The DNA polymerase of bacteriophage YerA41 replicates its T-modified DNA in a primer-independent manner

Abstract

Yersinia phage YerA41 is morphologically similar to jumbo bacteriophages. The isolated genomic material of YerA41 could not be digested by restriction enzymes, and used as a template by conventional DNA polymerases. Nucleoside analysis of the YerA41 genomic material, carried out to find out whether this was due to modified nucleotides, revealed the presence of a ca 1 kDa substitution of thymidine with apparent oligosaccharide character. We identified and purified the phage DNA polymerase (DNAP) that could replicate the YerA41 genomic DNA even without added primers. Cryo-electron microscopy (EM) was used to characterize structural details of the phage particle. The storage capacity of the 131 nm diameter head was calculated to accommodate a significantly longer genome than that of the 145 577 bp genomic DNA of YerA41 determined here. Indeed, cryo-EM revealed, in contrast to the 25 Å in other phages, spacings of 33-36 Å between shells of the genomic material inside YerA41 heads suggesting that the heavily substituted thymidine increases significantly the spacing of the DNA packaged inside the capsid. In conclusion, YerA41 appears to be an unconventional phage that packages thymidine-modified genomic DNA into its capsids along with its own DNAP that has the ability to replicate the genome.

Figure 1.

Electron microscopy analysis of YerA41 virions. Panel A. Negatively stained electron micrographs, left and central panels are YerA41-B, and right panel is YerA41-S. White triangles indicate tail fibers. Scale bar 100 nm. Panel B. Cryo-electron microscopy icosahedral reconstruction of YerA41-B capsid, coloured radially. Circles indicate h = 4 and k = 2 values that describe the T = 28 quasi-symmetry. Square panel shows fitting of HK97 major capsid protein (PDB 1OHG), in blue, into the YerA41-B map in grey. Panel C. Left, cross-sectional view along the z-axis (rotated 90° with respect to panel A), coloured radially. Capsid proteins are in purple and cyan, internal capsid proteins in blue, and genomic layers are in green, yellow, orange and red. Bottom right, central slice through the reconstruction in greyscale. Top right, rotationally averaged central slice in greyscale. Panel D. Density profile of rotationally averaged central slice, corresponding to top right section of panel C, plotted against radial distance from centre of the capsid. Colours correspond to radial layers in panel C.

Figure 2.

DNAP01 shows DNA polymerase activity also with YerA41 genome even without adding external primers. Panel A. DNA polymerase activity of DNAP01 compared with T4 DNA polymerase, measured with a commercial assay for 60 min. Each line represents the mean of three replicates. Negative control is represented by water. Panel B. Standard curve calculated with different concentrations of T4 DNA polymerase with known activity. Dashed lines represent the 95% confidence interval of the trend line created with the regression model shown in the same graphic. Panels C and D. 1% agarose gels of PCR results of the functional assay performed with YerA41 genomic material. 1. gp8 primers; 2. gp37 primers 3. dnap02 primers. At the top, the reagents added or any special condition applied during the treatment with DNAP01 is shown. All those samples were then used as template for PCR with Dream-Taq polymerase, as described in the methods section, and shown here are the products of those PCRs. The sizes of the bands are given in bp.

Figure 3.

RNase A treatment does not affect activity of DNAP01, which produces enough extension even in 1 min. Panel A. Influence of RNase A treatment on DNAP01 activity. Panel B. Influence of incubation time on DNAP01 activity. In both panels, the obtained PCR products were analysed in 1% agarose gel electrophoresis. Above the gel images are indicated the added reagents and any special conditions applied to the DNAP01 incubation. Aliquots of the samples were used as templates for PCR with Dream-taq using gp8 primers, as described in the methods section. The sizes of the bands are given in bp, and the incubation times, in min.