Nucleoprotein-based nanoscale assembly.
A system for addressing in the construction of macromolecular assemblies can be based on the biospecificity of DNA (cytosine-5) methyltransferases and the capacity of these enzymes to form abortive covalent complexes at targeted 5-fluorocytosine residues in DNA. Using this system, macromolecular assemblies have been created using two representative methyltransferases: M-HhaI and M x MspI. When 5-fluorocytosine (F) is placed at the targeted cytosine in each recognition sequence in a synthetic oligodeoxynucleotide (GFGC for M x HhaI or FCGG for M x MspI), we show that the first recognition sequence becomes an address for M x HhaI, while the second sequence becomes an address for M x MspI. A chimeric enzyme containing a dodecapeptide antigen linked to the C terminus of M-HhaI retained its recognition specificity. That specificity served to address the linked peptide to the GFGC recognition site in DNA. With this assembly system components can be placed in a preselected order on the DNA helix. Axial spacing for adjacent addresses can be guided by the observed kinetic footprint of each methyltransferase. Axial rotation of the addressable protein can be guided by the screw axis of the DNA helix. The system has significant potential in the general construction of macromolecular assemblies. We anticipate that these assemblies will be useful in the construction of regular protein arrays for structural analysis, in the construction of protein-DNA systems as models of chromatin and the synaptonemal complex, and in the construction of macromolecular devices.
Smith
S S
SS
Department of Cell and Tumor Biology, City of Hope National Medical Center, Duarte, CA 91010, USA.
Niu
L
L
Baker
D J
DJ
Wendel
J A
JA
Kane
S E
SE
Joy
D S
DS
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D015257:DNA-Cytosine Methylases; D005437:Flucytosine / Q000737:chemistry; D007700:Kinetics; D008832:Microchemistry / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009698:Nucleoproteins / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D013379:Substrate Specificity; D013816:Thermodynamics
D009698:Nucleoproteins; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D004247:DNA; D005437:Flucytosine; C037664:DNA modification methylase HhaI; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases; D004248:DNA (Cytosine-5-)-Methyltransferases