| Type II Methyl-directed restriction enzyme DpnI | |||||||
|---|---|---|---|---|---|---|---|
| Identifiers | |||||||
| Organism | |||||||
| Symbol | dpnC | ||||||
| PDB | 4ESJ | ||||||
| UniProt | P0A460 | ||||||
| Other data | |||||||
| EC number | 3.1.21.4 | ||||||
| |||||||
DpnI (pronounced "D-P-N one") is a Type IIM restriction endonuclease isolated from Streptococcus pneumoniae (formerly Diplococcus pneumonae). It recognizes and cuts methylated DNA at the sequence Gm6A↓TC.12
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.34
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.5 DpnI is widely available commercially, both alone and in "KLD" enzyme mixes containing kinase and ligase enzymes for treatment of site-directed mutagenesis reactions.6
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.78
See also
See also
- DpnII restriction endonuclease family, also isolated from S. pneumonae
- List of restriction enzyme cutting sites
- Restriction modification system
References
References
- Lacks, S.; Greenberg, B. (1975-06-10). "A deoxyribonuclease of Diplococcus pneumoniae specific for methylated DNA". The Journal of Biological Chemistry. 250 (11): 4060–4066. doi:10.1016/S0021-9258(19)41386-0. ISSN 0021-9258. PMID 236309.
- Roberts, R. J. (2003-04-01). "A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes". Nucleic Acids Research. 31 (7): 1805–1812. doi:10.1093/nar/gkg274. PMC 152790. PMID 12654995.
- Siwek, Wojciech; Czapinska, Honorata; Bochtler, Matthias; Bujnicki, Janusz M.; Skowronek, Krzysztof (August 2012). "Crystal structure and mechanism of action of the N6-methyladenine-dependent type IIM restriction endonuclease R.DpnI". Nucleic Acids Research. 40 (15): 7563–7572. doi:10.1093/nar/gks428. ISSN 1362-4962. PMC 3424567. PMID 22610857.
- Mierzejewska, Karolina; Siwek, Wojciech; Czapinska, Honorata; Kaus-Drobek, Magdalena; Radlinska, Monika; Skowronek, Krzysztof; Bujnicki, Janusz M.; Dadlez, Michal; Bochtler, Matthias (2014-07-29). "Structural basis of the methylation specificity of R.DpnI". Nucleic Acids Research. 42 (13): 8745–8754. doi:10.1093/nar/gku546. ISSN 0305-1048. PMC 4117772. PMID 24966351.
- Weiner, Michael P.; Costa, Gina L.; Schoettlin, Warren; Cline, Janice; Mathur, Eric; Bauer, John C. (December 1994). "Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction". Gene. 151 (1–2): 119–123. doi:10.1016/0378-1119(94)90641-6. PMID 7828859.
- "KLD Enzyme Mix | NEB". New England Biolabs. Retrieved 2024-09-07.
- Aughey, Gabriel N.; Southall, Tony D. (January 2016). "Dam it's good! DamID profiling of protein-DNA interactions". Wiley Interdisciplinary Reviews. Developmental Biology. 5 (1): 25–37. doi:10.1002/wdev.205. ISSN 1759-7684. PMC 4737221. PMID 26383089.
- Aughey, Gabriel N.; Cheetham, Seth W.; Southall, Tony D. (2019-03-15). "DamID as a versatile tool for understanding gene regulation". Development. 146 (6): dev173666. doi:10.1242/dev.173666. ISSN 0950-1991. PMC 6451315. PMID 30877125.