Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein binds the ribonucleotide substrates while the R2 protein contains a binuclear iron center and a tyrosyl free radical, essential for activity. The crystal structures of the corresponding Escherichia coli proteins suggest that the distance from the active site in R1 to the tyrosyl radical buried in R2 is about 35 A. Therefore, an electron pathway was suggested between the active site and the tyrosyl radical. Such a pathway could include a conserved tryptophan on the suggested R1 interaction surface of R2 and a conserved aspartic acid hydrogen bonded both to the tryptophan and to a histidine iron ligand. To find experimental support for such an electron pathway, we have replaced the conserved tryptophan in mouse R2 with phenylalanine or tyrosine and the aspartic acid with alanine. All the mutated R2 proteins were shown to bind metal with the same affinity as native R2 and to form the binuclear iron center. In addition, the W103Y and D266A proteins formed a normal tyrosyl free radical while only low amounts of radical were observed in the W103F protein. Neither the kinetic rate constants nor the equilibrium dissociation constant of the R1/R2 complex was affected by the mutations as shown by BIAcore biosensor technique. However, all mutant R2 proteins were completely inactive in the enzymatic assay, supporting the hypothesis that the tryptophan and aspartic acid residues are important links in an amino acid residue specific long-range electron transfer.

O6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein that removes the promutagenic O6-methylguanine lesion from DNA. In order to obtain more information about the mechanism of action of AGT, two conserved residues in a putative DNA binding domain were changed by site-directed mutagenesis, and the abilities of the mutant proteins to bind to DNA, to repair methylated DNA, and to convert O6-benzylguanine to guanine were examined. The alteration of arginine-128 to alanine (R128A) reduced the AGT activity toward methylated DNA substrates by a factor of more than 1000 but did not decrease the rate of reaction with O6-benzylguanine. The change of residue tyrosine-114 to glutamic acid (Y114E) completely abolished the ability to repair O6-methylguanine in DNA in the assays used showing that this was reduced by > 15,000-fold, but the ability to convert O6-benzylguanine to guanine was reduced by only 60-fold. Alteration of this residue to alanine (Y114A) reduced activity toward methylated DNA by > 1000-fold and toward O6-benzylguanine by about 80-fold. Neither the R128A nor the Y114E mutant AGT were able to compete with the control AGT for the repair of methylated DNA whereas the inactive mutant, C145A, in which the cysteine acceptor site is changed to alanine, competed effectively in this assay. These results suggest that the residues arginine-128 and tyrosine-114 are involved in the DNA binding properties of the AGT. The ability of the AGT proteins to form stable complexes with DNA was therefore examined by measuring the retardation of DNA during electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

O 6 -Methylguanine is removed from DNA via the transfer of the methyl group to a cysteine acceptor site present in the DNA repair protein O 6 -alkylguanine-DNA alkyltransferase. The human alkyltransferase is inactivated by the free base O 6 -benzylguanine, raising the possibility that substantially larger alkyl groups could also be accepted as substrates. However, the Escherichia coli alkyltransferase, Ada-C, is not inactivated by O 6 -benzylguanine. The Ada-C protein was rendered capable of reaction by the incorporation of two sitedirected mutations converting Ala 316 to a proline (A316P) and Trp 336 to alanine (W336A) or glycine (W336G). These changes increase the space at the active site of the protein where Cys 321 is buried and thus permit access of the O 6 -benzylguanine inhibitor. Reaction of the mutant A316P/W336A-Ada-C with O 6 -benzylguanine was greatly stimulated by the presence of DNA, providing strong support for the concept that binding of DNA to the Ada-C protein activates the protein. The Ada-C protein was able to repair O 6 -benzylguanine in a 16-mer oligodeoxyribonucleotide. However, the rate of repair was very slow, whereas the E. coli Ogt, the human alkyltransferase, and the mutant A316P/W336A-Ada-C alkyltransferases reacted very rapidly with this 16-mer substrate and preferentially repaired it when incubated with a mixture of the methylated and benzylated 16-mers. These results show that benzyl groups are better substrates than methyl groups for alkyltransferases provided that steric factors do not prevent binding of the substrate in the correct orientation for alkyl group transfer.

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