TY - JOUR
T1 - Restriction enzyme digestion of hemimethylated DNA
AU - Gruenbaum, Yosef
AU - Cedar, Howard
AU - Razin, Aharon
PY - 1981/6
Y1 - 1981/6
N2 - Hemimethylated duplex DNA of the bacteriophage ØX 174 was synthesized using primed repair synthesis in vitro with E. coli DNA polymerase I followed by ligation to produce the covalently closed circular duplex (RFI). Single-stranded ØX DNA was used as template, a synthetic oligonucleotide as primer and 5-methyldeoxycytidine-5′-triphosphate (5mdCTP) was used in place of dCTP. The hemimethylated product was used as substrate for cleavage by various restriction enzymes. Out of the 17 enzymes tested, only 5 (BstN I, Taq I, Hinc II, Hinf I and Hpa I) cleaved the hemimethylated DNA. Two enzymes (Msp I and Hae III) were able to produce nicks on the unmethylated strand of the cleavage site. Msp I, which is known to cleave at CCGG when the internal cytosine residue is methylated, does not cleave when both cytosines are methylated. Another enzyme, Apy I, cleaves at the sequence CCGG when the internal cytosine is methylated, but is inactive on hemimethylated DNA in which both cytosines are methylated. Hemimethylated molecules should be useful for studying DNA methylation both in vivo and in vitro.
AB - Hemimethylated duplex DNA of the bacteriophage ØX 174 was synthesized using primed repair synthesis in vitro with E. coli DNA polymerase I followed by ligation to produce the covalently closed circular duplex (RFI). Single-stranded ØX DNA was used as template, a synthetic oligonucleotide as primer and 5-methyldeoxycytidine-5′-triphosphate (5mdCTP) was used in place of dCTP. The hemimethylated product was used as substrate for cleavage by various restriction enzymes. Out of the 17 enzymes tested, only 5 (BstN I, Taq I, Hinc II, Hinf I and Hpa I) cleaved the hemimethylated DNA. Two enzymes (Msp I and Hae III) were able to produce nicks on the unmethylated strand of the cleavage site. Msp I, which is known to cleave at CCGG when the internal cytosine residue is methylated, does not cleave when both cytosines are methylated. Another enzyme, Apy I, cleaves at the sequence CCGG when the internal cytosine is methylated, but is inactive on hemimethylated DNA in which both cytosines are methylated. Hemimethylated molecules should be useful for studying DNA methylation both in vivo and in vitro.
UR - http://www.scopus.com/inward/record.url?scp=0019878040&partnerID=8YFLogxK
U2 - 10.1093/nar/9.11.2509
DO - 10.1093/nar/9.11.2509
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C2 - 6269052
AN - SCOPUS:0019878040
SN - 0305-1048
VL - 9
SP - 2509
EP - 2515
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 11
ER -