Cloning and expression of the ApaLI, NspI, NspHI, SacI, ScaI, and SapI restriction-modification systems in Escherichia coli.
The genes encoding the ApaLI (5'-GTGCAC-3'), NspI (5'-RCATGY-3'), NspHI (5'-RCATGY-3'), SacI (5'-GAGCTC-3'), SapI (5'-GCTCTTCN1-3', 5'-N4GAAGAGC-3') and ScaI (5'-AGTACT-3') restriction-modification systems have been cloned in E. coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5'-CATG-3') restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5'-GCTCTTC-3' blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene.
Xu
S Y
SY
New England Biolabs, Inc., Beverly, MA 01915, USA. xus@neb.com
Xiao
J P
JP
Ettwiller
L
L
Holden
M
M
Aliotta
J
J
Poh
C L
CL
Dalton
M
M
Robinson
D P
DP
Petronzio
T R
TR
Moran
L
L
Ganatra
M
M
Ware
J
J
Slatko
B
B
Benner
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003596:Cytosine / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics* / Q000378:metabolism; D015254:DNA Modification Methylases / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D003596:Cytosine; D015254:DNA Modification Methylases; C116423:NspI methylase protein, bacteria; D004248:DNA (Cytosine-5-)-Methyltransferases; C024408:endodeoxyribonuclease SacI; C100902:endodeoxyribonuclease SapI; C047020:endodeoxyribonuclease ScaI; D015252:Deoxyribonucleases, Type II Site-Specific; C056435:GTGCAC-specific type II deoxyribonucleases