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PMID:28009009
| Citation |
Wong, IN, Sayers, JR and Sanders, CM (2016) Bacteriophage T5 gene D10 encodes a branch-migration protein. Sci Rep 6:39414 |
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| Abstract |
Helicases catalyze the unwinding of double-stranded nucleic acids where structure and phosphate backbone contacts, rather than nucleobase sequence, usually determines substrate specificity. We have expressed and purified a putative helicase encoded by the D10 gene of bacteriophage T5. Here we report that this hitherto uncharacterized protein possesses branch migration and DNA unwinding activity. The initiation of substrate unwinding showed some sequence dependency, while DNA binding and DNA-dependent ATPase activity did not. DNA footprinting and purine-base interference assays demonstrated that D10 engages these substrates with a defined polarity that may be established by protein-nucleobase contacts. Bioinformatic analysis of the nucleotide databases revealed genes predicted to encode proteins related to D10 in archaebacteria, bacteriophages and in viruses known to infect a range of eukaryotic organisms. |
| Links |
PubMed PMC5180179 Online version:10.1038/srep39414 |
| Keywords |
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Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0008094: DNA-dependent ATPase activity |
ECO:0000315: |
F |
Figure 1b shows the mass of the eluted molecules to be consistent with those in DNA-dependent ATPase activity. Figure 1c shows enzymatic activity was only stimulated in the presence of duplex DNA. Figure 1d shows after cloning of D10 protein, D10-R389N (motif VI) was expressed and purified. When compared to wild type, a point mutation of the motif stopped all DNA-dependent ATPase activity. |
complete | ||||
| GO:1990163: ATP-dependent four-way junction helicase activity |
ECO:0000314: |
F |
The authors use a ATPase assay to determine if the helicase D10 can unwind complex branched DNA substrates. Figure 2b shows the ATP dependent unwinding of cruciform (four-way junction) DNA molecules by the T5 helicase gene D10. |
complete | ||||
| GO:0032508: DNA duplex unwinding |
ECO:0000314: |
P |
Figure 3 shows how d10 protein functions to unwind DNA. It uses partially homologous Holliday junction substrates and results in two products, including fork DNA and recombined duplex DNA. These gels prove that d10 can act as a DNA helicase. |
complete | ||||
Notes
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