M13 DNA Research Articles

Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

Analysis of Nucleoside Triphosphate Hydrolysis by Middle East Respiratory Syndrome Coronavirus Helicase

The purpose of this study is to measure the nucleoside triphosphate (NTP) hydrolysis by Middle East Respiratory Syndrome coronavirus (MERS‐CoV) helicase in the presence of different types of nucleic acids (NAs) for the determination of the optimal condition of hydrolysis activity. The steady‐state NTP hydrolysis analysis demonstrated that the MERS‐CoV helicase only (and considerably) hydrolyzed the ATP in the presence of poly(U). On the contrary, only UTP was considerably hydrolyzed in the presence of poly(C). When we used the DNAs as a substrate, the single‐stranded M13 DNA considerably stimulated the ATP and UTP hydrolysis. However, the ATP hydrolysis was not detected in the presence of ds DNA. The pH‐dependent ATP hydrolysis analysis demonstrated that the helicase optimally hydrolyzed ATP at a pH value of 7.0. The ATP hydrolysis was also performed in the presence of four different kinds of divalent cations, which demonstrated that only Mg2+ ions stimulated the ATP hydrolysis activity.

Strand Displacement and Unwinding Assays to Study the Concerted Action of the DNA Polymerase and SSB During Phi29 TP-DNA Replication.

The Bacillus subtilis phage Phi29 has a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5' end (TP-DNA). Phi29 single-stranded DNA-binding protein (SSB) is encoded by the viral gene 5 and binds the ssDNA generated during the Phi29 genome replication, stimulating the DNA elongation rate. Here, we describe some protocols to evaluate the effect of Phi29 SSB mutants on the DNA elongation rate and their unwinding activity during replication by Phi29 DNA polymerase using as substrate TP-DNA and also singly primed M13 DNA.

Quantification of single-strand DNA by sequence-specific counting in capillary flow cytometry

In this study, we report an approach to achieve sequence-specific counting of single DNA molecules, which is required for more versatile applications of the previously reported absolute DNA quantification technique based on flow cytometric DNA single molecule counting. While using the same capillary-based flow cytometric setup, fluorescence activation of a target DNA was made with a number of fluorescent oligonucleotide probes of complementary sequences to that of a target DNA. The feasibility of the proposed approach was tested with 7 kb single-strand M13 DNA as the target DNA for sequence specific counting for quantification. Sample preparation, the number of fluorescent oligonucleotide probes, and hybridization conditions mainly matter for the performance of the proposed method. Using a set of 30 sequence-specific fluorescent probes with a selected hybridization buffer, acceptable performance was confirmed through comparison with other conventional methods such as digital polymerase chain reaction (dPCR), UV spectrophotometry, and deoxyribonucleoside monophosphate analysis by mass spectrometry. Proven comparability to the dPCR method confirmed the feasibility of the proposed approach. With further improvement in instrumentation, the proposed method is expected to become established as a reference measurement procedure for sequence-specific quantification of nucleic acids working under a uniquely straightforward measurement principle.

Direct PCR detection, cloning, and characterization of fungi communities in soils and compost

A procedure was developed to detect and identify fungi in garden soil, forest soil, and compost samples. Microbial DNA was extracted using the Zymo Microbe DNA MiniPrep protocol. Fungi ribosomal internal transcribed space sequences (ITS) were amplified by PCR using primers ITS1 and ITS4. DNA fragments of approximately 640 base pair were detected in all positive samples. Clone libraries were constructed with amplified DNA by ligating the detected fragments with vector pCR®4-TOPO. Transformations were performed using competent Mix and Go Escherichia coli cells. Plasmids were isolated from each clone using the Zyppy plasmid miniprep protocol and inserts were screened by PCR using M13 DNA primers. Most of the identified fungal species were aligned to the phyla Ascomycota and Basidiomycota. BLAST analysis of clone libraries showed that sequences from compost samples were only comprised of the Ascomycota species Thermomyces lanuginosus. Clones from garden soils were mostly unidentified species closely aligned to Ascomycota and Zygomycota while the most abundant sequences in forest soils were related to the Basidiomycota species Cortinarius flexipes. In garden soil more than 50% of ITS sequences belonged to the Kingdom Plantae with species such as Cardamine hirsute, Stellaria media, and Cerastium dinaricum. However, there were no plant ITS sequences in compost and forest soil. Fungal species belonging to the phylum Ascomycota were widely distributed in the 3 environments studied with the forest soil showing the highest fungal diversity.

Jenis – jenis tumbuhan paku (Pteridofita) dari Hutan Universitas Riau, Provinsi Riaudan Pola Pita DNA berdasarkan Penanda DNA M13Primer

Kawasan hutan di Universitas Riau, Provinsi Riau merupakan salah satu daerah distribusi tumbuhan paku (Pteridofita). Kajian ini bertujuan mengidentifikasi jenis-jenis tumbuhan paku di Hutan Universitas Riau dan mengetahui pola pita DNA menggunakan penanda DNA universal M13. Sampel dikoleksi dari lapangan menggunakan metode eksplorasi dan kemudian diidentifikasi. Isolasi DNA menggunakan DNeasy Plant Mini Kit (Qiagen), dan diamplifikasi berdasarkan metode PCR menggunakan DreamTaq Green PCR Master Mix (2X) (Thermo Scientific) denganpenanda DNA M13 (GAGGGTGGCGGTTCT),. Hasil inventarisasi menunjukan 26 jenis paku dari 8 famili telah diidentifikasi pada kajian ini. Hasilelektroforesis PCR menunjukanbahwa pola pita DNA yang dihasilkan bervariasi dengan ukuran berkisar antara 200 bp sampai 2000 bp. Pola pita DNA menggunakan penanda universal M13 dapat dijadikan data pendukung dalam taksonomi tumbuhan paku.

DNA Origami as Seeds for Promoting Protein Crystallization.

This study reports the first experimental evidence of DNA origami as a seed resulting in the increase in probability of protein crystallization. Using the DNA origami constructed from long single-stranded M13 DNA scaffolds folded with short single-stranded DNA staples, it was found that the addition of the DNA origami in concentrations of 2-6 nM to mixtures of a well-characterized protein (catalase) solution (1.0-7.0 mg/mL) resulted in a higher proportion of mixtures with successful crystallization, up to 11× greater. The improvement in crystallization is evident particularly for mixtures with low concentrations of catalase (<5 mg/mL). DNA origami in different conformations of a flat rectangular sheet and a tubular hollow cylinder were examined. Both conformations improved the crystallization as compared to control experiments without M13 DNA or nonfolded M13 DNA but exhibited little difference in the extent of protein crystallization improvement. This work confirms the predictions of the potential use of DNA origami to promote protein crystallization, with potential application to systems with limited protein availability or difficulty in crystallization.

Quantification of M13 and T7 bacteriophages by TaqMan and SYBR green qPCR

TaqMan and SYBR Green quantitative PCR (qPCR) methods were developed as DNA-based approaches to reproducibly enumerate M13 and T7 phages from phage display selection experiments individually and simultaneously. The genome copies of M13 and T7 phages were quantified by TaqMan or SYBR Green qPCR referenced against M13 and T7 DNA standard curves of known concentrations. TaqMan qPCR was capable of quantifying M13 and T7 phage DNA simultaneously with a detection range of 2.75*101–2.75*108genome copies(gc)/μL and 2.66*101–2.66*108 genome copies(gc)/μL respectively. TaqMan qPCR demonstrated an efficient amplification efficiency (Es) of 0.97 and 0.90 for M13 and T7 phage DNA, respectively. SYBR Green qPCR was ten-fold more sensitive than TaqMan qPCR, able to quantify 2.75–2.75*107gc/μL and 2.66*101–2.66*107gc/μL of M13 and T7 phage DNA, with an amplification efficiency Es of 1.06 and 0.78, respectively. Due to its superior sensitivity, SYBR Green qPCR was used to enumerate M13 and T7 phage display clones selected against a cell line, and quantified titers demonstrated accuracy comparable to titers from traditional double-layer plaque assay. Compared to enzyme linked immunosorbent assay, both qPCR methods exhibited increased detection sensitivity and reproducibility. These qPCR methods are reproducible, sensitive, and time-saving to determine their titers and to quantify a large number of phage samples individually or simultaneously, thus avoiding the need for time-intensive double-layer plaque assay. These findings highlight the attractiveness of qPCR for phage enumeration for applications ranging from selection to next-generation sequencing (NGS).

Preparation of Double-Stranded (Replicative Form) Bacteriophage M13 DNA.

The double-stranded, closed-circular, replicative form (RF) of M13 DNA is present in high copy numbers in infected cells, and its physical characteristics are essentially identical to those of closed-circular plasmid DNAs. Any of the methods commonly used to purify plasmid DNA can therefore be used to isolate M13 RF DNA. This protocol describes the isolation of M13 RF DNA by alkaline lysis from small volumes (1-2 mL) of infected bacterial cultures. The yield of DNA (1-4 mg, depending on the size of the M13 clone) is more than enough for most purposes in molecular cloning. However, should more DNA be needed, the procedure can easily be scaled up.

Preparation of Single-Stranded Bacteriophage M13 DNA by Precipitation with Polyethylene Glycol.

Bacteriophage M13 single-stranded DNA is prepared from virus particles secreted by infected bacteria into the surrounding medium. Several methods are available to purify the polymorphic filamentous particles. In this protocol, the particles are concentrated by precipitation with polyethylene glycol (PEG) in the presence of high salt. Subsequent extraction with phenol releases the single-stranded DNA, which is then collected by precipitation with ethanol. The resulting preparation is pure enough to be used as a template for DNA sequencing. A yield of 5-10 µg of single-stranded DNA/mL of infected cells may be expected from recombinant bacteriophages bearing inserts of 300-1000 nt.

Guiding the Self-Assembly of RecA Proteinfilaments on DNA Scaffolds to Create Rationally Designed Nanostructures

Over the past three decades DNA self-assembly has been used to create increasingly complex nanoscale structures. DNA nanotubes with tunable persistence length have provided an attractive model system for protein filaments and structures with rationally defined nanoscale shapes, made using the “DNA origami” technique are promising for applications such as drug delivery and nano-plasmonics. However, the scale-up necessary for many DNA origami applications remains prohibited by cost and less than perfect yields. We seek to combine the ability of rational design afforded by DNA origami with the cost-efficient, high yield assembly of protein filaments to enable the construction of nanostructures with new functionalities. For a protein to be used in combination with DNA nanostructures, it must interact with DNA in a controllable way. RecA protein is an ideal choice because of its ability to form rigid filaments on DNA. Here we demonstrate the construction of a nanostructures including tetrahedra, rectangular shapes as well as micrometer scale filaments from DNA origami and RecA protein. We first fold specific sections of M13 single-stranded DNA, using the DNA origami technique, to create a mechanically flexible skeleton. Unfolded sections of the M13 DNA sequence are made double-stranded using a DNA polymerase gap-filling reaction. This skeleton is then rigidified by the addition of RecA, which self-assembles into filaments of defined length at specific double-stranded DNA target sites. Our method greatly improves upon conventional DNA origami in several ways. By reducing the number of distinct input components, we not only reduce cost but also the number of possible mis-folding events, allowing for higher yield nanostructures. Furthermore, the direct incorporation of proteins with origami adds a new level of functionality, thus broadening the scope of potential origami applications.

Genetic diversity in Iranian populations of Togninia minima, one of the causal agents of leaf stripe disease on grapevines

Background: Togninia minima Is the main fungal species commonly associated with grapevine leaf stripe disease (GLSD), worldwide. This species Is best known by Its conldlal state In nature. No data are available on the Incidence of an active sexual cycle within the populations of this species In many grapevine producing countries Including Iran. Material and Methods: Genetic variability within and among the populations of T. minima was analyzed using M13 DNA markers. Fifty one T. minima Isolates originating from symptomatic grapevines In north and northwestern regions of Iran were subjected to polymorphism analysis using M13 markers, namely M13mp 18F, M13mp 18R and M13 mlnlsatelllte. Correlation between the genetic similarity, the geographic origin and mating Identity of the Isolates was evaluated. Results: M13 mlnlsatelllte produced the 38 polymorphic bands, while M13 mp 18F and M13 mp 18R yielded 27 and 26 bands, respectively. Significant genetic distances were found among the Isolates from the same location and different parts of the country. No correlation was found among the genetic similarity groups with geographic origin and mating Identity of the Isolates. Isolates with different mating Identity sometimes clustered In the same group. Conclusion: Our data support the presence of a sexual cycle and the ongoing recombination within populations of the species In the sampled areas.

Single Molecule Force Spectroscopy of Hair-Cell Tip-Link Proteins

Mechanical stimuli from sound and head movements are converted into electrical signals by hair cells of the inner ear. Key to this process are tip links, fine protein filaments that convey mechanical force to hair-cell transduction channels. Tip links are made of two atypical cadherins: protocadherin-15 and cadherin-23, positioned at the lower and upper ends respectively. The tip link is held together by a non-covalent interaction between the N-termini of these two proteins. The crystal structure of the interacting domain has been solved, and molecular dynamics simulations have provided an estimate of the force it can withstand. However the lack of adequate technology has prevented direct measurement of the unbinding force.We have developed molecular tools to facilitate single-molecule force spectroscopy of tip-link proteins, based on self-assembled DNA nanoswitches (Halvorsen et al., 2011). The tips of protocadherin-15 and cadherin-23 are enzymatically conjugated to DNA oligos, which bind in selected locations to a single-stranded M13 DNA scaffold. The remaining single-stranded M13 is hybridized to complementary DNA oligomers. Once assembled, the nanoswitches are used in an optical tweezer system to measure the rupture forces of the tip link.Measured in this way, the unbinding force is about 35 piconewtons, a value consistent with extrapolations of the molecular dynamics simulations to slower loading rates, but disconcertingly low compared to what tip links must withstand physiologically. Tip links, however, have two strands, and thus two interaction domains in close proximity. Calculations suggest that this arrangement greatly increases the average lifetime of a tip link.

Interaction of Kaposi's sarcoma-associated herpesvirus ORF6 protein with single-stranded DNA.

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF6 is homologous to the herpes simplex virus 1 (HSV-1) ICP8 and Epstein-Barr virus (EBV) BALF2 proteins. Here, we describe its single-stranded DNA (ssDNA) binding properties. Based on previous findings with ICP8 and BALF2, a 60-amino-acid C-terminal deletion mutant of Orf6 was generated, and the protein was purified to explore the function of the C terminus in ssDNA binding. We showed that full-length ORF6 binds cooperatively to M13 ssDNA, disrupting its secondary structure and extending it to a length equivalent to that of duplex M13 DNA. The width of the ORF6-ssDNA filament is 9 nm, and a 7.3-nm repeat can be distinguished along the filament axis. Fluorescence polarization analysis revealed that the wild-type and C-terminal mutant ORF6 proteins bind equally well to short ssDNA substrates, with dissociation constant (Kd) values of 2.2 × 10(-7)M and 1.5 × 10(-7)M, respectively. These values were confirmed by electrophoretic mobility shift assay (EMSA) analysis, which also suggested that binding by the full-length protein may involve both monomers and small multimers. While no significant difference in affinities of binding between full-length ORF6 and the C-terminal deletion mutant were observed with the short DNAs, binding of the C-terminal mutant protein to M13 ssDNA showed a clear lack of cooperativity as seen by electron microscopy (EM). Incubation of a duplex DNA containing a long single-stranded tail with double-helical ORF6 protein filaments revealed that the ssDNA segment can be enveloped within the protein filament without disrupting the filament structure. This work describes the biochemical characterization of the single-stranded DNA binding protein of KSHV, ORF6, central to viral DNA replication in infected cells. A C-terminal deletion mutant protein was generated to aid in understanding the role of the C terminus in DNA binding. Here we analyze the binding of the wild-type and mutant proteins to short oligomeric and longer genomic ssDNA substrates. Although it is capable of interacting with the short substrates, the inability of mutant ORF6 to form oligomers in solution hindered it from fully covering the long genomic substrates. We previously showed that ORF6 forms long filaments in solution, and we showed here that these can absorb ssDNA without disruption of the filament structure. This work will provide an important basis for future studies by us and/or others.

Expression, purification, and enzymatic characterization of Bombyx mori nucleopolyhedrovirus DNA polymerase

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms. BmNPV DNA polymerase (Bm-DNAPOL), encoded by the ORF53 gene, plays a central role in viral DNA replication. In this work, a His-tagged Bm-DNAPOL fusion protein, constructed using a novel MultiBac expression system, was overexpressed in Sf-9 insect cells, purified to near homogeneity on Ni-NTA agarose beads and further purified by ion-exchange chromatography. About 0.4 mg of enzyme was obtained from about 1 × 10(9) infected Sf-9 cells in suspension culture. Characterization of the highly purified enzyme indicated that Bm-DNAPOL is a monomer with an apparent molecular mass of approximately 110,000 Da. It possessed a specific activity of 15,126.3 U/mg under optimal in vitro reaction conditions and behaved in the manner of a proliferating cell nuclear antigen (PCNA)-independent DNA polymerase on both poly(dA)/oligo(dT) primer/template and singly premiered M13 DNA. BmNPV viral replication may be independent of replication factor C and a PCNA complex, while single-stranded DNA binding protein might play an important role in BmNPV DNA replication. These findings will be significant in studies on BmNPV-based disease in silkworms and for using silkworms as a bioreactor for the production of biomolecules of commercial importance.

Thioredoxin, the Processivity Factor, Sequesters an Exposed Cysteine in the Thumb Domain of Bacteriophage T7 DNA Polymerase

Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase. It achieves processivity by binding to Escherichia coli thioredoxin (trx). gp5/trx complex binds tightly to a primer-DNA template enabling the polymerization of hundreds of nucleotides per binding event. gp5 contains 10 cysteines. Under non-reducing condition, exposed cysteines form intermolecular disulfide linkages resulting in the loss of polymerase activity. No disulfide linkage is detected when Cys-275 and Cys-313 are replaced with serines. Cys-275 and Cys-313 are located on loop A and loop B of the thioredoxin binding domain, respectively. Replacement of either cysteine with serine (gp5-C275S, gp5-C313S) drastically decreases polymerase activity of gp5 on dA(350)/dT(25). On this primer-template gp5/trx in which Cys-313 or Cys-275 is replaced with serine have 50 and 90%, respectively, of the polymerase activity observed with wild-type gp5/trx. With single-stranded M13 DNA as a template gp5-C275S/trx retains 60% of the polymerase activity of wild-type gp5/trx. In contrast, gp5-C313S/trx has only one-tenth of the polymerase activity of wild-type gp5/trx on M13 DNA. Both wild-type gp5/trx and gp5-C275S/trx catalyze the synthesis of the entire complementary strand of M13 DNA, whereas gp5-C313S/trx has difficulty in synthesizing DNA through sites of secondary structure. gp5-C313S fails to form a functional complex with trx as measured by the apparent binding affinity as well as by the lack of a physical interaction with thioredoxin during hydroxyapatite-phosphate chromatography. Small angle x-ray scattering reveals an elongated conformation of gp5-C313S in comparison to a compact and spherical conformation of wild-type gp5.

Characterization of Human DNA Polymerase Delta and Its Subassemblies Reconstituted by Expression in the Multibac System

Mammalian DNA polymerase δ (Pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and DNA repair processes. We have reconstituted human Pol δ complexes in insect cells infected with a single baculovirus into which one or more subunits were assembled. This system allowed for the efficient expression of the tetrameric Pol δ holoenzyme, the p125/p50 core dimer, the core+p68 trimer and the core+p12 trimer, as well as the p125 catalytic subunit. These were isolated in milligram amounts with reproducible purity and specific activities by a highly standardized protocol. We have systematically compared their activities in order to gain insights into the roles of the p12 and p68 subunits, as well as their responses to PCNA. The relative specific activities (apparent k cat) of the Pol δ holoenzyme, core+p68, core+p12 and p125/p50 core were 100, 109, 40, and 29. The corresponding apparent K d's for PCNA were 7.1, 8.7, 9.3 and 73 nM. Our results support the hypothesis that Pol δ interacts with PCNA through multiple interactions, and that there may be a redundancy in binding interactions that may permit Pol δ to adopt flexible configurations with PCNA. The abilities of the Pol δ complexes to fully extend singly primed M13 DNA were examined. All the subassemblies except the core+p68 were defective in their abilities to completely extend the primer, showing that the p68 subunit has an important function in synthesis of long stretches of DNA in this assay. The core+p68 trimer could be reconstituted by addition of p12.

A simple and sensitive method for determining the strand orientation of single-stranded viral genomes

Determining the nature of the viral genome is one of the first steps in characterization of any new virus. However, in the case of viruses with a single-stranded genome, it is not always simple to identify its orientation. In this study, a rapid, sensitive and simple PCR-based method is described to identify the strand orientation of single-stranded viral genomes. This method has been tested on the single-stranded DNA viruses, M13 and phiX174.

Role of damage-specific DNA polymerases in M13 phage mutagenesis induced by a major lipid peroxidation product trans-4-hydroxy-2-nonenal

One of the major lipid peroxidation products trans-4-hydroxy-2-nonenal (HNE), forms cyclic propano- or ethenoadducts bearing six- or seven-carbon atom side chains to G>C≫A>T. To specify the role of SOS DNA polymerases in HNE-induced mutations, we tested survival and mutation spectra in the lacZα gene of M13mp18 phage, whose DNA was treated in vitro with HNE, and which was grown in uvrA−Escherichia coli strains, carrying one, two or all three SOS DNA polymerases. When Pol IV was the only DNA SOS polymerase in the bacterial host, survival of HNE-treated M13 DNA was similar to, but mutation frequency was lower than in the strain containing all SOS DNA polymerases. When only Pol II or Pol V were present in host bacteria, phage survival decreased dramatically. Simultaneously, mutation frequency was substantially increased, but exclusively in the strain carrying only Pol V, suggesting that induction of mutations by HNE is mainly dependent on Pol V. To determine the role of Pol II and Pol IV in HNE induced mutagenesis, Pol II or Pol IV were expressed together with Pol V. This resulted in decrease of mutation frequency, suggesting that both enzymes can compete with Pol V, and bypass HNE-DNA adducts in an error-free manner. However, HNE-DNA adducts were easily bypassed by Pol IV and only infrequently by Pol II.Mutation spectrum established for strains expressing only Pol V, showed that in uvrA− bacteria the frequency of base substitutions and recombination increased in relation to NER proficient strains, particularly mutations at adenine sites. Among base substitutions A:T→C:G, A:T→G:C, G:C→A:T and G:C→T:A prevailed.The results suggest that Pol V can infrequently bypass HNE-DNA adducts inducing mutations at G, C and A sites, while bypass by Pol IV and Pol II is error-free, but for Pol II infrequent.

Production of Recombinant Human DNA Polymerase Delta in a Bombyx mori Bioreactor

Eukaryotic DNA polymerase δ (pol δ) plays a crucial role in chromosomal DNA replication and various DNA repair processes. It is thought to consist of p125, p66 (p68), p50 and p12 subunits. However, rigorous isolation of mammalian pol δ from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. While recombinant pol δ isolated from infected insect cells have some problems of consistency in the quality of the preparations, and the yields are much lower. To address these deficiencies, we have constructed recombinant BmNPV baculoviruses using MultiBac system. This method makes the generation of recombinant forms of pol δ containing mutations in any one of the subunits or combinations thereof extremely facile. From about 350 infected larvae, we obtained as much as 4 mg of pol δ four-subunit complex. Highly purified enzyme behaved like the one of native form by rigorous characterization and comparison of its activities on poly(dA)/oligo(dT) template-primer and singly primed M13 DNA, and its homogeneity on FPLC gel filtration. In vitro base excision repair (BER) assays showed that pol δ plays a significant role in uracil-intiated BER and is more likely to mediate LP BER, while the trimer lacking p12 is more likely to mediate SN BER. It seems likely that loss of p12 modulates the rate of SN BER and LP BER during the repair process. Thus, this work provides a simple, fast, reliable and economic way for the large-scale production of human DNA polymerase δ with a high activity and purity, setting up a new platform for our further research on the biochemical properties of pol δ, its regulation and the integration of its functions, and how alterations in pol δ function could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability.