory effect' in DNA polymerase rebinding, i.e., the enzyme tends to preserve its prior activity upon reassociation. This effect, potentially linked to the ssDNA/dsDNA junction's conformation, might play a role in regulating binding preference enabling high processivity amidst rapid protein exchange. Taken together, our findings support an autonomous replication model that includes rapid protein exchange, burst of activity, and a 'memory effect' while moving processively forward.