FAITHFUL CLEAVAGE OF THE P1 PACKAGING SITE (PAC) REQUIRES 2 PHAGE PROTEINS, PACA AND PACB, AND 2 ESCHERICHIA-COLI PROTEINS, IHF AND HU

The PacA and PacB subunits of the bacteriophage P1 DNA packaging enzym e (pacase) are necessary for cleavage of the phage packaging site (pac ). In the accompanying paper, we show that the PacB subunit of the enz yme specifically binds to pac in the absence of PacB, but requires fac tors present in an Escherichia coli extract to do so. We show here tha t 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 footprintin g experiments show that IHF protects approximately 40 bp of DNA around an IHF consensus sequence adjacent to the cleavage site. DNase I foot printing experiments also show that in the presence of either IHF or H U, PacB 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 fur ther demonstrated by the severe reduction in both the fidelity and eff iciency 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 mode l that can account for the role of the various phage and host proteins , and for DNA bending in the pac cleavage reaction.