Takuya Torizawa

Structural details on mdm2-p53 interaction

Mdm2 is a cellular antagonist of p53 that keeps a balanced cellular level of p53. The two proteins are linked by a negative regulatory feedback loop and physically bind to each other via a putative helix formed by residues 18-26 of p53 transactivation domain (TAD) and its binding pocket located within the N-terminal 100-residue domain of mdm2 (Kussie, P. H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A. J., and Pavletich, N. P. (1996) Science 274, 948-953). In a previous report we demonstrated that p53 TAD in the mdm2-freee state is mostly unstructured but contains two nascent turns in addition to a “preformed” helix that is the same as the putative helix mediating p53-mdm2 binding. Here, using heteronuclear multidimensional NMR methods, we show that the two nascent turn motifs in p53 TAD, turn I (residues 40-45) and turn II (residues 49-54), are also capable of binding to mdm2. In particular, the turn II motif has a higher mdm2 binding affinity (∼20 μm) than the turn I and targets the same site in mdm2 as the helix. Upon mdm2 binding this motif becomes a well defined full helix turn whose hydrophobic face formed by the side chains of Ile-50, Trp-53, and Phe-54 inserts deeply into the helix binding pocket. Our results suggest that p53-mdm2 binding is subtler than previously thought and involves global contacts such as multiple “non-contiguous” minimally structured motifs instead of being localized to one small helix mini-domain in p53 TAD.

31P NMR study of the interactions between oligodeoxynucleotides containing (6-4) photoproduct and Fab fragments of monoclonal antibodies specific for (6-4) photoproduct

A 31P nuclear magnetic resonance (NMR) study of the interactions between oligonucleotides containing the (6‐4) photoproduct and the Fab fragments of monoclonal antibodies (64M3 and 64M5) recognizing the (6‐4) photoproduct is reported. The 31P chemical shift data indicate that backbone conformation of (6‐4) adduct is affected by the presence of flanking oligodeoxynucleotides, and (6‐4) adducts with different backbone conformations are accommodated in the antigen binding sites of these antibodies. It was also revealed that epitopes for these antibodies consist of not only the (6‐4) adduct but the flanking di‐ or tri‐deoxynucleotides on both the 5′ and 3′ sides as well.

DNA-binding properties of the antibody specific for the Dewar photoproduct of thymidylyl-(3-5')-thymidine.

A monoclonal antibody (DEM-1) specific for the Dewar photoproduct is used for detection and quantification of photolesions in DNA. To help understand the molecular recognition of damaged DNA by the antibody protein, we have cloned and sequenced the variable region genes of DEM-1. We have also prepared Fab fragments of DEM-1 (DEM1Fab), and synthesized two kinds of 3′-biotinylated oligonucleotides of different lengths containing a central Dewar photoproduct of TpT to analyze the effects of the antigen size on the binding rates by means of surface plasmon resonance (SPR). Results obtained from SPR analyses suggest that DEM1Fab may recognize tetranucleotide unit as the epitope.

Efficient chemical synthesis of a pyrimidine (6–4) pyrimidone photoproduct analog and its properties

Abstract We synthesized an analog of a pyrimidine (6–4) pyrimidone photoproduct with a formacetal linkage instead of the phosphodiester in the (6–4) photoproduct, and investigated the interaction between a monoclonal antibody specific to the natural (6–4) photoproduct and a DNA containing the photoproduct analog.