How the Bfi I restriction enzyme uses one active site to cut two DNA strands

Abstract

Unlike other restriction enzymes, BfiI functions without metal ions. It recognizes an asymmetric DNA sequence, 5 -ACTGGG-3 , and cuts top and bottom strands at fixed positions downstream of this sequence. Many restriction enzymes are dimers of identical subunits, with one active site for each DNA strand. Others, like FokI, dimerize transiently during catalysis. BfiI is also a dimer but it has only one active site, at the dimer interface. We show here that BfiI remains a dimer as it makes double-strand breaks in DNA and that its single active site acts sequentially, first on the bottom and then the top strand. Hence, after cutting the bottom strand, a rearrangement of either the protein and͞or the DNA in the BfiI-DNA complex must switch the active site to the top strand. Low pH values selectively block top-strand cleavage, converting BfiI into a nicking enzyme that cleaves only the bottom strand. The switch to the top strand may depend on the ionization of the cleaved 5 phosphate in the bottom strand. BfiI thus uses a mechanism for making double-strand breaks that is novel among restriction enzymes.S equence-specific cleavage of double-stranded DNA underpins many genetic events, including recombination, transposition, viral DNA integration, and the restriction of DNA (1-4). The enzymes that make double-strand breaks use many different strategies to achieve this end. The simplest may be the orthodox type II restriction enzymes such as EcoRI or EcoRV, dimers of identical subunits that recognize palindromic DNA sequences. In the presence of Mg 2ϩ , they cleave both strands at fixed positions in their recognition sites (3). They bind their target sites symmetrically, so that one active site from the dimer is positioned against one DNA strand and likewise the second active site on the other strand. Independent reactions in each active site then generate the double-strand break. Some homing endonucleases, such as I-CreI, also act in this way (4), as do the enzymes that resolve Holliday junctions (2).This strategy cannot apply to enzymes that recognize nonpalindromic sites and cut both strands away from the recognition site (3). For example, FokI, a type IIS restriction enzyme, recognizes the sequence 5Ј-GGATG-3Ј and cuts top and bottom strands 9 and 13 nt downstream of this site, respectively (5). FokI is a monomer with separate domains for DNA recognition and catalysis. The catalytic domain has a single active site, which is like that in each subunit of an orthodox enzyme, so the FokI monomer cannot cleave both DNA strands (6). To make doublestrand breaks, the monomer of FokI bound to its recognition site associates transiently with a second monomer to form a dimer with two active sites (7-9). Many type IIS enzymes operate in this way (10,11).An alternative to using a homodimeric enzyme to make double-strand breaks is an enzyme with different subunits. For example, the transposition of Tn7 requires two proteins, TnsA and TnsB, that each cleave one strand of the DNA (12). Interestingly, the structure of TnsA is similar...

Site-specific DNA transesterification catalyzed by a restriction enzyme

“…Buffers for reactions in Tris were: 0.3 M Tris-acetate, pH 8.3/0.3 M KCl/8 mM EDTA or 0.1 M Tris/0.1 M KCl/2.5 mM EDTA. The samples were collected at timed intervals and quenched with phenol/ chloroform or loading dye solution (25). Rapid DNA hydrolysis and transesterification reactions were studied in a quench-flow device (KinTek, Austin, TX): equal volumes of radiolabeled oligonucleotide (1 nM) and BfiI enzyme (50-100 nM dimer) were mixed and quenched with 1.0 M NaOH.…”

“…Separation of DNA hydrolysis and transesterification products was performed by denaturing PAGE as described in ref. 25. Radiolabeled DNA was detected and quantified by PhosphorImager (PerkinElmer, Wellesley, MA) (25).…”

“…25. Radiolabeled DNA was detected and quantified by PhosphorImager (PerkinElmer, Wellesley, MA) (25).…”

“…It first cuts the bottom strand 4 nt downstream from its recognition sequence, 5Ј-ACTGGG-3Ј. Then it slowly cuts the top strand at varied positions 5-7 nt downstream from the site (25). To study the reactions of BfiI, a minimal substrate, 14/15 (Table 1 and Fig.…”

“…BfiI has a single active site, which it uses sequentially to cut both DNA strands (24,25). It first cuts the bottom strand 4 nt downstream from its recognition sequence, 5Ј-ACTGGG-3Ј.…”