Abstract
The nuclear factor I (NFI) family of site-specific DNA-binding proteins plays a role in both transcription and adenovirus DNA replication. The DNA binding domain of NFI family members contains 4 cysteine residues (Cys-2, Cys-3, Cys-4, and Cys-5) that are conserved in all NFI proteins. Mutation of the Cys-2, Cys-4, and Cys-5 residues in the human NFI-C protein to several other amino acids abolished DNA binding, while 8 of 10 mutations of the Cys-3 residue had little or no effect on binding. Wild-type NFI-C was inactivated by N-ethylmaleimide in vitro, while the active Cys-3 mutant proteins were resistant to N-ethylmaleimide. Treatment of wild-type NFI in vitro with the oxidizing agent diamide also inactivated DNA binding, and subsequent reduction with dithiothreitol restored binding activity. The active Cys-3 mutant NFI proteins were resistant to diamide-inactivation, indicating that the Cys-3 residue is required for modulation of DNA-binding by oxidation state. These studies indicate that oxidative-inactivation can play an important role in the modifying NFI-DNA-protein interactions. The presence of this nonessential Cys-3 residue in all known NFI proteins raises the possibility that it may function in a manner similar to redox-sensitive cysteine residues found in other site-specific DNA-binding proteins.
Highlights
Previous studies demonstrated two forms of covalent modiamino acids abolishedDNA binding, while8 of 10 muta- fication of NFI, N-glycosylation and phosphorylation, that tions of the Cys-3 residue halidttle or noeffect on bind- could potentially affect NFI function in vivo
Wild-type NFI-C was inactivated by N-ethylmaleimsp-ecies purified fromhuman HeLacells were shown t o contain rNittdhievFesiaoIiisttintehnadrneDtviutNtiiootAtrlrwNowobr,h-eiientsithltdehoiyrnttlhehgmde,eabaolaenixcnidimtddiviiisnzdeuigenbC.gasTyecrsqat-eigu3vaeeittnnmmyttT.euhdnrteaetiadnoamutfcctwipidtviireoleodnt-aCetlyiywsnpossi-et3hiwnaede0lacravip--e-tlepeirned,katfrehreodedmDcidHaNereAbnLotbiahciyncaddel lirtlnaosgt(et1ah9cwet,ii2vnt0hiot)ny.t-NegorlfFmyItchiopnesuassyreliilafamilteiecodddafifcfroioiedrmdmrehfssouiordmmfuaNsensFo;fHIhNeoisLwFoaI-mutant NFI proteins were resistant to diamide-inactivac-ells is a substrate for the dsDNA-dependent protein kinase tion, indicating that the Cys-3 residue is required for invitro [21],althoughagain no differences in the activities modulationofDNA-bindingbyoxidation state
These results suggested that low temperature induction of NFI protein might reduce any potentialtoxicity of the wild-type and mutant NFI proteins in E. coli
Summary
Schematicdiagramshowingthe non-conserved cysteine 1 residue (Cys-1) and thefour conserved cysteine residues, and Cys-5) present in the DNA binding domain of hNFI-C220. Mutations were generateda t each of these positions adsescribed under "Materials and Methods," and substitutions that were shown to be activeand inactive for DNA binding a s assessed by gel mobility shift assays are shown above and below the sequence line, respectively. The designations Cys-1 to C y s d correspond to absolute residue positions Cys-79. (Cy&)of the hNFI-C/CTF1 protein [4] (Cy&)of the hNFI-C/CTF1 protein [4] (NCBI sequence identification no. 30266)
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