Excision of the oxidatively formed 5-hydroxyhydantoin and 5-hydroxy-5-methylhydantoin pyrimidine lesions by Escherichia coli and Saccharomyces cerevisiae DNA N-glycosylases.
BACKGROUND
(5R) and (5S) diastereomers of 1-[2-deoxy-beta-D-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-beta-D-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2'-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes.


METHODS
Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements.


RESULTS
In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the kcat/Km ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase.


CONCLUSIONS
The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates.


CONCLUSIONS
The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.
Gasparutto
Didier
D
Laboratoire des Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique UMR-E3 CEA-UJF, INAC, DSM, CEA-Grenoble, F-38054 Grenoble Cedex 9, France. didier.gasparutto@cea.fr
Muller
Evelyne
E
Boiteux
Serge
S
Cadet
Jean
J
D004249:DNA Damage; D045647:DNA Glycosylases / Q000737:chemistry*; D004260:DNA Repair; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006801:Humans; D006827:Hydantoins / Q000737:chemistry*; D007700:Kinetics; D009841:Oligonucleotides / Q000737:chemistry; D010084:Oxidation-Reduction; D011743:Pyrimidines / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013237:Stereoisomerism; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D006827:Hydantoins; D009841:Oligonucleotides; D011743:Pyrimidines; D029701:Saccharomyces cerevisiae Proteins; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D045647:DNA Glycosylases