TY - JOUR
T1 - The modified DNA base β-D-glucosylhydroxymethyluracil confers resistance to micrococcal nuclease and is incompletely recovered by 32P- postlabeling
AU - van Leeuwen, Fred
AU - de Kort, Martin
AU - van der Marel, Gijs A.
AU - van Boom, Jacques H.
AU - Borst, Piet
PY - 1998
Y1 - 1998
N2 - The hypermodified DNA base β-D-glucosylhydroxy-methyluracil, also called J, is a naturally occurring DNA modification. J was initially detected by 32P-postlabeling in Trypanosoma brucei and was recently also found in several other eukaryotic parasites. To use 32P-postlabeling as a method to quantitate the absolute levels of J in DNA we have tested the postlabeling efficiency of J using various synthesized standard oligonucleotides containing J. It is known that modified nucleotides, especially bulky ones, are often partially recovered by postlabeling and they are poor substrates for some of the enzymes used. We found that on average only 50% of J is recovered, which shows that the amount of J in T. brucei DNA has been twofold underestimated. Experiments with a short oligomer and defined pyrimidine tracts showed that the incomplete recovery of J is caused at least in part by resistance of J-containing DNA to degradation by micrococcal nuclease.
AB - The hypermodified DNA base β-D-glucosylhydroxy-methyluracil, also called J, is a naturally occurring DNA modification. J was initially detected by 32P-postlabeling in Trypanosoma brucei and was recently also found in several other eukaryotic parasites. To use 32P-postlabeling as a method to quantitate the absolute levels of J in DNA we have tested the postlabeling efficiency of J using various synthesized standard oligonucleotides containing J. It is known that modified nucleotides, especially bulky ones, are often partially recovered by postlabeling and they are poor substrates for some of the enzymes used. We found that on average only 50% of J is recovered, which shows that the amount of J in T. brucei DNA has been twofold underestimated. Experiments with a short oligomer and defined pyrimidine tracts showed that the incomplete recovery of J is caused at least in part by resistance of J-containing DNA to degradation by micrococcal nuclease.
UR - https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=0032079902&origin=inward
UR - https://www.ncbi.nlm.nih.gov/pubmed/9570833
U2 - https://doi.org/10.1006/abio.1998.2587
DO - https://doi.org/10.1006/abio.1998.2587
M3 - Article
C2 - 9570833
SN - 0003-2697
VL - 258
SP - 223
EP - 229
JO - Analytical biochemistry
JF - Analytical biochemistry
IS - 2
ER -