CHROMOSOME END FORMATION IN PHAGE-LAMBDA, CATALYZED BY TERMINASE, IS CONTROLLED BY 2 DNA ELEMENTS OF COS, COSN AND R3, AND BY ATP

The terminase enzyme of phage lambda is a site-specific endonuclease t hat nicks DNA concatemers to regenerate the 12 nucleotide cohesive end s of the mature chromosome. The enzyme's DNA target, cos, consists of a nicking domain, cosN, and a binding domain, cosB. cosB, situated to the right of cosN, comprises three 16 bp repeat sequences, R1, R2 and R3. A similar sequence, R4, is present to the left of cosN. It is show n here that terminase has an intrinsic specificity for cosN which is i ndependent of the R sites. The interaction with cosN is mediated by bi nding to target sites that include 12 bp on the 5', and 2-7 bp on the 3' side of the nick. Of the four R sites, only R3 is required for the proper formation of ends. When R3 is present, an ATP-charged terminase system correctly catalyzes the production of staggered nicks in cosN, at sites N1 and N2 on the bottom and top strands, respectively. When ATP is omitted, the bottom strand is nicked incorrectly, at the site N x, 8 bp to the left of N1. If R3 is removed or disabled by a point mut ation, nicking in cosN becomes dependent upon ATP but, even in the pre sence of ATP, bottom strand nicking is divided between sites N1, the c orrect site, and Nx, the incorrect one. Thus, R3 is an important regul atory element and must reside in cis in respect to cosN. Furthermore, cosN substrates bearing point mutations at N1 and N2 are nicked at sit es Nx and Ny, 8 bp to the left of N1 and N2, respectively. When R3 is present and ATP is added, nicking is redirected to the N1 and N2 posit ions despite the mutations present. Thus, terminase binding to R3, on one side of cosN, regulates the rotationally symmetric nicking reactio ns on the bottom and top strands within cosN.