Overexpression, purification, sequence analysis, and characterization of the T4 bacteriophage dda DNA helicase.
The bacteriophage T4 dda protein is a 5'-3' DNA helicase that stimulates DNA replication and recombination reactions in vitro and seems to play a role in the initiation of T4 DNA replication in vivo. Oligonucleotide probes based on NH2-terminal amino acid sequence were used to precisely map the location of the dda gene on the T4 chromosome. Using polymerase chain reaction techniques, the dda gene was then cloned into an expression vector, and the overproduced protein was purified in two chromatography steps. Both the genomic and cloned dda genes were sequenced and found to be identical, encoding a protein of 439 amino acids. The dda protein contains amino acid sequences resembling those of other known helicases, and is most homologous to the Escherichia coli recD protein. Protein affinity chromatography was used to show a direct interaction between the dda protein and the T4 uvsX protein (a rec A-type DNA recombinase).
Hacker
K J
KJ
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Alberts
B M
BM
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins
D004279:DNA, Viral; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D004265:DNA Helicases; C077401:dda DNA helicase protein, Bacteriophage T4