Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA.
BspQI is a thermostable Type IIS restriction endonuclease (REase) with the recognition sequence 5'GCTCTTC N1/N4 3'. Here we report the cloning and expression of the bspQIR gene for the BspQI restriction enzyme in Escherichia coli. Alanine scanning of the BspQI charged residues identified a number of DNA nicking variants. After sampling combinations of different amino acid substitutions, an Nt.BspQI triple mutant (E172A/E248A/E255K) was constructed with predominantly top-strand DNA nicking activity. Furthermore, a triple mutant of BspQI (Nb.BspQI, N235A/K331A/R428A) was engineered to create a bottom-strand nicking enzyme. In addition, we demonstrated the application of Nt.BspQI in optical mapping of single DNA molecules. Nt or Nb.BspQI-nicked dsDNA can be further digested by E. coli exonuclease III to create ssDNA for downstream applications. BspQI contains two potential catalytic sites: a top-strand catalytic site (Ct) with a D-H-N-K motif found in the HNH endonuclease family and a bottom-strand catalytic site (Cb) with three scattered Glu residues. BlastP analysis of proteins in GenBank indicated a putative restriction enzyme with significant amino acid sequence identity to BspQI from the sequenced bacterial genome Croceibacter atlanticus HTCC2559. This restriction gene was amplified by PCR and cloned into a T7 expression vector. Restriction mapping and run-off DNA sequencing of digested products from the partially purified enzyme indicated that it is an EarI isoschizomer with 6-bp recognition, which we named CatHI (CTCTTC N1/N4).
Zhang
Penghua
P
New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Too
Priscilla Hiu-Mei
PH
Samuelson
James C
JC
Chan
Siu-Hong
SH
Vincze
Tamas
T
Doucette
Stephanie
S
Bäckström
Stefan
S
Potamousis
Konstantinos D
KD
Schramm
Timothy M
TM
Forrest
Dan
D
Schwartz
David C
DC
Xu
Shuang-yong
SY
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D053904:DNA Breaks, Single-Stranded; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D004277:DNA, Single-Stranded; D004262:DNA Restriction Enzymes