DpnI (pronounced "''D-P-N one''") is a Type IIM
restriction endonuclease
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
isolated from
''Streptococcus pneumonae'' (formerly ''Diplococcus pneumonae''). It recognizes and cuts
methylated DNA at the sequence G
m6A↓TC.
Structure
The structure of DpnI comprises an N-terminal catalytic domain and a C-terminal
winged helix DNA binding domain, both of which show specificity for the methylated GATC sequence. The catalytic domain is disordered in solution and becomes ordered upon binding DNA.
Uses in molecular biology
DpnI is commonly used to digest template DNA after
site-directed mutagenesis
Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenes ...
. Most strains of
''E. coli'' used in molecular biology express
Dam methylase, a protein that methylates DNA at the sequence GATC. Adding DpnI to the product of a PCR reaction digests only the template DNA, as the template DNA was isolated from ''E. coli'' and will have methylation at this sequence while the newly synthesized DNA will not. DpnI is widely available commercially, both alone and in "KLD" enzyme mixes containing
kinase
In biochemistry, a kinase () is an enzyme that catalyzes the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates. This process is known as phosphorylation, where the high-energy ATP molecule don ...
and
ligase
In biochemistry, a ligase is an enzyme that can catalyze the joining ( ligation) of two molecules by forming a new chemical bond. This is typically via hydrolysis of a small pendant chemical group on one of the molecules, typically resulting i ...
enzymes for treatment of site-directed mutagenesis reactions.
DpnI is also used to digest methylated GATC sequences in
DamID
DNA adenine methyltransferase identification, often abbreviated DamID, is a molecular biology protocol used to map the binding sites of DNA- and chromatin-binding proteins in eukaryotes. DamID identifies binding sites by expressing the proposed DNA ...
, a technique that uses Dam methylation combined with sequencing to identify protein-DNA interactions.
See also
*
DpnII restriction endonuclease family, also isolated from ''S. pneumonae''
*
List of restriction enzyme cutting sites
A restriction enzyme or restriction endonuclease is a special type of biological macromolecule that functions as part of the "immune system" in bacteria. One special kind of restriction enzymes is the class of " homing endonucleases", these bein ...
*
Restriction modification system
The restriction modification system (RM system) is found in bacteria and archaea, and provides a defense against foreign DNA, such as that borne by bacteriophages.
Bacteria have restriction enzymes, also called restriction endonucleases, which ...
References
{{reflist
Enzymes
Restriction enzymes
EC 3.1.21
Bacterial enzymes