Introduction

Recently, we showed that a presumably triple-stranded DNA structure at the terminus of double-stranded DNA molecules, formed with deoxyoligonucleotides complementary to the 5′ terminus of one of the strands in the presence of RecA protein, is unusually stable even after removal of the protein (1). Formation of the triple-stranded structure was quite efficient, as approximately 80% of the specific termini of double-stranded DNA molecules were converted to this structure. Interestingly, the stable structure is not formed with deoxyoligonucleotides whose sequence is complementary to the 3′ terminal sequence of one of the strands (1).

Here we report that the triple-stranded DNA structure (2,3,4,5) can serve as a template for DNA polymerase reaction, which incorporates deoxynucleotides into one of the specific stands (complementary to the deoxyoligonucleotides), apparently replacing preexisting nucleotides in the strand with those of the substrate for DNA polymerase. Although the nature of the structure and the precise mechanism of the nucleotide incorporation are unknown, the unique strand specificity required to form such a stable structure and its ability to serve as a template for DNA polymerase reaction prompted us to study whether the finding can be used as a way to directly label a specific terminus of linear DNA molecules. Despite its various potentially useful application, such as in fixing DNA molecules to a solid support in a specific orientation, no simple and efficient method is currently available to label only one specific terminus of linear DNA molecules, particularly when one wants to directly label or modify DNA molecules of interest and/or to deal with DNA molecules of large molecular size for which PCR cannot be applied. Here we show that this approach indeed can be used for labeling (modifying) a specific terminus of DNA molecules directly regardless of its size and thus can be applied for fixing such DNA molecules to a solid support in a specific orientation.

Materials and Methods

Preparation of DNA and Deoxyoligonucleotides

To prepare linear DNA molecules, M13 mp 18RF DNA (Takara-Bio, Shiga, Japan) was digested by SnaBI or HincII, which produce a single linear DNA molecule with blunt ends. Deoxyoligonucleotides were custom-synthesized. 5′ end-labeled deoxyoligonucleotides labeled with ³²P were prepared using a MEGALABEL™ kit (Takara-Bio) and T4 polynucleotide kinase in the presence of [γ-³²P]ATP. Unincorporated [γ-³²P]ATP was removed by gel filtration (Sephadex® G50, GE-Healthcare, Piscataway, NJ, USA). The sequences of the deoxyoligonucleotide used here are shown in Table 1.

Table 1. Sequences of the Deoxyoligonucleotides Used (Click to enlarge)

Formation of Triple-Stranded Terminal Structure

Formation of triple-stranded terminal structure for DNA polymerase reaction was carried out by combining 20 µL reaction mixture A, containing a 10 pmol deoxyoligonucleotide, 3 µg RecA protein (Epicenter Technologies, Omaha, NE, USA), 0.48mM ATP-γS (Roche Diagnostics, Indianapolis, IN, USA), 1.0 mM magnesium-acetate, and 30 mM Tris-acetate, pH 7.2, with 20 µL reaction mixture B, containing 200 ng double-stranded DNA, 20 mM magnesium-acetate, and 30 mM Tris-acetate, pH 7.2, and the combined mixture was incubated for 30 min at 37°C. Reaction mixtures A and B had been preincubated for 5 min at 37°C prior to the reaction. After the reaction, 2.0 µL 10% (w/v) sodium dodecyl sulfate (SDS), 2.0 µL 0.5 M EDTA, pH 8.0, and 0.2 µL proteinase K (22 mg/mL; Roche Diagnostics) were added and incubated for 30 min at 37°C. Unincorporated deoxyoligonucleotides were removed by gel filtration through Sephacryl™ S-400 HR Micro columns (GE Healthcare).

Labeling of the Terminal Sequence

DNA (200 ng) with a terminally located structure with a 60-mer deoxyoligonucleotide formed as described above. Then 40 µL was diluted with 60 µL TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), subjected to phenol: chloroform:isoamyl alcohol (25:24:1, pH 7.9) treatment, and ethanol-precipitated after addition of 10 µL 3 M sodium-acetate and 1 µL glycogen (Roche Diagnostics). The precipitates were dried in vacuo, dissolved in 10 µL Milli-Q® water (Millipore, Billerica, MA, USA), and incubated with 2 U exo-Klenow fragment in a 30-µL reaction mixture that consisted of 10 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 7.5 mM dithiothreitol, 2 U Klenow fragment, 0.02 mM [α-³²P]dCTP, 0.02 mM dGTP, 0.02 mM dATP, and 0.02 mM dTTP. The mixture was then incubated for 15 min at 37°C. After addition of 10 µL TE buffer, excess [α-³²P]dCTP was removed by gel filtration over Sephadex G50. The sample was then subjected to electrophoresis through an agarose gel, stained with ethidium bromide, and autoradiographed using X-ray film (Hyperfilm-MP™; Eastman Kodak, Rochester, NY, USA).