Neighbouring bases in template influence base-pairing of isoguanine.
Assuming that the efficiency of the incorporation of 5-methyl-2'-deoxyisocytosine-5' triphosphate (dMiCTP) and dTTP opposite isoguanine (iG) is a measure of the proportion of the keto and enol tautomers of iG in the Thermus aquaticus DNA polymerase active centre, we studied the influence of temperature and iG-neighbouring bases in the template on base-pairing of iG. On the basis of experiments with four sequences (3'-TXT-5', 3'-GXG-5', 3'-CXC-5', 3'-CXT-5', where X=iG) at 37, 50, 65 and 80 degrees C, we found that 3'-neighbours decrease the fraction of the keto tautomer in the order C>G>or=T, whereas temperature apparently does not influence the tautomeric equilibrium of iG. The variability of the ratio of incorporation of dMiCTP versus dTTP (5-20) primarily reflects the variability of K (m) values, since V (max) values are roughly similar, which indicates that the iG.MiC and iG.T pairs fit the polymerase active centre equally well. The altering of the base-pairing of iG by sequence context is discussed in relation to tautomerism and miscoding of this oxidized adenine derivative. A key derivative for preparation oligodeoxynucleotides, O (2)-diphenylcarbamoyl- N (6)-[(dimethylamino)ethylidene]-2'-deoxyisoguanosine, is extremely labile (t (1/2)=3.5 min) in 3% trichloroacetic acid/dichloromethane, i.e. under the conditions of automated DNA synthesis, which results in low yield and length heterogeneity of templates.
Maciejewska
Agnieszka M
AM
Department of Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawińskiego Str., 02 106 Warsaw, Poland.
Lichota
Katarzyna D
KD
Kuśmierek
Jarosław T
JT
D020029:Base Pairing; D001665:Binding Sites; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006147:Guanine / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism*; D019914:Taq Polymerase / Q000378:metabolism*; D013696:Temperature; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000378:metabolism
C472029:5-methyl-2'-deoxyisocytosine-5'-triphosphate; D009841:Oligonucleotides; D013942:Thymine Nucleotides; D006147:Guanine; D003570:Cytidine Triphosphate; C008183:isoguanine; D019914:Taq Polymerase; C024157:thymidine 5'-triphosphate