DpnI
{{Short description|Restriction enzyme}}
{{Infobox nonhuman protein
| Name = Type II Methyl-directed restriction enzyme DpnI
| Symbol = dpnC
| UniProt = P0A460
| Organism = Streptococcus pneumoniae
| ECnumber = 3.1.21.4
| PDB = 4ESJ
}}
DpnI (pronounced "D-P-N one") is a Type IIM restriction endonuclease isolated from Streptococcus pneumonae (formerly Diplococcus pneumonae). It recognizes and cuts methylated DNA at the sequence Gm6A↓TC.{{Cite journal |last1=Lacks |first1=S. |last2=Greenberg |first2=B. |date=1975-06-10 |title=A deoxyribonuclease of Diplococcus pneumoniae specific for methylated DNA |journal=The Journal of Biological Chemistry |volume=250 |issue=11 |pages=4060–4066 |doi=10.1016/S0021-9258(19)41386-0 |doi-access=free |issn=0021-9258 |pmid=236309}}{{Cite journal |last=Roberts |first=R. J. |date=2003-04-01 |title=A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes |journal=Nucleic Acids Research |volume=31 |issue=7 |pages=1805–1812 |doi=10.1093/nar/gkg274 |pmc=152790 |pmid=12654995}}
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.{{Cite journal |last1=Siwek |first1=Wojciech |last2=Czapinska |first2=Honorata |last3=Bochtler |first3=Matthias |last4=Bujnicki |first4=Janusz M. |last5=Skowronek |first5=Krzysztof |date=August 2012 |title=Crystal structure and mechanism of action of the N6-methyladenine-dependent type IIM restriction endonuclease R.DpnI |journal=Nucleic Acids Research |language=en |volume=40 |issue=15 |pages=7563–7572 |doi=10.1093/nar/gks428 |issn=1362-4962 |pmc=3424567 |pmid=22610857}}{{Cite journal |last1=Mierzejewska |first1=Karolina |last2=Siwek |first2=Wojciech |last3=Czapinska |first3=Honorata |last4=Kaus-Drobek |first4=Magdalena |last5=Radlinska |first5=Monika |last6=Skowronek |first6=Krzysztof |last7=Bujnicki |first7=Janusz M. |last8=Dadlez |first8=Michal |last9=Bochtler |first9=Matthias |date=2014-07-29 |title=Structural basis of the methylation specificity of R.DpnI |journal=Nucleic Acids Research |language=en |volume=42 |issue=13 |pages=8745–8754 |doi=10.1093/nar/gku546 |issn=0305-1048 |pmc=4117772 |pmid=24966351}}
Uses in molecular biology
DpnI is commonly used to digest template DNA after site-directed mutagenesis. 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.{{Cite journal |last1=Weiner |first1=Michael P. |last2=Costa |first2=Gina L. |last3=Schoettlin |first3=Warren |last4=Cline |first4=Janice |last5=Mathur |first5=Eric |last6=Bauer |first6=John C. |date=December 1994 |title=Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction |url=https://linkinghub.elsevier.com/retrieve/pii/0378111994906416 |journal=Gene |language=en |volume=151 |issue=1–2 |pages=119–123 |doi=10.1016/0378-1119(94)90641-6|pmid=7828859 }} DpnI is widely available commercially, both alone and in "KLD" enzyme mixes containing kinase and ligase enzymes for treatment of site-directed mutagenesis reactions.{{Cite web |title=KLD Enzyme Mix {{!}} NEB |url=https://www.neb.com/en-us/products/m0554-kld-enzyme-mix |access-date=2024-09-07 |website=New England Biolabs}}
DpnI is also used to digest methylated GATC sequences in DamID, a technique that uses Dam methylation combined with sequencing to identify protein-DNA interactions.{{Cite journal |last1=Aughey |first1=Gabriel N. |last2=Southall |first2=Tony D. |date=January 2016 |title=Dam it's good! DamID profiling of protein-DNA interactions |journal=Wiley Interdisciplinary Reviews. Developmental Biology |volume=5 |issue=1 |pages=25–37 |doi=10.1002/wdev.205 |issn=1759-7684 |pmc=4737221 |pmid=26383089}}{{Cite journal |last1=Aughey |first1=Gabriel N. |last2=Cheetham |first2=Seth W. |last3=Southall |first3=Tony D. |date=2019-03-15 |title=DamID as a versatile tool for understanding gene regulation |journal=Development |volume=146 |issue=6 |pages=dev173666 |doi=10.1242/dev.173666 |issn=0950-1991 |pmc=6451315 |pmid=30877125}}
See also
- DpnII restriction endonuclease family, also isolated from S. pneumonae
- List of restriction enzyme cutting sites
- Restriction modification system