Table 2.
Genetic and biochemical classification of LlaGI
| Type I | Type III | Type IIL | Type ISP | |
|---|---|---|---|---|
| e.g. EcoR124I | e.g. EcoPI | e.g. MmeI | e.g. LlaGI | |
| Genes/protein | ||||
| Genes | HsdR, hsdM, hsdS | Res, mod | RM | RM |
| Nuclease Complex | R2M2S1 | Res2Mod2 | n.d. | RM1 (RM2 for cleavage?) |
| Recognition sequence | Asymmetric, bipartite GAAnnnnnnRTCG | Short, asymmetric AGACC | Short, asymmetric, degenerate TCCrAC | Short, asymmetric, degenerate CTnGAyG |
| DNA methylation | ||||
| Methylation | Both strands | One strand | One strand | One strand |
| DNA cleavage | ||||
| Cofactors | ATP, Mg2+, AdoMet | ATP, Mg2+ | Mg2+ | ATP, Mg2+ |
| Multiple sites required? | Yes, Linear DNANo, circular DNA | Yes (+AdoMet) No (-AdoMet)a | Yes | Yes (dsDNA break) No (nicking) |
| Sites required in cis? | Yes | Yes | No | Yes |
| Communication mode | 1D | 1D | 3D | 1D |
| Preference for site directionality? | None | Indirect repeatb | None | Indirect Repeat (head-to-head only) |
| Cleavage loci? | Non-specific random | Non-specific | Non-specific | Non-specific random |
| Distant and proximal | Fixed proximal | Fixed proximal | Distant and proximal | |
| Helicase motor activity | No helicase domain | |||
| DNA Loop translocation required | Yes | No | Yes | |
| DNA Supercoiling | Yes | No | Yes | |
| Directionality | Bidirectional (two unidirectional motors per complex) | Bidirectionalb | Unidirectional | |
| Translocation rate | 463 bp/s (20°C) | n.d. | 173 ATP/s (20°C) | |
| ATPase rate | 503 ATP/s/motor(20°C) | ∼0.2 ATP/s/per complex (20°C) | 250 ATP/s (20°C) | |
| ATP per bp | 1–1.5 | «1 | 1.5–2 | |
| Step size | 1–2 | n.d. | n.d. | |
| Average distance moved per event | ∼5000 bp | n.d. | n.d. | |
| Motor polarity | 3′–5′ | n.d. | n.d. | |
| Triplex displacement | Yes | No | Yes |
Data for EcoR124I was collated from refs (14,27,35,39–41). Data for EcoPI was collated from refs (30,42–45). Data for MmeI was collated from refs (2,9,34). Data for LlaGI was from this paper and refs. 1,10,11. n.d. is where data is not determined.
aRelaxed cleavage conditions with high enzyme:DNA ratios.
bDogma states that Type III enzymes only cut sites in head-to-head repeat (33) but more recent analysis suggests that tail-to-tail repeats are cut equally well (Kara van Aelst, M.D.S. and Ralf Seidel, unpublished data).