Chemical mapping of cytosines enzymatically flipped out of the DNA helix.
Haloacetaldehydes can be employed for probing unpaired DNA structures involving cytosine and adenine residues. Using an enzyme that was structurally proven to flip its target cytosine out of the DNA helix, the HhaI DNA methyltransferase (M.HhaI), we demonstrate the suitability of the chloroacetaldehyde modification for mapping extrahelical (flipped-out) cytosine bases in protein-DNA complexes. The generality of this method was verified with two other DNA cytosine-5 methyltransferases, M.AluI and M.SssI, as well as with two restriction endonucleases, R.Ecl18kI and R.PspGI, which represent a novel class of base-flipping enzymes. Our results thus offer a simple and convenient laboratory tool for detection and mapping of flipped-out cytosines in protein-DNA complexes.
Daujotyte
Dalia
D
Institute of Biotechnology, Graiciūno 8, LT-02241 Vilnius, Lithuania.
Liutkeviciūte
Zita
Z
Tamulaitis
Gintautas
G
Klimasauskas
Saulius
S
D000079:Acetaldehyde / Q000031:analogs & derivatives / Q000737:chemistry; D001671:Biochemistry / Q000379:methods; D003596:Cytosine / Q000032:analysis* / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism
D003596:Cytosine; D004247:DNA; C004656:chloroacetaldehyde; C054926:DNA modification methylase AluI; C037664:DNA modification methylase HhaI; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases; D004248:DNA (Cytosine-5-)-Methyltransferases; D004262:DNA Restriction Enzymes; C114636:endodeoxyribonuclease Ecl18kI; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D000079:Acetaldehyde