Function of domains of human O6-alkylguanine-DNA alkyltransferase.
O6-Alkylguanine-DNA alkyltransferase (AGT) is an important DNA repair protein that protects from alkylating agents by converting O6-alkylguanine to guanine forming S-methylcysteine in the AGT protein. The crystal structure of human AGT shows clearly the presence of two domains. The N-terminal domain contains a bound zinc atom, and zinc binding confers a mechanistic enhancement to repair activity, but this domain has no known function. The C-terminal domain contains all residues so far implicated in alkyl transfer including the cysteine acceptor site (Cys145), the O6-alkylguanine binding pocket, and a DNA binding domain. We have expressed and purified the two domains of human AGT separately. The C-terminal domain was totally inactive in vitro, but good activity forming S-alkylcysteine at Cys145 was obtained after recombination with the N-terminal domain via a freeze-thawing procedure. This suggests that the N-terminal domain plays a critical structural role in maintaining an active configuration of the C-terminal domain. However, this C-terminal domain alone had activity in protecting against the cytotoxic and mutagenic activity of the methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) when expressed in Escherichia coli cells lacking endogenous AGT, suggesting that other proteins can fulfill this function. Remarkably, the free N-terminal domain of hAGT was able to repair O6-alkylguanine in vitro via alkyl transfer provided that zinc ions were present. The N-terminal domain was also able to produce moderate protection from MNNG when expressed in E. coli. This cryptic Zn2+-dependent DNA repair activity may be relevant to the evolution and function of AGTs.
Fang
Qingming
Q
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Kanugula
Sreenivas
S
Pegg
Anthony E
AE
D004260:DNA Repair; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008769:Methylnitronitrosoguanidine; C015558:O(6)-methyl-2'-deoxyguanosine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D003849:Deoxyguanosine