Efficient cleavage of RNA at high temperatures by a thermostable DNA-linked ribonuclease H.
To construct a DNA-linked RNase H, which cleaves RNA site-specifically at high temperatures, the 15-mer DNA, which is complementary to the polypurine-tract sequence of human immunodeficiency virus-1 RNA (PPT-RNA), was cross-linked to the unique thiol group of Cys135 in the Thermus thermophilus RNase HI variant. The resultant DNA-linked enzyme (d15-C135/TRNH), as well as the d15-C135/ERNH, in which the RNase H portion of the d15-C135/TRNH is replaced by the Escherichia coli RNase HI variant, cleaved the 15-mer PPT-RNA site-specifically. The mixture of the unmodified enzyme and the unlinked 15-mer DNA also cleaved the PPT-RNA but in a less strict manner. In addition, this mixture cleaved the PPT-RNA much less effectively than the DNA-linked enzyme. These results indicate that the cross-linking limits but accelerates the interaction between the enzyme and the DNA/RNA substrate. The d15-C135/TRNH cleaved the PPT-RNA more effectively than the d15-C135/ERNH at temperatures higher than 50 degrees C. The d15-C135/TRNH showed the highest activity at 65 degrees C, at which the d15-C135/ERNH showed little activity. Such a thermostable DNA-linked RNase H may be useful to cleave RNA molecules with highly ordered structures in a sequence-specific manner.
Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan.
M
M
Haruki
T
T
Nogawa
N
N
Hirano
H
H
Chon
Y
Y
Tsunaka
M
M
Morikawa
S
S
Kanaya
D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000096:biosynthesis; D015497:HIV-1 / Q000201:enzymology; D006358:Hot Temperature; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism; D011687:Purines / Q000737:chemistry; D012313:RNA / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism*; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000201:enzymology*
D017931:DNA Primers; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; D011687:Purines; D012367:RNA, Viral; D012313:RNA; D004247:DNA; D016914:Ribonuclease H; D003545:Cysteine