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During lagging strand chromatin replication, multiple Okazaki fragments (OFs) require processing and nucleosome assembly, but the mechanisms linking these processes remain unclear. Here, using transmission electron microscopy and rapid degradation of DNA ligase Cdc9, we observed flap structures accumulated on lagging strands, controlled by both Pol δ's strand displacement activity and Fen1's nuclease digestion. The distance between neighboring flap structures exhibits a regular pattern, indicative of matured OF length. While fen1 Δ or enhanced strand displacement activities by polymerase δ (Pol δ; pol3 exo- ) minimally affect inter-flap distance, mutants affecting replication-coupled nucleosome assembly, such as cac1 , do significantly alter it. Deletion of Pol32, a subunit of DNA Pol δ, significantly increases this distance. Mechanistically, Pol32 binds to histone H3-H4 and is critical for nucleosome assembly on the lagging strand. Together, we propose that Pol32 establishes a connection between nucleosome assembly and the processing of OFs on lagging strands.mcm2-3A , do significantly alter it. Deletion of Pol32, a subunit of DNA Pol δ, significantly increases this distance. Mechanistically, Pol32 binds to histone H3-H4 and is critical for nucleosome assembly on the lagging strand. Together, we propose that Pol32 establishes a connection between nucleosome assembly and the processing of OFs on lagging strands.

A type of modified nucleotide, deoxynucleotide γ-amidotriphosphates (dNTPγNH 2 s), exhibited around five times higher stability than dNTPs. These phosphamide nucleotides can be utilized by several DNA polymerases, and the amplification of a 10 kb DNA fragment through the polymerase chain reaction (PCR) can be accomplished even under conditions of high temperature, extended storage, or repeated freeze-thaw cycles. However, the control PCR with standard dNTPs was unsuccessful. These results indicate that dNTPγNH 2 s have the potential to substitute dNTPs in PCR.

Single-stranded DNA (ssDNA) is the foundation of modern biology, with wide applications in gene editing, sequencing, DNA information storage, and materials science. However, synthesizing ssDNA with high efficiency, high throughput, and low error rate in vitro remains a major challenge. Various methods have been developed for ssDNA synthesis, and some significant results have been achieved. In this review, six main methods were introduced, including solid-phase oligonucleotide synthesis, terminal deoxynucleotidyl transferase-based ssDNA synthesis, reverse transcription, primer exchange reaction, asymmetric polymerase chain reaction, and rolling circle amplification. The advantages and limitations of each method were compared, as well as illustrate their representative achievements and applications. Especially, rolling circle amplification has received significant attention, including ssDNA synthesis, assembly, and application based on recent work. Finally, the future challenges and opportunities of ssDNA synthesis were summarized and discussed. Envisioning the development of new methods and significant progress will be made in the near future with the efforts of scientists around the world., (© 2024 Wiley‐VCH GmbH.)

Nonspecific amplification is a serious issue in DNA detection as it can lead to false-positive results and reduce specificity. It is very important to well understand its mechanism through sequencing nonspecific products. Here, an approach is developed using a nanopore sequencing technique after acquiring the long repetitive sequence of DNA products from nonspecific amplification. Based on the sequencing results, a new mechanism of nonspecific amplification designated as dynamic mismatched primer binding (DMPB) with the background DNA (bgDNA) is proposed. Unexpectedly, our findings show that the primers (∼20 nt) can bind to bgDNA for primer extension when only 6-11 fully matched (9-14 mismatched) base pairs are formed. After the single-stranded DNAs (ssDNAs) attached to the first primer are produced, more interestingly, with the aid of DNA polymerase, the other primer can bind to these ssDNAs in the case that the fully matched base pairs formed between them are even shorter than 6 bp. As a result, perfect "seeds" for polymerase chain reaction with information on both primers are produced so that exponential nonspecific amplification can occur. The DMPB mechanism can explain nonspecific amplification in other approaches as well. Finally, a mini-hairpin DNA is used to effectively inhibit nonspecific amplification by preventing the formation of an unexpected primer-bgDNA complex.

The use of terminal deoxynucleotidyl transferase for the first time in a mechanistic exploration-through colorimetric sensing and isothermal titration calorimetric studies-has provided direct experimental evidence of a boronic acid moiety bridging two DNA duplexes via the 3' hydroxyl groups, offering new opportunities and insights into the domain of DNA (nano)biotechnology.

Developing dye-based isothermal nucleic acid amplification (INAA) at low temperatures such as 37 °C remains a technical challenge. Here, we describe a nested phosphorothioated (PS) hybrid primer-mediated isothermal amplification (NPSA) assay which only utilizes EvaGreen (a DNA-binding dye) to achieve specific and dye-based subattomolar nucleic acid detection at 37 °C. The success of low-temperature NPSA essentially depends on employing Bacillus smithii DNA polymerase, a strand-displacing DNA polymerase with wide range of activation temperature. However, the NPSA's high efficiency entails nested PS-modified hybrid primers and the additives of urea and T4 Gene 32 Protein. To address the inhibition of urea on reverse transcription (RT), one-tube two-stage recombinase-aided RT-NPSA (rRT-NPSA) is established. By targeting human Kirsten rat sarcoma viral ( KRAS ) oncogene, NPSA (rRT-NPSA) stably detects 0.2 aM of KRAS gene (mRNA) within 90 (60) min. In addition, rRT-NPSA possesses subattomolar sensitivity to detect human ribosomal protein L13 mRNA. The NPSA/rRT-NPSA assays are also validated to obtain consistent results with PCR/ RT -PCR methods on qualitatively detecting DNA/mRNA targets extracted from cultured cells and clinical samples. As a dye-based, low-temperature INAA method, NPSA inherently facilitates the development of miniaturized diagnostic biosensors.

DNA detection, which includes the measurement of variants in sequences or the presence of certain genes, is widely used in research and clinical diagnosis. Both require DNA-dependent DNA polymerase-catalyzed strand extension. Currently, these techniques rely heavily on the instruments used to visualize the results. This study introduced a simple and direct colorimetric method to measure polymerase-directed elongation. First, pyrophosphate (PPi), a by-product of strand extension, is converted into phosphate (Pi). Phosphate levels were measured using either Mo-Sb or BIOMOL Green reagent. This study showed that this colorimetry can distinguish single-base variants and detect PCR products in preset stringent conditions, implicating the potential value of this strategy to detect DNA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Han Yang et al.)

Nucleosides and 2'-deoxyribonucleoside triphosphates (dNTPs) bearing 3,3'-dimethoxy-2,2'-diphenyl-6-(4-hydroxyphenyl)-bodipy fluorophore attached through a propargyl or propargyl-triethylene glycol linker to position 5 of 2'-deoxycytidine were designed and synthesized. They exerted bright red fluorescence and good sensitivity to viscosity changing their lifetime from 1.6 to 4.5 ns. The modifed dNTPs were substrates for DNA polymerases and were used in enzymatic synthesis of labeled DNA through primer extension. The modified DNA probes served as viscosity sensors responding to protein binding by changes of lifetime. The nucleotide with longer linker ( dC ) was transported to live cells and incorporated into the genomic DNA, which can be useful for staining of DNA and imaging of DNA synthesis. pegMOB TP ) was transported to live cells and incorporated into the genomic DNA, which can be useful for staining of DNA and imaging of DNA synthesis.

Microorganisms in the environment can be distinguished into dominant and rare microbial species based on their genes. It is difficult to obtain genetic information derived from rare microbial species (rare genes) because of the differences in relative abundance. DNA normalization is an approach that is used to obtain genetic information derived from rare microbial species from an environmental sample. This method involves the addition of adapter sequences for the amplification, denaturation, and reassociation of the DNA fragments and single-stranded DNA (ssDNA)/double-stranded DNA (dsDNA) separation. In this method, the amount of a high-copy-number of DNA fragments and a low-copy-number of DNA fragments can be equalized. Improvements in this technique are expected to provide novel genetic information or genes in rare microbial species. However, few model experimental systems have been reported to validate the DNA normalization techniques. This study is aimed to improve the DNA normalization technique used to obtain genetic information of rare genes from rare microbial species. An experimental study was constructed with two antibiotic resistance genes, whose copy numbers differed up to a million-fold. Both genes were mixed and the mixture of DNA fragments, of high- and low-copy-number, containing these genes was normalized by separating ssDNA/dsDNA fragments using hydroxyapatite. Normalized DNA fragments were introduced into Escherichia coli and DNA normalization was evaluated by counting colonies. Moreover, we improved the method to amplify a low-copy-number of DNA fragments by the addition of adapter sequences to DNA fragments using HiDi DNA polymerase to increase the efficiency of DNA normalization. This normalization method was achieved with a 100,000-fold difference. These methods allowed for quantitative evaluation of the DNA normalization efficiency. The experimental data and methods obtained in this study are expected to improve the DNA normalization efficiency to obtain novel genetic information or genes., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Clinically and biologically, it is essential to detect rare DNA-sequence variants for early cancer diagnosis or drug-resistance mutation identification. Some of the common quantitative polymerase chain reaction (qPCR)-based variant detection methods are restricted in the limit of detection (LoD) because the DNA polymerases used for these methods have a high polymerase misincorporation rate; thus, the detection sensitivity is sometimes unsatisfactory. With the proofreading activity, high-fidelity (HiFi) DNA polymerases have a 50- to 250-fold higher fidelity. However, there are currently no proper probe-based designs functioning as the fluorescence indicator allowing multiplexed HiFi qPCR reactions, thus restricting the application of HiFi DNA polymerases like the variant detection. We presented the occlusion system, composed of a 5'-overhanged primer with a fluorophore modification and a probe with a short-stem hairpin and a 3' quencher modification. We demonstrated that the occlusion system allowed multiplexing HiFi qPCR reaction, and it was compatible with the current variant-enrichment method to improve the LoD up to 10-fold. Thus, the occlusion system satisfactorily functioned as an efficient fluorescence indicator in HiFi qPCR reactions and allowed the application of HiFi DNA polymerases in variant detection methods to improve detection sensitivity.

Natural DNA is exquisitely evolved to store genetic information. The chirally inverted L-DNA, possessing the same informational capacity but resistant to biodegradation, may serve as a robust, bioorthogonal information repository. Here we chemically synthesize a 90-kDa high-fidelity mirror-image Pfu DNA polymerase that enables accurate assembly of a kilobase-sized mirror-image gene. We use the polymerase to encode in L-DNA an 1860 paragraph by Louis Pasteur that first proposed a mirror-image world of biology. We realize chiral steganography by embedding a chimeric D-DNA/L-DNA key molecule in a D-DNA storage library, which conveys a false or secret message depending on the chirality of reading. Furthermore, we show that a trace amount of an L-DNA barcode preserved in water from a local pond remains amplifiable and sequenceable for 1 year, whereas a D-DNA barcode under the same conditions could not be amplified after 1 day. These next-generation mirror-image molecular tools may transform the development of advanced mirror-image biology systems and pave the way for the realization of the mirror-image central dogma and exploration of their applications., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

In vitro molecular circuits, based on DNA-programmable chemistries, can perform an increasing range of high-level functions, such as molecular level computation, image or chemical pattern recognition and pattern generation. Most reported demonstrations, however, can only accept nucleic acids as input signals. Real-world applications of these programmable chemistries critically depend on strategies to interface them with a variety of non-DNA inputs, in particular small biologically relevant chemicals. We introduce here a general strategy to interface DNA-based circuits with non-DNA signals, based on input-translating modules. These translating modules contain a DNA response part and an allosteric protein sensing part, and use a simple design that renders them fully tunable and modular. They can be repurposed to either transmit or invert the response associated with the presence of a given input. By combining these translating-modules with robust and leak-free amplification motifs, we build sensing circuits that provide a fluorescent quantitative time-response to the concentration of their small-molecule input, with good specificity and sensitivity. The programmability of the DNA layer can be leveraged to perform DNA based signal processing operations, which we demonstrate here with logical inversion, signal modulation and a classification task on two inputs. The DNA circuits are also compatible with standard biochemical conditions, and we show the one-pot detection of an enzyme through its native metabolic activity. We anticipate that this sensitive small-molecule-to-DNA conversion strategy will play a critical role in the future applications of molecular-level circuitry., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Polymerase chain reaction (PCR) analysis using DNA from dried blood spot (DBS) samples on filter paper is a critical technique for spinal muscular atrophy (SMA) newborn screening. However, DNA extraction from DBS is time-consuming, and elimination of PCR inhibitors from DBS is almost impossible., Methods: Exon 7 of the two homologous SMA-related genes, survival motor neuron (SMN) 1 and SMN2, of five SMA patients and five controls were amplified by PCR with a punched-out circle of the DBS paper. Two types of DNA preparation methods were tested; DNA-extraction (extracted DNA was added in a PCR tube) and non-DNA-extraction (a punched-out DBS circle was placed in a PCR tube). As for the DNA polymerases, two different enzymes were compared; TaKaRa Ex Taq™ and KOD FX Neo™. To test the diagnostic quality of PCR products, RFLP (Restriction fragment length polymorphism) analysis with DraI digestion was performed, differentiating SMN1 and SMN2., Results: In PCR using extracted DNA, sufficient amplification was achieved with TaKaRa Ex Taq™ and KOD FX Neo™, and there was no significant difference in amplification efficiency between them. In direct PCR with a punched-out DBS circle, sufficient amplification was achieved when KOD FX Neo™ polymerase was used, while there was no amplification with TaKaRa Ex Taq™. RFLP analysis of the direct PCR products with KOD FX Neo™ clearly separated SMN1 and SMN2 sequences and proved the presence of both of SMN1 and SMN2 in controls, and only SMN2 in SMA patients, suggesting that the direct PCR products with KOD FX Neo™ were of sufficient diagnostic quality for SMA testing., Conclusion: Direct PCR with DNA polymerases like KOD FX NeoTM has potential to be widely used in SMA newborn screening in the near future as it obviates the DNA extraction process from DBS and can precisely amplify the target sequences in spite of the presence of PCR inhibitors.

DNA-encoded chemical libraries (DELs) have become an important ligand discovery technology in biomedical research and drug discovery. DELs can be comprised of hundreds of millions to billions of candidate molecules and provide outstanding chemical diversity for discovering novel ligands and inhibitors for a large variety of biological targets. However, in most cases, DELs are selected against purified and immobilized proteins based on binding affinity. The development and application of DELs to more complex biological targets requires selection methods compatible with nonimmobilized and unpurified proteins. Here, we describe an approach using polymerase-based extension and target-directed photo-cross-linking and its application to the interrogation of a solution-phase protein target, carbonic anhydrase II.

DNA-damage tolerance (DDT) is employed by eukaryotes to deal with replication blocks on the template strand, and is divided into two parallel pathways that are activated by sequential ubiquitination of proliferating cell nuclear antigen (PCNA) at the Lys164 residue. Rad6-Rad18-mediated PCNA monoubiquitination promotes translesion DNA synthesis (TLS) and the monoubiquitinated PCNA can be further polyubiquitinated by an Mms2-Ubc13-Rad5 complex, leading to error-free lesion bypass. We previously reported that the DNA helicase Sgs1 is required for error-free lesion bypass, probably through the double-Holliday junction migration and subsequent resolution. Surprisingly, a synthetic genetic array (SGA) screen using rev1 and rev3 as baits did not reveal an anticipated synthetic effect with sgs1, indicating a possible involvement of Sgs1 in TLS. Here, we report detailed genetic analyses demonstrating that Sgs1 plays a key role in efficient TLS and that it is probably required for the signaling of DNA damage leading to PCNA monoubiquitination. These studies collectively illustrate that Sgs1 participates in both branches of DDT and possibly plays a role in pathway choice.

Nucleotide incorporation by DNA polymerase forms the basis of DNA sequencing-by-synthesis. In current platforms, either the single-stranded DNA or the enzyme is immobilized on a solid surface to locate the incorporation of individual nucleotides in space and/or time. Solid-phase reactions may, however, hinder the polymerase activity. We demonstrate a device and a protocol for the enzymatic labeling of genomic DNA arranged in a dense array of single molecules without attaching the enzyme or the DNA to a surface. DNA molecules accumulate in a dense array of pits embedded within a nanoslit due to entropic trapping. We then perform ϕ29 polymerase extension from single-strand nicks created on the trapped molecules to incorporate fluorescent nucleotides into the DNA. The array of entropic traps can be loaded with λ-DNA molecules to more than 90% of capacity at a flow rate of 10 pL min -1 . The final concentration can reach up to 100 μg mL -1 , and the DNA is eluted from the array by increasing the flow rate. The device may be an important preparative module for carrying out enzymatic processing on DNA extracted from single-cells in a microfluidic chip.

We proposed a terminal deoxynucleotidyl transferase (TdT)-assisted rolling circle amplification (RCA) strategy for the amplified detection of genome-containing biological targets. The synergetic signal amplification of DNase I-mediated genomic DNA fragmentation and subsequent RCA allow the sensing platform to have extraordinarily high sensitivity.

Competing Interests: The author declares no conflict of interest.

As biosensing devices shrink smaller and smaller, they approach a scale in which single molecule electronic sensing becomes possible. Here, we review the operation of single-enzyme transistors made using single-walled carbon nanotubes. These novel hybrid devices transduce the motions and catalytic activity of a single protein into an electronic signal for real-time monitoring of the protein's activity. Analysis of these electronic signals reveals new insights into enzyme function and proves the electronic technique to be complementary to other single-molecule methods based on fluorescence. As one example of the nanocircuit technique, we have studied the Klenow Fragment (KF) of DNA polymerase I as it catalytically processes single-stranded DNA templates. The fidelity of DNA polymerases makes them a key component in many DNA sequencing techniques, and here we demonstrate that KF nanocircuits readily resolve DNA polymerization with single-base sensitivity. Consequently, template lengths can be directly counted from electronic recordings of KF's base-by-base activity. After measuring as few as 20 copies, the template length can be determined with <1 base pair resolution, and different template lengths can be identified and enumerated in solutions containing template mixtures., Competing Interests: The authors declare no conflict of interest.

Mammalian RAD51 paralogs are implicated in the repair of collapsed replication forks by homologous recombination. However, their physiological roles in replication fork maintenance prior to fork collapse remain obscure. Here, we report on the role of RAD51 paralogs in short-term replicative stress devoid of DSBs. We show that RAD51 paralogs localize to nascent DNA and common fragile sites upon replication fork stalling. Strikingly, RAD51 paralogs deficient cells exhibit elevated levels of 53BP1 nuclear bodies and increased DSB formation, the latter being attributed to extensive degradation of nascent DNA at stalled forks. RAD51C and XRCC3 promote the restart of stalled replication in an ATP hydrolysis dependent manner by disengaging RAD51 and other RAD51 paralogs from the halted forks. Notably, we find that Fanconi anemia (FA)-like disorder and breast and ovarian cancer patient derived mutations of RAD51C fails to protect replication fork, exhibit under-replicated genomic regions and elevated micro-nucleation. Taken together, RAD51 paralogs prevent degradation of stalled forks and promote the restart of halted replication to avoid replication fork collapse, thereby maintaining genomic integrity and suppressing tumorigenesis., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In this work we present the use of a silicon-on-insulator (SOI) chip featuring an array of 64 optical ring resonators used as refractive index sensors for real-time and label-free DNA detection. Single ring functionalisation was achieved using a click reaction after precise nanolitre spotting of specific hexynyl-terminated DNA capture probes to link to an azido-silanised chip surface. To demonstrate detectability using the ring resonators and to optimise conditions for solid-phase amplification, hybridisation between short 25-mer single stranded DNA (ssDNA) fragments and a complementary capture probe immobilised on the surface of the ring resonators was carried out and detected through the shift in the resonant wavelength. Using the optimised conditions demonstrated via the solid-phase hybridisation, a 144-bp double stranded DNA (dsDNA) was then detected directly using recombinase and polymerase proteins through on-chip target amplification and solid-phase elongation of immobilised forward primers on specific rings, at a constant temperature of 37°C and in less than 60min, achieving a limit of detection of 7.8·10(-13)M (6·10(5) copies in 50µL). The use of an automatic liquid handler injection instrument connected to an integrated resealable chip interface (RCI) allowed programmable multiple injection protocols. Air plugs between different solutions were introduced to prevent intermixing and a proportional-integral-derivative (PID) temperature controller minimised temperature based drifts., (Published by Elsevier B.V.)

DNA intercalators are widely used as fluorescent probes to visualize DNA and DNA transactions in vivo and in vitro. It is well known that they perturb DNA structure and stability, which can in turn influence DNA-processing by proteins. Here we elucidate this perturbation by combining single-dye fluorescence microscopy with force spectroscopy and measuring the kinetics of DNA intercalation by the mono- and bis-intercalating cyanine dyes SYTOX Orange, SYTOX Green, SYBR Gold, YO-PRO-1, YOYO-1 and POPO-3. We show that their DNA-binding affinity is mainly governed by a strongly tension-dependent dissociation rate. These rates can be tuned over a range of seven orders of magnitude by changing DNA tension, intercalating species and ionic strength. We show that optimizing these rates minimizes the impact of intercalators on strand separation and enzymatic activity. These new insights provide handles for the improved use of intercalators as DNA probes with minimal perturbation and maximal efficacy.

Objective: To investigate single-molecule detection and characterization of DNA replication., Methods: Single-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication., Results: The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis., Conclusions: The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.

Up to now the mechanism of formation of frameshift mutations caused by cyclobutane pyrimidine dimers has not been yet explained satisfactorily. Mechanisms of different mutations are usually considered in polymerase model. Here, the alternative polymerase-tautomer model of ultraviolet mutagenesis is developed. The mechanism of targeted insertion formation caused by cis-syn cyclobutane thymine dimers is proposed. Insertions are mutations when one or several DNA bases are inserted.Targeted insertions are mutations of a frameshift type--when one or severalnucleotides are inserted opposite damageswhich may stop synthesis of DNA. Targeted insertions are induced bycyclobutane pyrimidine dimmers. Ultraviolet irradiation may result in a change of tautomer state of DNA bases. A thymine base may form 5 rare tautomer forms that are stable if the base is a part of cyclobutane dimer. As it was shown by structural analysis, one rare tautomeric form of thymine forms hydrogen bonds with no one canonical DNA base. Therefore, under SOS or error-prone synthesis of DNA containing cis-syn cyclobutane thymine dimers with such rare tautomeric&#95;form a specialize or modified DNA polymerase leaves a single nucleotide gap opposite the cis-syn cyclobutane thymine dimer. According to Streisinger model, if the DNA composition within this region is homogeneous, the end of the growing DNA strand can slip and form complementary pairs with a template nucleotide neighboring to the dimer of such type a loop is formed. Further elongation of the daughter strand leads to the appearance of targeted insertion in the daughter strand. Here, it is first shown that cis-syn cyclobutane thymine dimers with one or both bases in the specific tautomer conformation--opposite which it is impossible to insert a canonical base with a hydrogen bond formation--results in targeted insertions. Moreover, the model of forming targeted single--and several-base insertions is developed. The polymerase-tautomer model of ultraviolet mutagenesis can explain a nature and mechanism of appearing not only hot and cold spots, targeted and untargeted base substitution mutations but also targeted frameshift mutations.

Genomic integrity is crucial for correct chromosome segregation and physiological rates of cell proliferation. Mutations, deletions and translocations, hallmarks of human tumors, drive the aberrant proliferation and metastatic behavior of cancer cells. These chromosomal rearrangements often occur at genomic sites susceptible to breakage during DNA replication, including regions with G-quadruplex (G4)-forming potential. G4s are stable secondary structures that guanine-rich single-stranded DNA can readily adopt in vitro. However, their formation in eukaryotic cells has remained elusive and thus a subject of debate ever since they were first described. Recent work has more convincingly implicated G4s in a variety of biological processes including telomere maintenance, gene expression, epigenetic regulation and DNA replication. However, the downside of employing thermodynamically very stable alternative DNA structures as regulatory entities lies in their potential to also interfere with normal DNA metabolic processes, such as transcription and replication, which require readability of each base to faithfully transmit genetic information. Indeed, it has become clear that G4 structures can pose prominent barriers to replication fork progression and that they are also intrinsically recombinogenic. Here, we discuss mechanisms that cells evolved to counteract these detrimental effects, thereby ensuring the faithful inheritance of G4-containing genomes., (© 2013.)

In addition to the canonical right-handed double helix, DNA molecule can adopt several other non-B DNA structures. Readily formed in the genome at specific DNA repetitive sequences, these secondary conformations present a distinctive challenge for progression of DNA replication forks. Impeding normal DNA synthesis, cruciforms, hairpins, H DNA, Z DNA and G4 DNA considerably impact the genome stability and in some instances play a causal role in disease development. Along with previously discovered dedicated DNA helicases, the specialized DNA polymerases emerge as major actors performing DNA synthesis through these distorted impediments. In their new role, they are facilitating DNA synthesis on replication stalling sites formed by non-B DNA structures and thereby helping the completion of DNA replication, a process otherwise crucial for preserving genome integrity and concluding normal cell division. This review summarizes the evidence gathered describing the function of specialized DNA polymerases in replicating DNA through non-B DNA structures., (© 2013. Published by Elsevier Ltd. All rights reserved.)

By diversifying the biological effector functions of antibodies, class switch DNA recombination (CSR) plays a critical role in the maturation of the immune response. It is initiated by activation-induced cytidine deaminase (AID)-mediated deoxycytosine deamination, yielding deoxyuridine (dU), and dU glycosylation by uracil DNA glycosylase (Ung) in antibody switch (S) region DNA. Here we showed that the translesion DNA synthesis polymerase Rev1 directly interacted with Ung and targeted in an AID-dependent and Ung-independent fashion the S regions undergoing CSR. Rev1(-/-)Ung(+/+) B cells reduced Ung recruitment to S regions, DNA-dU glycosylation, and CSR. Together with an S region spectrum of mutations similar to that of Rev1(+/+)Ung(-/-) B cells, this suggests that Rev1 operates in the same pathway as Ung, as emphasized by further decreased CSR in Rev1(-/-)Msh2(-/-) B cells. Rescue of CSR in Rev1(-/-) B cells by a catalytically inactive Rev1 mutant shows that the important role of Rev1 in CSR is mediated by Rev1's scaffolding function, not its enzymatic function., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

The development of cancer drug resistance is a persistent clinical problem limiting the successful treatment of disseminated malignancies. However, the molecular mechanisms by which initially chemoresponsive tumors develop therapeutic resistance remain poorly understood. Error-prone translesional DNA synthesis (TLS) is known to underlie the mutagenic effects of numerous anticancer agents, but little is known as to whether mutation induced by this process is ultimately relevant to tumor drug resistance. Here, we use a tractable mouse model of B-cell lymphoma to interrogate the role of error-prone translesional DNA synthesis in chemotherapy-induced mutation and resistance to front-line chemotherapy. We find that suppression of Rev1, an essential TLS scaffold protein and dCMP transferase, inhibits both cisplatin- and cyclophosphamide-induced mutagenesis. Additionally, by performing repeated cycles of tumor engraftment and treatment, we show that Rev1 plays a critical role in the development of acquired cyclophosphamide resistance. Thus, chemotherapy not only selects for drug-resistant tumor population but also directly promotes the TLS-mediated acquisition of resistance-causing mutations. These data provide an example of an alteration that prevents the acquisition of drug resistance in tumors in vivo. Because TLS also represents a critical mechanism of DNA synthesis in tumor cells following chemotherapy, these data suggest that TLS inhibition may have dual anticancer effects, sensitizing tumors to therapy as well as preventing the emergence of tumor chemoresistance.

Current forensic DNA typing is conducted in approximately 8-10h. Steps include DNA extraction, quantification, polymerase chain reaction (PCR) amplification of multiple short tandem repeat (STR) loci, capillary electrophoresis separation with fluorescence detection, data analysis and DNA profile interpretation. The PCR amplification portion of the workflow typically takes approximately 3h with standard thermal cycling protocols. Here we demonstrate a rapid cycling protocol that amplifies 15 STR loci and the sex-typing marker amelogenin from the Identifiler STR typing kit in less than 36 min. This rapid protocol employs commercially available polymerases and the widely used GeneAmp 9700 thermal cycler. Complete concordance of STR allele calls (for 60 samples) between the rapid and standard thermal cycling protocols were observed although there was incomplete adenylation at several of the loci examined and some PCR artifacts were detected. Using less than 750 pg of template DNA and 28 cycles, STR peaks for all loci were above a 150 relative fluorescent unit (RFU) detection threshold with fully adequate inter-locus balance and heterozygote peak height ratios of greater than 0.84.

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.

Two unusual enzymatic activities are required for the realization of a single molecule sequencing: a polymerase for copying a deoxyribonuclease (DNA) target into complementary flurophore-labeled DNA, and an exonuclease for the successive hydrolysis of the completely dye-labeled DNA. Recently, we found that the wild-type Klenow fragment of Escherichia coli DNA polymerase I is well-suited for the synthesis of DNA in a reaction set-up that contains exclusively specific rhodamine-labeled analogs of the natural pyrimidine nucleotides (dCTP and dTTP). This protocol describes the procedure used for the preparation of DNA that is labeled at all pyrimidine bases of one strand, as well as an example of enzymatic downstream processing of the DNA product.

The Drosophila Mus308 gene is unusual in encoding both a family A DNA polymerase domain and a DNA/RNA helicase domain. A mus308 mutation was shown to result in increased sensitivity to DNA cross-linking agents, leading to the hypothesis that Mus308 functions in the repair of DNA interstrand cross-links. Recently a mammalian ortholog of Mus308, POLQ, has been identified. We report here the identification, cloning, and characterization of POLN and its gene product, a new mammalian DNA polymerase also related to Mus308. The human cDNA encodes a protein of 900 amino acid residues. The region starting from residue 419 shares 33% identity (48% similarity) with the equivalent region of Escherichia coli DNA polymerase I. POLN is expressed in human cell lines with numerous alternatively spliced transcripts, and a full-length human coding region that comprises 24 exons within 160 kilobases of genomic DNA. Expression analysis by northern blotting and in situ hybridization showed highest expression of full-length POLN in human and mouse testis. POLN localized to the nucleus when expressed as a enhanced green fluorescent protein (GFP)-tagged protein in human fibroblasts. GFP-tagged recombinant POLN had DNA polymerase activity on activated calf thymus DNA and on a singly primed template.

A number of different energy transfer dye labeled-cassettes were synthesized using aminoacid based trifunctional linkers and coupled to the propargylamino-substituted dideoxynucleoside-5'-triphosphates (ddNTPs). These terminators were evaluated for their energy transfer efficiency and DNA sequencing potential using thermostable DNA polymerase.

Human polymerase kappa (polkappa), the product of the human POLK (DINB1) gene, is a member of the Y superfamily of DNA polymerases that support replicative bypass of chemically modified DNA bases (Ohmori, H., Friedberg, E. C., Fuchs, R. P., Goodman, M. F., Hanaoka, F., Hinkle, D., Kunkel, T. A., Lawrence, C. W., Livneh, Z., Nohmi, T., Prakash, L., Prakash, S., Todo, T., Walker, G. C., Wang, Z., and Woodgate, R. (2001) Mol. Cell 8, 7-8; Gerlach, V. L., Aravind, L., Gotway, G., Schultz, R. A., Koonin, E. V., and Friedberg, E. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11922-11927). Polkappa is shown here to bypass 5,6-dihydro-5,6-dihydroxythymine (thymine glycol) generated in two different DNA substrate preparations. Polkappa inserts the correct base adenine opposite thymine glycol in preference to the other three bases. Additionally, the enzyme correctly extends beyond the site of the thymine glycol lesion when presented with adenine opposite thymine glycol at the primer terminus. However, steady state kinetic analysis of nucleotides incorporated opposite thymine glycol demonstrates different misincorporation rates for guanine with each of the two DNA substrates. The two substrates differ only in the relative proportions of thymine glycol stereoisomers, suggesting that polkappa distinguishes among stereoisomers and exhibits reduced discrimination between purines when incorporating a base opposite a 5R thymine glycol stereoisomer. When extending beyond the site of the lesion, the misincorporation rate of polkappa for each of the three incorrect nucleotides (adenine, guanine, and thymine) is dramatically increased. Our findings suggest a role for polkappa in both nonmutagenic and mutagenic bypass of oxidative damage.

Several recently discovered human DNA polymerases are associated with translesion synthesis past DNA adducts. These include human DNA polymerase kappa (pol kappa), a homologue of Escherichia coli pol IV, which enhances the frequency of spontaneous mutation. Using a truncated form of pol kappa (pol kappa Delta C), translesion synthesis past dG-(+)- or dG-(-)-anti-N(2)-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) adducts was explored. Site-specifically-modified oligodeoxynucleotides containing a single stereoisomeric dG-N(2)-BPDE lesion were used as DNA templates for primer extension reactions catalyzed by pol kappa Delta C. Primer extension was retarded one base prior to the dG-N(2)-BPDE lesion; when incubated for longer times or with higher concentration of enzyme, full primer extension was observed. Quantitative analysis of fully extended products showed preferential incorporation of dCMP, the correct base, opposite all four stereoisomeric dG-N(2)-BPDE lesions. (+)-trans-dG-N(2)-BPDE, a major BPDE-DNA adduct, promoted small amounts of dTMP, dAMP, and dGMP misincorporation opposite the lesion (total 2.7% of the starting primers) and deletions (1.1%). Although (+)-cis-dG-N(2)-BPDE was most effective in blocking translesion synthesis, its miscoding properties were similar to other dG-N(2)-BPDE isomers. Steady-state kinetic data indicate that dCMP is efficiently inserted opposite all dG-N(2)-BPDE adducts and extended past these lesions. The relative frequency of translesion synthesis (F(ins) x F(ext)) of dC.dG-N(2)-BPDE pairs was 2-6 orders of magnitude higher than that of other mismatched pairs. Pol kappa may play an important role in translesion synthesis by incorporating preferentially the correct base opposite dG-N(2)-BPDE. Its relatively low contribution to mutagenicity suggests that other newly discovered DNA polymerase(s) may be involved in mutagenic events attributed to dG-N(2)-BPDE adducts in human cells.

Humans have three DNA polymerases, Poleta, Polkappa, and Poliota, which are able to promote replication through DNA lesions. However, the mechanism by which these DNA polymerases are targeted to the replication machinery stalled at a lesion site has remained unknown. Here, we provide evidence for the physical interaction of human Polkappa (hPolkappa) with proliferating cell nuclear antigen (PCNA) and show that PCNA, replication factor C (RFC), and replication protein A (RPA) act cooperatively to stimulate the DNA synthesis activity of hPolkappa. The processivity of hPolkappa, however, is not significantly increased in the presence of these protein factors. The efficiency (V(max)/K(m)) of correct nucleotide incorporation by hPolkappa is enhanced approximately 50- to 200-fold in the presence of PCNA, RFC, and RPA, and this increase in efficiency is achieved by a reduction in the apparent K(m) for the nucleotide. Although in the presence of these protein factors, the efficiency of the insertion of an A nucleotide opposite an abasic site is increased approximately 40-fold, this reaction still remains quite inefficient; thus, it is unlikely that hPolkappa would bypass an abasic site by inserting a nucleotide opposite the site.

This unit describes how PCR can be used to exponentially amplify segments of DNA located between two specified primer hybridization sites. A single-sided PCR method is used that initially requires specification of only one primer hybridization site; the second is defined by the ligation-based addition of a unique DNA linker. This linker, together with the flanking gene-specific primer, allows exponential amplification of any fragment of DNA. Because a defined, discrete-length sequence is added to every fragment, complex populations of DNA such as sequence ladders can be amplified intact with retention of single-base resolution. The ligation-based protocol was specifically designed for genomic footprinting and direct sequencing reactions, and is described in this context; it can, however, be used for other applications.

Nested deletions useful for dideoxy DNA sequencing are a set of deletions originating at one end of a target DNA fragment and extending various lengths along the target DNA. Each successively longer deletion brings "new" regions of the target DNA into sequencing range (about 300 bp for normal sequencing gels) of the primer site for a general discussion of nested deletions in DNA sequencing). Two protocols for generating nested subclones via enzymatic digestion are included in this unit. In the first, a set of nested deletions is generated by exonuclease III. The primary advantage of this method is that the deletion products generated from the original clone can be recircularized to generate functional plasmids and thus do not require subcloning into another vector. An alternate method utilizes Bal 31 nuclease to generate the deletions. This method requires subcloning of the deletion fragments into a separate vector for subsequent use. Both methods require the presence of unique restriction sites in the vector that are not present in the insert DNA. Bal 31 can also be used to generate nested deletions for chemical sequencing in conjunction with specialized chemical sequencing vectors.

Standard dideoxy DNA sequencing can be performed easily and efficiently with nonisotopic, chemiluminescent detection by utilizing primers labeled with biotin in the sequencing reactions. As described in this unit, reaction products are separated by denaturing gel electrophoresis, transferred to a nylon membrane, and detected by first binding a streptavidin-alkaline phosphatase conjugate, then incubating with a chemiluminescent 1,2-dioxetane substrate. The emitted light signal is imaged on standard X-ray film, producing high-resolution DNA sequencing ladders. Indirect alkaline phosphatase-labeling of biotinylated DNA with free streptavidin and biotinylated alkaline phosphatase is also detailed, Finally, the detection of sequencing reactions labeled with other haptens using specific antibody-alkaline phosphatase conjugates is described.

Damage and repair of DNA isolated from brain and spleen of gamma-irradiated rats were assayed using the polymerase chain reaction (PCR) method. Damage produced by gamma-radiation in DNA in cells of these tissues of exposed animals was shown to block PCR with the Tth polymerase. This blockage was noted as a decrease in the level of amplification of the fragments of a transcribed gene (beta-actin), an inducible gene (p53), and a nontranscribed one (IgE, heavy chain). The most pronounced decrease in the amplification of the gene fragments was observed on the DNA template isolated from rats immediately after their gamma-irradiation. When DNA was isolated 0.5-5.0 h after exposure, the amplification level was restored, no matter what transcription activity the genes possessed. For comparison, we used in PCR in vitro gamma-irradiated DNA as well as DNA templates with UV-damage, 8-oxy-2;-deoxyguanosine (8-O-dG), and apurinic sites (AP-sites). We found that gamma- and UV-irradiated DNA as well as DNA with AP-sites blocked the Tth polymerase in PCR, whereas 8-O-dG did not effect the level of PCR amplification of gene fragments. The observed changes in the level of PCR amplification of genes on the DNA template from tissues of gamma-irradiated animals are due to various radiation-induced lesions capable of blocking the Tth polymerase. The results show that the PCR method can be used for assaying the integral DNA damage and repair in cells from irradiated animals.

The influence of sequence context on the ability of DNA polymerase to bypass sites of base loss was addressed using an in vitro selection system. Oligonucleotides containing either an aldehydic abasic site or tetrahydrofuran surrounded by four randomized bases on both the 5' and 3' sides were used as templates for synthesis by phage T4 DNA polymerase holoenzyme proficient or deficient in the 3'-->5' proofreading exonuclease activity. Successful bypass products were purified, subcloned and the sequences of approximately 100 subclones were determined for each of the four polymerase/lesion combinations tested. Between 7 and 19 % of the bypass products contained deletions of one to three nucleotides in the randomized region. In bypass products not containing deletions, biases for and against certain nucleotides were readily noticeable across the entire randomized region. Template strands from successful bypass products of abasic sites had a high frequency of T in most of the randomized positions, while those from bypass products of tetrahydrofuran had a high frequency of G at the positions immediately to the 3' and 5' side of the lesion. Consensus sequences were shared by successful bypass products of the same lesion but not between bypass products of the two lesions. The consensus sequence for efficient bypass of tetrahydrofuran was over-represented in several frames relative to the lesion. T4 DNA polymerase inserted A opposite abasic sites 63 % of the time in the presence of proofreading and 79 % of the time in its absence, followed by G>T>C, while the insertion of A opposite tetrahydrofuran ranged between 93 % and 100 % in the presence and absence of proofreading, respectively. Finally, sequence context influenced the choice of nucleotide inserted opposite abasic sites and consensus sequences which favored the incorporation of nucleotides other than A were defined., (Copyright 1999 Academic Press.)

Two nucleoside triphosphates, a pyrimidine modified with an ammonium functionality and a purine modified with an imidazolyl functionality are compatible with all conditions for a combinatorial selection of nucleic-acid catalysts. We believe that this work is the first to demonstrate the potential for using not one but two modified nucleotides in tandem. The potential for an enriched catalytic repertoire is envisioned.

Under anaerobic conditions neocarzinostatin chromophore, an enediyne antibiotic, forms a covalent drug-DNA adduct on the 5' carbon of deoxyribose at a specific single site in a 2-nucleotide bulge, rather than strand cleavage, by a mechanism involving general base-catalyzed intramolecular drug activation to a reactive radical species. We have taken advantage of the selectivity of this reaction to prepare a single-stranded oligonucleotide containing a single drug adduct at a T residue and to study its effect on the template properties of the oligonucleotide in replicative synthesis, as followed by 5'-32P-labeled primer extension by several DNA polymerases. With the Klenow fragment of Escherichia coli DNA polymerase I, synthesis stops at the base immediately 3' to the adduct. The same enzyme, but lacking 3' to 5' exonuclease activity, permits synthesis to proceed by one additional nucleotide. This effect is enhanced when Mn2+ is substituted for Mg2+. T4, herpes simplex virus, and cytomegalovirus DNA polymerases all act like Klenow polymerase. Sequenase (exo-minus T7 DNA polymerase) is qualitatively similar to exo-minus Klenow polymerase but is more efficient in inserting a nucleotide opposite the lesion. With the small-gap-filling human DNA polymerase beta, which lacks intrinsic exonucleolytic activity, primer extension proceeds to the nucleotide opposite the lesion. However, when a gap was created opposite the lesion, polymerase beta adds as many as two additional nucleotides 5' to the adduct site. The fidelity of base incorporation opposite the lesion was not impaired, in contrast with adducts on DNA bases.