Urea lesion formation in DNA as a consequence of 7,8-dihydro-8-oxoguanine oxidation and hydrolysis provides a potent source of point mutations.
The DNA oxidation product 7,8-dihydro-8-oxoguanine (8-oxoG) forms several mutagenic oxidation products, including a metastable oxaluric acid (Oa) derivative. We report here that a synthetic oligonucleotide containing Oa hydrolyzes under simulated "in vivo" conditions to form a mutagenic urea (Ua) lesion. Using the Oa 2'-deoxyribonucleoside as a model, the hydrolysis rate depended strongly upon the concentrations of bicarbonate and divalent magnesium. In buffered solutions containing physiologically relevant levels of these species, the half-life of Oa nucleoside was approximately 40 h at 37 degrees C. The mutagenic properties of Ua in DNA were investigated using a M13mp7L2 bacteriophage genome containing Ua at a specific site. Transfection of the lesion-containing genome into wild-type AB1157 Escherichia coli allowed determination of the mutation frequency and DNA polymerase bypass efficiency from the resulting progeny phage. Ua was bypassed with an efficiency of 11% as compared to a guanine control and caused a 99% G-->T mutation frequency, assuming the lesion originated from G, which is at least an order of magnitude higher than the mutation frequency of 8-oxoG under the same conditions. SOS induction of bypass DNA polymerase(s) in the bacteria prior to transfection caused the mutation frequency and type to shift to 43% G-->T, 46% G-->C, and 10% G-->A mutations. We suggest that Ua is instructional, meaning that the shape of the lesion and its interactions with DNA polymerases influence which nucleotide is inserted opposite the lesion during replication and that the instructional nature of the lesion is modulated by the size of the binding pocket of the DNA polymerase. Replication past Ua, when formed by hydrolysis of the 8-oxoG oxidation product Oa, denotes a pathway that nearly quantitatively generates point mutations in vivo.
Henderson
Paul T
PT
Department of Chemistry and Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Neeley
William L
WL
Delaney
James C
JC
Gu
Feng
F
Niles
Jacquine C
JC
Hah
Sang Soo
SS
Tannenbaum
Steven R
SR
Essigmann
John M
JM
D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D004926:Escherichia coli / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry*; D006868:Hydrolysis; D016296:Mutagenesis; D009153:Mutagens / Q000737:chemistry; D010072:Oxamic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D010084:Oxidation-Reduction; D017354:Point Mutation; D013014:SOS Response, Genetics / Q000235:genetics; D014162:Transfection; D014508:Urea / Q000737:chemistry*
C453560:7,8-dihydro-8-oxoguanine; D009153:Mutagens; C008727:oxaluric acid; D006147:Guanine; D014508:Urea; D004247:DNA; D010072:Oxamic Acid