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.[1][2]
| Type II Methyl-directed restriction enzyme DpnI | |||||||
|---|---|---|---|---|---|---|---|
| Identifiers | |||||||
| Organism | |||||||
| Symbol | dpnC | ||||||
| PDB | 4ESJ | ||||||
| UniProt | P0A460 | ||||||
| Other data | |||||||
| EC number | 3.1.21.4 | ||||||
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Structure
editThe 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.[3][4]
Uses in molecular biology
editDpnI 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.[7][8]
See also
edit- DpnII restriction endonuclease family, also isolated from S. pneumonae
- List of restriction enzyme cutting sites
- Restriction modification system
References
edit- ^ 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.