Structure and operation of the DNA-translocating type I DNA restriction enzymes

Abstract

Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes.

Structural and Functional Analysis of the Symmetrical Type I Restriction Endonuclease R.EcoR124INT

“…Our SANS results demonstrate that the subunit architecture and overall shape of the engineered, symmetrical endonuclease closely resembles the wild-type enzyme [12]. The two modified S NT subunits, corresponding to just over half an intact S subunit, are able to dimerise effectively via the coiled-coil interface to form a structure that is homologous to the native S subunit containing two TRD’s, but with dyad symmetry.…”

Impact of target site distribution for Type I restriction enzymes on the evolution of methicillin-resistant Staphylococcus aureus (MRSA) populations

“…The S subunit contains conserved regions (cr, orange) around the two TRDs (various colours). Ribbon cartoon models of each subunit of the EcoR124I Type I RM enzyme (13) with domains coloured as above are also shown. The EcoR124I amino acid sequences show homology with those of the S. aureus Type I RM enzymes (Supplementary Figures S3 and S4).…”

Crystal structure of a novel domain of the motor subunit of the Type I restriction enzyme EcoR124 involved in complex assembly and DNA binding

“…Another known crystal structure of HsdR from Vibrio vulnificus also lacks ~220 C-terminal residues and folds into three domains (endonuclease and two RecA-like helicases), providing no further insight into the 3D structure of the C-terminus. A structure of the full EcoR124I complex was derived using electron microscopy and small-angle scattering (neutron and X-ray) (26). However, the ring-like shape together with a resolution of only 21 Å unfortunately does not allow to orient HsdR in the EM map unambiguously.…”