Restriction endonucleases that resemble a component of the bacterial DNA repair machinery

Abstract

It has long been known that most Type II restriction endonucleases share a conserved core fold and similar active-sites. The same core folding motif is also present in the MutH protein, a component of the bacterial DNA mismatch repair machinery. In contrast to most Type II restriction endonucleases, which assemble into functional dimers and catalyze double-strand breaks, MutH is a monomer and nicks hemimethylated DNA. Recent biochemical and crystallographic studies demonstrate that the restriction enzymes BcnI and MvaI share many additional features with MutH-like proteins, but not with most other restriction endonucleases. The structurally similar monomers all recognize approximately symmetric target sequences asymmetrically. Differential sensitivities to slight substrate asymmetries, which could be altered by protein engineering, determine whether the enzymes catalyze only single-strand nicks or double-strand breaks.

The Type II restriction endonuclease MvaI has dual specificity

“…Further support for the nicking mechanism comes from observations with MvaI, BcnI and the monomeric DNA mismatch repair protein MutH. There is structural similarity between the three proteins (15), and MutH was shown to be a nicking enzyme, making single-strand scissions on the unmethylated strand of hemimethylated Gm6ATC sites (29). Against this background, the nicking activity of MvaI detected in this study is not surprising.…”

Degenerate sequence recognition by the monomeric restriction enzyme: single mutation converts BcnI into a strand-specific nicking endonuclease

“…In the first structure (accession code 2ODI), the enzyme is bound to the DNA exclusively in the orientation that brings the C-strand (5′-CC C GG-3′) close to the active site (8). Nearly all H-bond donor and acceptor sites at the central C:G base pair are involved in H-bond interactions with amino acid residues of BcnI.…”

“…In the major groove, the Nε atom of H77 forms a hydrogen bond with the N4 atom of cytosine while the Nε atom of H219 forms a hydrogen bond with the O6 atom of guanine (Figure 1A, bottom). As the Nδ atoms of both H77 and H219 are in contact with H-bond acceptors, the assignment implies that H77 is neutral while H219 is in the charged form (8). …”

“…The H-bond network to the central base pair in the minor groove is nearly fully conserved in both binding orientations. In the major groove, however, the change in the central base pair brings a proton acceptor (N7 of guanine) close to H77 and a proton donor (N4 of cytosine) close to H219 (8). To accommodate the different pattern of proton donors and acceptors in the alternative binding mode, H219 residue most likely becomes deprotonated, while the heterocyclic ring of H77 rotates by 180° to make a H-bond between its Nε and the N7 atom of the central guanine (Figure 1A, bottom).…”

SssI DNS metiltranszferáz által katalizált citozin dezamináció

“…Ismert ugyanis, hogy a reakció sebessége a hőmérséklet és a biszulfit koncentráció emelésével jelentősen növelhető (Hayatsu, 2008;Hayatsu és mtsai., 1970Hayatsu és mtsai., , 2004Shiraishi és Hayatsu, 2004;Sono és mtsai., 1973 Sokolowska et al, 2007;Xu et al, 2004;Yang, 2005). Mind a négy enzim palindróm, vagy közel palindróm szubsztráthellyel rendelkezik, melyeket aszimmetrikusan ismernek fel, kapcsolatot létesítve a DNS mindkét szálával.…”