Bacillus subtilis bacteriophage SPP1 G40P helicase lacking the n-terminal domain unwinds DNA bidirectionally.
Bacillus subtilis bacteriophage SPP1 G40P hexameric replicative DNA helicase unidirectionally translocates with a 5'-->3' polarity while separating the DNA strands. A G40P mutant derivative lacking the N-terminal domain (containing amino acid residues 110-442 from G40P, G40PDeltaN109) was purified and characterized. G40PDeltaN109 showed an ATPase activity that was dependent on the presence of single-stranded (ss) DNA. Unlike G40P, G40PDeltaN109 was shown to bind with similar affinity both ssDNA arms of forked structures by nuclease protection assays. In a pH-dependent manner, G40PDeltaN109 unwound a branched double-arm substrate preferentially with a 3'-->5' polarity. Our results show that the linker region and the C-terminal domain of G40P are sufficient to render an enzyme capable of encircling the ssDNA tails of the forked DNA and to unwind DNA with both 5'-->3' and 3'-->5' polarity. The presence of the N-terminal domain, which does not play an essential role in helicase action, might be required indirectly for strand discrimination and polarity of translocation.
Mesa
Pablo
P
Department of Microbial Biotechnology, Centro Nacional de Biotecnología, CSIC, Darwin 3, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Alonso
Juan C
JC
Ayora
Silvia
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D001412:Bacillus subtilis; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D009690:Nucleic Acid Conformation; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D004265:DNA Helicases; C115901:G40P helicase protein, Bacteriophage SPP1