Type IIB restriction-modification systems, such as BcgI, feature a single protein with both endonuclease and methyltransferase activities. Type IIB nucleases require two recognition sites and cut both strands on both sides of their unmodified sites. BcgI cuts all eight target phosphodiester bonds before dissociation. The BcgI protein contains A and B polypeptides in a 2:1 ratio: A has one catalytic centre for each activity; B recognizes the DNA. We show here that BcgI is organized as A2B protomers, with B at its centre, but that these protomers self-associate to assemblies containing several A2B units. Moreover, like the well known FokI nuclease, BcgI bound to its site has to recruit additional protomers before it can cut DNA. DNA-bound BcgI can alternatively be activated by excess A subunits, much like the activation of FokI by its catalytic domain. Eight A subunits, each with one centre for nuclease activity, are presumably needed to cut the eight bonds cleaved by BcgI. Its nuclease reaction may thus involve two A2B units, each bound to a recognition site, with two more A2B units bridging the complexes by protein–protein interactions between the nuclease domains.

The Type IIB restriction–modification protein BcgI contains A and B subunits in a 2:1 ratio: A has the active sites for both endonuclease and methyltransferase functions while B recognizes the DNA. Like almost all Type IIB systems, BcgI needs two unmethylated sites for nuclease activity; it cuts both sites upstream and downstream of the recognition sequence, hydrolyzing eight phosphodiester bonds in a single synaptic complex. This complex may incorporate four A2B protomers to give the eight catalytic centres (one per A subunit) needed to cut all eight bonds. The BcgI recognition sequence contains one adenine in each strand that can be N6-methylated. Although most DNA methyltransferases operate at both unmethylated and hemi-methylated sites, BcgI methyltransferase is only effective at hemi-methylated sites, where the nuclease component is inactive. Unlike the nuclease, the methyltransferase acts at solitary sites, functioning catalytically rather than stoichiometrically. Though it transfers one methyl group at a time, presumably through a single A subunit, BcgI methyltransferase can be activated by adding extra A subunits, either individually or as part of A2B protomers, which indicates that it requires an assembly containing at least two A2B units.

DISF is an RNA polymerase II (pol II) elongation factor. It can bind to pol II and regulates the synthesis of mRNA both positively and negatively. Biochemical analyses show that DISF can interfere the action of TFIIS, a mRNA cleaving enzyme at 3 0 end but stabilize the ternary complex when mRNA is greater than 35 nt. To resolve these puzzle, we set out to use various biophysical means, including single molecule FRET ( and electron microscopy reconstruction techniques, to characterize the complex formed by DISF and pol II. Here, we report a low resolution 3D structure of yeast pol II yeast pol II complexed with DISF and also smFRET constraints toward depicting the geometric relationship between mRNA and DISF.