Role of His-16 in turnover of T4 pyrimidine dimer glycosylase.
Previously, the histidine residue at position 16 in the mature T4 pyrimidine dimer glycosylase (T4-PDG) protein has been suggested to be involved in general (non-target) DNA binding. This interpretation is likely correct, but, in and of itself, cannot account for the most dramatic phenotype of mutants at this position: their inability to restore ultraviolet light resistance to a DNA repair-deficient Escherichia coli strain. Accordingly, this residue has been mutated to serine, glutamic, aspartic acid, lysine, cysteine, and alanine. The mutant proteins were expressed, purified, and their abilities to carry out several functions of T4-PDG were assessed. The mutant proteins were able to perform most functions tested in vitro, albeit at reduced rates compared with the wild type protein. The most likely explanation for the biochemical phenotypes of the mutants is that the histidine residue is required for rapid turnover of the enzyme. This role is interpreted and discussed in the context of a reaction mechanism able to account for the complete spectrum of products generated by T4-PDG during a single turnover cycle.
Meador
Michael G
MG
Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, Texas 77555-1071, USA.
Rajagopalan
Lavanya
L
Lloyd
R Stephen
RS
Dodson
M L
ML
D000409:Alanine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D002470:Cell Survival; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D045647:DNA Glycosylases / Q000737:chemistry* / Q000378:metabolism; D004260:DNA Repair; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014466:Ultraviolet Rays
D009841:Oligonucleotides; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine; D006639:Histidine; D004247:DNA; D045647:DNA Glycosylases; C036401:deoxyribopyrimidine endonucleosidase; D008239:Lysine; D003545:Cysteine; D000409:Alanine