The cleavage site for the restriction endonucleasesBanI andHgiC I is 5′ …GIGPyPuCC …3′

We have determined the cleavage site for the restriction endonucleases Ban I (1) and HgiC I to be Identical. Both cleaving the sequence 5'... GiGPyPuCC ...3' leaving a 4 base 5' extension. This is In contrast to Kroger et al (2), who reported the cleavage for HgiC I as 5'...iGGPyPuCC...3' leaVng a 6 base 5'extension. To determine the cleavage site of Ban I and HgiC I. the following experiment was performed: single-stranded M13 mpl8 DNA. which contains the Banl/HgiC I site In the the polylinker region (the Kpn I site), was used as a template for a primer extension reaction. This template and a M13 sequencing primer were Incubated with the Klenow fragment of E. coll DNA polymerase I, dGTP, dCTP, dTTP, and la-^S) dATP, such that the primer was extended through and beyond the Ban I site. Following elongation, the polymerase was Inactivated by heat treatment and the extended chains were cleaved with Ban I or HgiC I. To one half of the sample , an additional amount of DNA polymerase plus all four deoxynucleotide triphosphates were added (lane+). To the other half no additions were made (lane-). After further incubation , these samples were subject to electrophoresls on a DNA sequencing gel alongside the product of a set of standard dldeoxysequencing reactions which were produced with the same primer. Both Ban I and HgfC I treatment of the product of the initial DNA polymerase reaction (lane -) generates a fragment which co-migrates with the band in the G channel corresponding to the 5' G in the sequence GGTACC. Subseauent treatment of the Ban I or HaiC I cleaved product with DNA polymerase . __ removed the band co-migrating with the B 0 PI I n Q I C l G residue and generated a band four nucleotides longer co-mlgratlng with the A f* a. f i A T r + f i 5 C residue in the sequence GGTACC M ^ T o « • ^ ^ N* (lane + ), which corresponds to cleavage " between the two G residues on the unlabelled template strand. These results " ^ —— mm indicate that HgC I and Ban I cleave the sequence GGPyPuCC symmetrically be" * ^ M * * ^ W tween the two G residues on each strand » ^ ^ " to yield a four base 5' extension. The ^ ^ ,m ^m cleavage for both Ban I and HgC I is ^ ^ ^Z ^0 ^ 5'... GlGPyPuC C...3 • ' *~ 3'... C CPuPyGTG...5\ ttg