Strand Extension Research Articles

Enhancing the Biomimetic Mechanics of Bottlebrush Graft-Copolymers through Selective Solvent Annealing.

Self-assembled networks of bottlebrush copolymers are promising materials for biomedical applications due to a unique combination of ultra-softness and strain-adaptive stiffening, characteristic of soft biological tissues. Transitioning from ABA linear-brush-linear triblock copolymers to A-g-B bottlebrush graft copolymer architectures allows significant increasing the mechanical strength of thermoplastic elastomers. Using real-time synchrotron small-angle X-ray scattering, it is shown that annealing of A-g-B elastomers in a selective solvent for the linear A blocks allows for substantial network reconfiguration, resulting in an increase of both the A domain size and the distance between the domains. The corresponding increases in the aggregation number and extension of bottlebrush strands lead to a significant increase of the strain-stiffening parameter up to 0.7, approaching values characteristic of the brain and skin tissues. Network reconfiguration without disassembly is an efficient approach to adjusting the mechanical performance of tissue-mimetic materials to meet the needs of diverse biomedical applications.

Target-responsive triplex aptamer nanoswitch enables label-free and ultrasensitive detection of antibody in human serum via lighting-up RNA aptamer transcriptions

Accurate and sensitive monitoring of the concentration change of anti-digoxigenin (Anti-Dig) antibody is of great importance for diagnosing infectious and immunological diseases. Combining a novel triplex aptamer nanoswitch and the high signal-to-noise ratio of lighting-up RNA aptamer signal amplification, a label-free and ultrasensitive fluorescent sensing approach for detecting Anti-Dig antibodies is described. The target Anti-Dig antibodies recognize and bind with the nanoswitch to open its triplex helix stem structure to release Taq DNA polymerase and short ssDNA primer simultaneously, which activates the Taq DNA polymerase to initiate downstream strand extension of ssDNA primer to yield specific dsDNA containing RNA promoter sequence. T7 RNA polymerase recognizes and binds to these promoter sequences to initiate RNA transcription reaction to produce many RNA aptamer sequences. These aptamers can recognize and bind with Malachite Green (MG) dye specifically and produce highly amplified fluorescent signal for monitoring Anti-Dig antibodies from 50 pM to 50 nM with a detection limit down to 33 pM. The method also exhibits high selectivity for Anti-Dig antibodies and can be used to discriminate trace Anti-Dig antibodies in diluted serum samples. Our method is superior to many immunization-based Anti-Dig antibody detection methods and thus holds great potential for monitoring disease progression and efficacy.

Construction of a Quantum-Dot-Based FRET Nanosensor through Direct Encoding of Streptavidin-Binding RNA Aptamers for N6-Methyladenosine Demethylase Detection.

N6-Methyladenosine (m6A) demethylases can catalyze the removal of the methyl modification on m6A, and it is closely associated with the occurrence, proliferation, differentiation, and metastasis of malignancies. The m6A demethylases (e.g., fat mass and obesity-associated protein (FTO)) may act as a cancer biomarker and are crucial for anticancer drug screening and early clinical diagnosis. Herein, we demonstrate the construction of a quantum-dot-based Förster resonance energy-transfer (FRET) nanosensor through direct encoding of streptavidin-binding RNA aptamers (SA aptamers) for m6A demethylase detection. This nanosensor employs multiple Cy5-molecule-labeled SA aptamers as the building materials to construct the 605QD-RNA-Cy5 nanoassembly, and it exploits the hinder effect of m6A upon elongation and ligation reactions to distinguish m6A-containing RNA probes from demethylated RNA probes. When m6A demethylase is present, the m6A-containing RNA probes are demethylated to generate the demethylated RNA probes, initiating strand extension and ligation reactions to yield a complete transcription template for SA aptamers. Subsequently, a T7-assisted cascade transcription amplification reaction is activated to transcribe abundant SA aptamers with the incorporation of multiple Cy5 fluorophores. The Cy5-incorporated SA aptamers can self-assembly onto the streptavidin-coated 605QD surface to obtain the 605QD-SA aptamer-Cy5 nanoassemblies, resulting in the generation of distinct FRET signals. This nanosensor exhibits ultrahigh sensitivity and excellent specificity, and it can detect endogenous FTO at the single-cell level. Furthermore, this nanosensor can precisely measure enzyme kinetic parameters, screen m6A demethylase inhibitors, and differentiate the FTO expression between breast cancer patients and healthy individual tissues, offering a versatile platform for clinical diagnostic and drug discovery.

A Constitutive Model for Mechanical Behaviors of Novel Double Network Hydrogels with Mechanophores

Double network hydrogels (DN hydrogels) with high stretchability and toughness have attracted broad research interest. Recently, a kind of novel tough DN hydrogels was designed by means of the reactive strand extension strategy, which introduced mechanophores into the first network. When the strands of the first network reach their nominal stretching limit, the mechanophores allow the strands to survive through force-coupled reactions instead of fracture. As a consequence, the novel hydrogels can achieve a better mechanical performance compared with the conventional DN hydrogels. In this work, we aim to develop a constitutive model for the novel DN hydrogels. The model is based on the worm-like single-chain model by introducing bond deformation. The network alteration theory is used to account for the damage behaviors. The theoretical framework is capable of clarifying the difference in mechanical behaviors between conventional and novel DN hydrogels, which demonstrates the importance of bond deformation on the mechanical behaviors of DN hydrogels.

Colorimetry-Based Phosphate Measurement for Polymerase Elongation.

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.

Superstretchable Elastomer from Cross-linked Ring Polymers.

The stretchability of polymeric materials is critical to many applications such as flexible electronics and soft robotics, yet the stretchability of conventional cross-linked linear polymers is limited by the entanglements between polymer chains. We show using molecular dynamics simulations that cross-linked ring polymers are significantly more stretchable than cross-linked linear polymers. Compared to linear polymers, the entanglements between ring polymers do not act as effective cross-links. As a result, the stretchability of cross-linked ring polymers is determined by the maximum extension of polymer strands between cross-links, rather than between trapped entanglements as in cross-linked linear polymers. The more compact conformation of ring polymers before deformation also contributes to the increase in stretchability.

Nanoscale dynamics of actin filaments in the red blood cell membrane skeleton.

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15–18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature, and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ are not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200–300 nm apart interspersed with dimmer F-actin staining regions. Single molecule stochastic optical reconstruction microscopy imaging of Alexa 647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, nonrandom distribution of F-actin nodes. Treatment of RBCs with latrunculin A and cytochalasin D indicates that F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape, and membrane deformability.

STI PCR: An efficient method for amplification and de novo synthesis of long DNA sequences

Despite continuous improvements, it is difficult to efficiently amplify large sequences from complex templates using current PCR methods. Here, we developed a suppression thermo-interlaced (STI) PCR method for the efficient and specific amplification of long DNA sequences from genomes and synthetic DNA pools. This method uses site-specific primers containing a common 5′ tag to generate a stem-loop structure, thereby repressing the amplification of smaller non-specific products through PCR suppression (PS). However, large target products are less affected by PS and show enhanced amplification when the competitive amplification of non-specific products is suppressed. Furthermore, this method uses nested thermo-interlaced cycling with varied temperatures to optimize strand extension of long sequences with an uneven GC distribution. The combination of these two factors in STI PCR produces a multiplier effect, markedly increasing specificity and amplification capacity. We also developed a webtool, calGC, for analyzing the GC distribution of target DNA sequences and selecting suitable thermo-cycling programs for STI PCR. Using this method, we stably amplified very long genomic fragments (up to 38 kb) from plants and human and greatly increased the length of de novo DNA synthesis, which has many applications such as cloning, expression, and targeted genomic sequencing. Our method greatly extends PCR capacity and has great potential for use in biological fields.

Design Features to Accelerate the Higher-Order Assembly of DNA Origami on Membranes

Nanotechnology often exploits DNA origami nanostructures assembled into even larger superstructures up to micrometer sizes with nanometer shape precision. However, large-scale assembly of such structures is very time-consuming. Here, we investigated the efficiency of superstructure assembly on surfaces using indirect cross-linking through low-complexity connector strands binding staple strand extensions, instead of connector strands binding to scaffold loops. Using single-molecule imaging techniques, including fluorescence microscopy and atomic force microscopy, we show that low sequence complexity connector strands allow formation of DNA origami superstructures on lipid membranes, with an order-of-magnitude enhancement in the assembly speed of superstructures. A number of effects, including suppression of DNA hairpin formation, high local effective binding site concentration, and multivalency are proposed to contribute to the acceleration. Thus, the use of low-complexity sequences for DNA origami higher-order assembly offers a very simple but efficient way of improving throughput in DNA origami design.

Convenient and highly sensitive electrochemical biosensor for monitoring acid phosphatase activity

Acid phosphatase (ACP) is a potential biomarker for different diseases. Using the DNA extension-based biological signal amplification and redox recycling of electrochemical species on reduced graphene oxide (rGO)-modified electrode, we established here a highly sensitive, label-free and electrode immobilization-free electrochemical assay for ACP in diluted human serum samples. Target analyte of ACP catalyzes the de-phosphorylation of the 3′-PO4 termini of a ssDNA into 3′−OH on the magnetic beads, which subsequently initiates strand extension into long guanine-rich ssDNAs by the terminal deoxynucleotidyl transferase with pre-defined ratio of dGTP to dATP. The magnetic accumulation of the beads on the rGO-modified electrode thus yield largely enhanced current signals, due to redox recycling oxidation of the many guanine bases in the extended DNAs mediated by [Ru(bpy)3]2+. Such significant current amplification can therefore lead to sensitive electrochemical detection of ACP in the range from 5 to 100 U/L with a low detection limit of 0.52 U/L. High selectivity of the sensing method can also be obtained and the monitoring of ACP in diluted serum samples is demonstrated. With the significant advantages of simplicity and sensitivity, this assay approach holds great potential for convenient detection of other biomolecules such as alkaline phosphatase at low levels.

Optical characterization and tunable antibacterial properties of gold nanoparticles with common proteins

The interaction of three proteins, viz. Bovine Serum Albumin (BSA), Human Serum Albumin (HSA) and Hen Egg White Lysozyme (HEWL) with gold nanoparticles (GNPs) is investigated using surface plasmon resonance (SPR) spectroscopy, fluorescence spectroscopy and circular dichroism (CD). Size and morphology of the samples was established using Transmission Electron Microscopy (TEM) and stability studies was established using zeta potential analysis. The stability of protein-GNP complex was found to be greater than that of individual protein as well as individual GNPs. Also HEWL-GNP complex was more stable compared to the other protein complexes. Absorbance of proteins increases with increase in gold nanoparticle concentration due to the extension of peptide strands of protein and decrease in hydrophobicity of gold nanoparticles. A ground state complex is also formed which is evident from the moderate shift observed in the absorbance peaks. Apparent association constant was also determined from the absorption spectra and was found to be maximum for HEWL and minimum for HSA. Gold nanoparticles were found to act as quenchers and reduced the protein fluorescence intensity. Binding constant and number of binding sites were found to be maximum for HEWL and minimum for HSA. The temperature dependent fluorescence studies were also performed to calculate the thermodynamic parameters and to determine the nature of interaction between the proteins and gold nanoparticles. The circular dichroism studies elucidate the reason behind the maximum binding for HEWL and minimum binding for HSA. TGA analysis determined the thermal stability of the samples. Fluorescence lifetime studies indicate static quenching of proteins. Antibacterial activity of protein-gold nanoparticles was studied against four pathogens, viz. Bacillus pumilus, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. HEWL exhibits a tunable antimicrobial activity against Pseudomonas aeruginosa due to the maximum binding of HEWL with gold nanoparticles. The study proposes a novel method for adjusting the antibacterial activity of HEWL against Pseudomonas aeruginosa when the resistance of this pathogen is a major issue in the chemotherapy of many infectious diseases. Thus the combination therapy of protein-gold nanoparticles could prove to be a new approach in medical field in the near future.

Translesion synthesis genes POLI and REV1 in breast tumors and response to neoadjuvant chemotherapy.

e12642 Background: Translesion synthesis (TLS) of DNA by specialized polymerases (POLs) and REV1, a scaffold protein that recruits other TLS DNA POLs for nascent strand extension, experimentally enable tumor cells to replicate through DNA damage. TLS POLs are hypothesized to contribute to treatment resistance. Methods: We conducted a discovery gene expression study using NanoString technology with a bespoke codeset enriched for DNA damage response genes and RNA from 12 TN, 10 HER2+ and 29 ER+/HER2- breast cancer patients before neoadjuvant chemotherapy (NACT) and when available, in the matching residual tumor. Eleven patients achieved a pathologic complete response (pCR), 12 had < 1 cm residual disease (partial responders) and 28 patients had > 1cm residual disease (non-responders). RNA counts were normalized and log-transformed for differential expression analyses corrected for false discovery. Results: DNA polymerase Iota ( POLI) was the top overexpressed gene in the pretreatment biopsy of non-responders independent of tumor subtype (p corrected = 0.0002). Expression of REV1 was also significantly higher in non-responders and positively correlated with POLI (r = 0.577, p < 0.0001). Despite a small sample size, pretreatment expression levels of POLI and REV1 were significantly higher in ER+ (n = 38) than ER- (n = 13) tumors (p = 0.0006). When restricted to ER+ tumors, POL1 and REV1 expression levels were significantly higher in pretreatment biopsy of non-responders (n = 25) than partial and complete responders (n = 13). For ER+ patients, response by median POL1/REV1 expression levels are shown in Table. Pre/post treatment POL1 and REV1 expression did not change in patients with residual disease. Conclusions: In discovery, two genes involved in TLS of DNA (POLI and REV1) were found overexpressed in pretreatment biopsies from breast cancers poorly responsive to NACT, independent of tumor subtype. Overexpression was more common among ER+ tumors, which exhibit a high inherent resistance to chemotherapy. If confirmed, expressed levels of POLI and REV1 may identify a subgroup of breast cancers resistant to NACT for which targeted inhibition of mutagenic TLS may be beneficial. [Table: see text]

A New Enclosed Method for Transverse Mechanical Testing on CICC Conductors

CFETR, the China Fusion Engineering Test Reactor, is a new tokamak reactor under preliminary design, where the toroidal field coils were designed to generate a magnetic field of 14.3 T. Here the TF conductors are required to operate under a Lorentz force of 1200 kN/m while keeping stable performance. For these requirements, the main goal is to limit the degradation of the conductor. According to the previous study, the conductor can be analyzed with a mechanical method to evaluate the conductor-performance in the electromagnetic field. In this method, the force was distributed equally from the up and downside of the press modules, which has notches in the middle of the conduit. The stress distribution is quite different from the accumulated stress in the Lorentz force direction. Moreover, the strand extension has been found in the notches. Considering these conditions, a new method where the cable surrounded by an enclosed module was designed to simulate this kind of accumulative stress in the cable. The new module is defined as an enclosed jacket press method compared to the former method (jacket with notches) developed at the University of Twente. In this study, the cable's mechanical properties tested by two different modules were compared. The results were analyzed to evaluate the similarity of different press methods for EM simulation. The result shows that the cable pressed with an enclosed module jacket provided more similar deformation trends when comparing to the distribution of electromagnetic stress within the cable.

Coupling strand extension/excision amplification with target recycling enables highly sensitive and aptamer-based label-free sensing of ATP in human serum.

Detection of aberrant ATP concentrations with high sensitivity and selectivity is of critical importance for monitoring many biological processes and disease stages. By coupling extension/excision amplification with target recycling, we have established an aptamer-based method for label-free fluorescence ATP detection in human serum with high sensitivity. The ATP target molecules associate with the aptamer-containing double hairpin probes and cause conformational changes of the probes to initiate the cyclic strand extension/excision processes in the presence of polymerase, endonuclease and assistance sequences for the recycling of ATP and the production of a large number of G-quadruplex sequences. The organic dye thioflavin T subsequently binds these G-quadruplex sequences to yield substantially enhanced fluorescence emission for achieving highly sensitive detection of ATP down to 2.2 nM in the range of 5 to 200 nM without using any labels. The developed aptamer sensing method also exhibits high selectivity and allows the monitoring of ATP at low concentrations in diluted real samples, which offers promising opportunities to establish effective signal magnification means for the detection of various biomolecules at trace levels.

Target-dependent dual strand extension recycling amplifications for non-label and ultrasensitive sensing of serum microRNA

MicroRNAs (miRNAs) are important biomarkers because their abnormal expressions or mutations can usually indicate the development of cancers or other diseases. However, their high family sequence homology and low abundance pose a major challenge for the analysis of miRNAs. In the present work, we developed a dual strand extension recycling signal amplification strategy for non-label detection of miRNA-155 with high sensitivity on the basis of DNA polymerase and the G-quadruplex/thioflavin T (ThT) complexes. The specific miRNA-155 target sequences could initiate two strand extension-based independent recycling cycles under the function of the DNA polymerase, resulting in the production of many active G-quadruplex segments. The dye of ThT further bound the G-quadruplexes to induce greatly enhanced fluorescence for ultra-sensitively detecting miRNA-155 sequences at the low concentration of 35 fM in the dynamic range from 0.1 pM to 100 pM. The proposed detection strategy used the completely unmodified and synthetic DNA as probes and could be performed in homogeneous solutions. This method with a high selectivity could also be used for diluted serum samples. The demonstration of our new method for miRNA detection thus offers it with high potentials for miRNA biomarker analyses with the purpose of clinical diagnostics and biomedical applications.

Dual-Priming Isothermal Amplification (DAMP) for Highly Sensitive and Specific Molecular Detection with Ultralow Nonspecific Signals.

Nucleic acid amplification tests have been widely used in clinical diagnostics, food safety monitoring, and molecular biology. Loop-mediated isothermal amplification (LAMP) is a prevailing nucleic acid isothermal amplification method. It has become a powerful alternative to conventional polymerase chain reaction (PCR) for pathogen detection because of its simplicity, rapidity, and high sensitivity. However, the current LAMP methods, especially LAMP with two loop primers, suffer from undesired nonspecific amplification with strong background signals due to the increasing target sites. This nonspecific amplification substantially reduced the reliability of LAMP and limited its applications in clinical diagnostics. Here, we report a "dual-priming" ("self-priming" and "pairing-priming") isothermal amplification (DAMP) assay for rapid nucleic acid detection with ultralow nonspecific signals. This method takes advantage of the "dual-priming" strand extension strategy by adding two pairing-competition primers and designing unique inner primers, enabling highly sensitive and specific molecular detection. As an application demonstration, the DAMP assay was used to detect HIV-1 DNA/RNA and Escherichia coli DNA, showing equal or better sensitivity with shorter detection time compared to conventional LAMP and PCR methods. More importantly, the DAMP assay showed ultralow background signals without false positive signals even after a 2 h incubation. Such a simple, reliable, sensitive, and specific DAMP assay can be well suited for rapid nucleic acid detection as point-of-care testing, particularly in resource-limited settings.

Replication-Coupled DNA-Protein Crosslink Repair by SPRTN and the Proteasome in Xenopus Egg Extracts

SummaryDNA-protein crosslinks (DPCs) are bulky lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S phase removal of DPCs, but how SPRTN is targeted to DPCs during DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is partially dependent on the ubiquitin ligase activity of TRAIP. In contrast, SPRTN-mediated DPC degradation does not require DPC polyubiquitylation but instead depends on nascent strand extension to within a few nucleotides of the lesion, implying that polymerase stalling at the DPC activates SPRTN on both leading and lagging strand templates. Our results demonstrate that SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms that promote replication across immovable protein barriers.

Accurate calculation and measurement method for elongation of pre-stressed strand of Bridges

In the pre-stressed construction of bridge strands, tension stress is used as the control and strand elongation is used as the calibration and the elongation of strands directly affects the performance of the bridge structure. So, it must be very precise. In actual construction, due to inadequate grasp of basic theory by technicians, improper measurement methods or inaccurate selection of calculation formulas, neglecting the relationship between the actual elongation value and the tool/job clip withdrawal amount, resulting in the deviation between the actual measured value and the theoretical design value is beyond the standard, which is very unfavorable to the quality of the engineering structure. This article starts with the basic science theory, through numerical calculation and theoretical analysis, explains the work stress compensation retracting amount of the Work anchor and tool anchor, and the compensation stress calculation method due to the stress loss caused by the retraction. It shows the working principle of the pre-stressed equipment, and proposes and validates accurate calculation and measurement method of strand extension.

Sequence-Specific Solution NMR Assignments of the β-Barrel Insertase BamA to Monitor Its Conformational Ensemble at the Atomic Level.

β-barrel outer membrane proteins (Omps) are key functional components of the outer membranes of Gram-negative bacteria, mitochondria, and plastids. In bacteria, their biogenesis requires the β-barrel-assembly machinery (Bam) with the central insertase BamA, but the exact translocation and insertion mechanism remains elusive. The BamA insertase features a loosely closed gating region between the first and last β-strand 16. Here, we describe ∼70% complete sequence-specific NMR resonance assignments of the transmembrane region of the BamA β-barrel in detergent micelles. On the basis of the assignments, NMR spectra show that the BamA barrel populates a conformational ensemble in slow exchange equilibrium, both in detergent micelles and lipid bilayer nanodiscs. Individual conformers can be selected from the ensemble by the introduction of a C-terminal strand extension, single-point mutations, or specific disulfide cross-linkings, and these modifications at the barrel seam are found to be allosterically coupled to sites at the entire barrel circumference. The resonance assignment provides a platform for mechanistic studies of BamA at atomic resolution, as well as for investigating interactions with potential antibiotic drugs and partner proteins.

Human DNA polymerase η accommodates RNA for strand extension

Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During replication, DNA polymerases tolerate patches of ribonucleotides on the parental strands to different extents. The majority of human DNA polymerases have been reported to misinsert ribonucleotides into genomes. However, only PrimPol, DNA polymerase α, telomerase, and the mitochondrial human DNA polymerase (hpol) γ have been shown to tolerate an entire RNA strand. Y-family hpol η is known for translesion synthesis opposite the UV-induced DNA lesion cyclobutane pyrimidine dimer and was recently found to incorporate ribonucleotides into DNA. Here, we report that hpol η is able to bind DNA/DNA, RNA/DNA, and DNA/RNA duplexes with similar affinities. In addition, hpol η, as well as another Y-family DNA polymerase, hpol κ, accommodates RNA as one of the two strands during primer extension, mainly by inserting dNMPs opposite unmodified templates or DNA lesions, such as 8-oxo-2'-deoxyguanosine or cyclobutane pyrimidine dimer, even in the presence of an equal amount of the DNA/DNA substrate. The discovery of this RNA-accommodating ability of hpol η redefines the traditional concept of human DNA polymerases and indicates potential new functions of hpol η in vivo.