Table 3.
Summary of the sensitivity of different REases to DNA modifications introduced by the Hia5 enzyme
| REase | Recognition site | Number of sites in pHia5ET | Sensitivity to m6A | Cleavage |
|---|---|---|---|---|
| BglII | AGaTCT | 1 | S | N |
| Bsh1285I | CGRYCG R = A/G | 5 | S | N |
| Bsu15I | ATCGAT | 2 | S | N |
| CviQI | GTAC | 3 | S | N |
| DraI | TTTAAA | 2 | S | N |
| Eco32I | GATATC | 0 (1)b | S | N |
| EcoRI | GAATTC | 0 (1)b | S | N |
| HincII | GTYRAC | 1 (GTTAAC) | S | N |
| HindIII | AaGCTT | 0 (1)b | S | N |
| HinfI | GANTC | 19 | S | N |
| HphI | GGTGA/TCACC | 16 | S | N |
| KspAI | GTTAAC | 1 | S | N |
| MboIa | GATC | 24 | S | N |
| MluI | db ACGCGT | 1 | S | N |
| MlyI | GAGTC/GACTC | 9 | S | N |
| MunI | CAATTG | 1 | S | N |
| NlaIII | CATG | 24 | S | N |
| PaeI | GCATGC | 1 | S | N |
| PstI | CTGCAG | 0 (2)b | S | N |
| PvuII | dbCAGCTG | 3 | S | N |
| SspI | aATATT | 3 | S | N |
| VspI | ATTAAT | 5 | S | N |
| XbaI | TCTaGA | 1 | S | N |
| XhoI | CTCGAG | 1 | S | N |
| TaiI | ACGT | 16 | S | N |
| TaqI | TCGA | 16 | S | N |
| TfiI | GAWTC | 10 | S | N |
| AluI | AGCT | 24 | S | P, L |
| TasI | AATT | 28 | S | P, L |
| Tru1I | TTAA | 29 | S | P, L |
| CaiI | CAGNNNCTG | 1 | U | N |
| CseI | GACGC | 12 | U | N |
| Eam1104I | GAAGAG | 3 | U | N |
| Eco147I | AGGCCT | 1 | U | N |
| Eco47III | AGCGCT | 3 | U | N |
| Eco91I | GGTNACC | 1 | U | N |
| LweI | GCATC | 24 | U | N |
| Mph1103I | ATGCAT | 3 | U | N |
| NdeI | CaTATG | 1 | U | N |
| PfeI | GAWTC | 10 | U | N |
| PscI | ACATGT | 1 | U | N |
| SchI | GAGTC/GACTC | 9 | U | N |
| XmiI | GTMKAC | 1 | U | N |
| XapI | RAATTY | 6 | U | N |
| AdeI | CACNNNGTG | 1 | U | N |
| BpiI | GAAGAC | 5 | U | N |
| Bsp119I | TTCGAA | 1 | U | N |
| BcnI | CCSGG | 12 | (−) | Y |
| BglI | GGCN5GCC | 1 | (−) | Y |
| Bme1390I | CCNGG | 21 | (−) | Y |
| Bpu1102I | GCTNAGC | 2 | U | Y |
| Bsh1236I | CGCG | 37 | (−) | Y |
| Bsp143Ia | GATC | 24 | Not | Y |
| Bsp120I | GGGCCC | 1 | (−) | Y |
| Bsp68I | TCGCGA | 1 | Not | Y |
| BspLI | GGNNCC | 20 | (−) | Y |
| BspOI | GCTAGC | 0 (2)b | U | Y |
| Bst1107I | GTATAC | 1 | U | Y |
| BsuRI | GGCC | 22 | (−) | Y |
| Csp6I | GTAC | 3 | U | Y |
| DpnIa | Gm6ATC | 24 | Not | Y |
| Eco130I | CCWWGG | 1 | (−) | Y |
| Eco88I | CYCGRG | 2 | (−) | Y |
| EcoO109I | RGGNCCY | 3 | (−) | Y |
| FspBI | CTAG | 6 | U | Y |
| Hin1I | GRCGYC | 5 | (−) | Y |
| Hin6I | GCGC | 32 | (−) | Y |
| HpyF10VI | GCN7GC | 41 | (−) | Y |
| MbiI | GAGCGG/CCGCTC | 4 | U | Y |
| MspI | CCGG | 29 | (−) | Y |
| NheI | GCTAGC | 0 (2)b | U | Y |
| PvuI | CGATCG | 1 | Not | Y |
| SmaI | CCCGGG | 1 | (−) | Y |
aTested in λ DNA dam- in vitro methylated with the Hia5 enzyme. bnumber of recognition sites in plasmid pAltOKI.
Y: complete cleavage; N: no digestion; P: partial digestion, L: plasmid linearized; S: sensitive to m6A; U: unknown sensitivity to m6A; Not: not sensitive to m6A; (−): absence of adenine residues in recognition sequence; db: methylation of both strands required to block cleavage; A: the enzyme will not cut if the marked adenine is methylated; a: the enzyme is not sensitive to methylation of the marked adenine; A: there are no data on the sensitivity of the enzyme to methylation of the unmarked adenine.