BtsCI and BseGI display sequence preference in the nucleotides flanking the recognition sequence.

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science

Base Sequence* ; Binding Sites* ; Inverted Repeat Sequences*; DNA/*chemistry ; DNA/*metabolism ; Deoxyribonucleases, Type II Site-Specific/*metabolism; DNA/genetics ; Protein Binding

Restriction enzymes are the bread and butter of Molecular Biology. Nonetheless, how restriction enzymes recognize and cleave their target is not always clear. When developing a method for the enzymatic production of oligonucleotides, we noticed that type II endonucleases BtsCI and BseGI, which recognize the sequence GGATGNN^, perform incomplete digestions of DNA hairpins, with the top strand nick not always occurring correctly. We tested the cutting of synthetic hairpins containing all possible combinations of dinucleotides following the recognition site and our results show that all sequences containing one adenine following GGATG were digested more efficiently. We further show that the same sequence preference is also observable in double stranded DNA at higher Mg2+ concentrations and even in optimal conditions. Kinetic results show that BtsCI has a noteworthy difference in the first-rate constants between different sequences and between the two catalytic domains. An increase in Mg2+ resulted in a drastic decrease in the catalytic activity of the top (sense) strand that wasn't always accompanied by a nick in the bottom strand (antisense).

The authors have declared that no competing interests exist.

Proc Natl Acad Sci U S A. 2005 Apr 26;102(17):5905-8. (PMID: 15840723)
Nucleic Acids Res. 2014 Jan;42(1):45-55. (PMID: 23863841)
Nat Protoc. 2008;3(9):1476-84. (PMID: 18772875)
Nat Methods. 2013 Jul;10(7):647-52. (PMID: 23727986)
J Biol Chem. 2002 Feb 8;277(6):4024-33. (PMID: 11729187)
J Mol Biol. 1962 Jul;5:18-36. (PMID: 13862047)
Nat Protoc. 2013 Apr;8(4):737-48. (PMID: 23493070)
Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10570-5. (PMID: 9724744)
Nucleic Acids Res. 2014;42(16):10596-604. (PMID: 25120268)
J Mol Biol. 1962 Jul;5:37-49. (PMID: 13888713)
J Biol Chem. 2005 Dec 16;280(50):41584-94. (PMID: 16223716)
J Mol Biol. 1975 Jan 25;91(3):315-28. (PMID: 1102702)
Nucleic Acids Res. 2014 Jan;42(1):20-44. (PMID: 24068554)
Proc Natl Acad Sci U S A. 1976 Feb;73(2):293-7. (PMID: 1061131)
Nucleic Acids Res. 2014 Jan;42(1):56-69. (PMID: 23990325)
Science. 2010 Jan 1;327(5961):78-81. (PMID: 19892942)
Nucleic Acids Res. 2014;42(16):10618-31. (PMID: 25120263)
PLoS One. 2015 Jan 28;10(1):e0117059. (PMID: 25629514)
Nucleic Acids Res. 2017 Oct 13;45(18):e160. (PMID: 28977490)
Mol Biotechnol. 2003 Mar;23(3):225-43. (PMID: 12665693)
Biochemistry. 2011 Oct 4;50(39):8264-9. (PMID: 21888353)
Annu Rev Microbiol. 2017 Sep 8;71:233-261. (PMID: 28657885)
Bioinformatics. 2003 Jan 22;19(2):295-6. (PMID: 12538257)
Eur J Biochem. 1997 May 15;246(1):1-22. (PMID: 9210460)
Nucleic Acids Res. 2010 Mar;38(4):1294-303. (PMID: 19955230)
Nature. 2017 Dec 6;552(7683):84-87. (PMID: 29219963)
Science. 2005 Sep 9;309(5741):1728-32. (PMID: 16081699)

9007-49-2 (DNA)
EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific)

Date Created: 20180818 Date Completed: 20190208 Latest Revision: 20190215

20260130

PMC6097692

10.1371/journal.pone.0202057

30118487