Faithful cleavage of the P1 packaging site (pac) requires two phage proteins, PacA and PacB, and two Escherichia coli proteins, IHF and HU.
The PacA and PacB subunits of the bacteriophage P1 DNA packaging enzyme (pacase) are necessary for cleavage of the phage packaging site (pac). In the accompanying paper, we show that the PacA subunit of the enzyme specifically binds to pac in the absence of PacB, but requires factors present in an Escherichia coli extract to do so. We show here that either of two E. coli DNA binding proteins, integration host factor (IHF) or HU, can replace this extract and promote the binding of PacA to pac. IHF binds to pac independently of PacA and DNase I footprinting experiments show that IHF protects approximately 40 bp of DNA around an IHF consensus sequence adjacent to the cleavage site. DNase I footprinting experiments also show that in the presence of either IHF or HU, PacA binds to the hexanucleotide sequences (5'-TGATCA/G) that flank the cleavage site and that have been previously shown to be essential for pac cleavage. The importance of IHF and HU in pac cleavage is further demonstrated by the severe reduction in both the fidelity and efficiency of pac cleavage in vitro with extracts lacking both IHF and HU. Addition of either IHF or HU to the deficient extracts renders them fully proficient for pac cleavage. Finally, we show that IHF bends DNA at the IHF site within pac. Based on these results, we propose a model that can account for the role of the various phage and host proteins, and for DNA bending in the pac cleavage reaction.
Skorupski
K
K
DuPont Merck Pharmaceutical Co. Glenolden Laboratory, PA 19036.
Sauer
B
B
Sternberg
N
N
D001426:Bacterial Proteins / Q000378:metabolism*; D017101:Bacteriophage P1 / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004706:Endodeoxyribonucleases; D004926:Escherichia coli / Q000378:metabolism* / Q000821:virology; D034982:Integration Host Factors; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014764:Viral Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D014764:Viral Proteins; C040330:histone-like protein HU, bacteria; C073163:PacB protein, Enterobacteria phage P1; D004706:Endodeoxyribonucleases; C073162:pacase protein, Enterobacteria phage P1