The cellular functions of NM (nonmetastatic) 23, a regulator of metastasis and normal development, is not known. The recent finding that NM23 binds DNA and activates c-myc gene transcription was the first direct evidence of a gene regulatory activity for NM23. NM23 also exhibits nucleoside diphosphate kinase (NDPK) activity in vitro, but this lacks correlation with the in vivo functions, and it is also distinct from the DNA binding activity. New evidence indicates that the DNA binding activity of NM23 is related to a novel enzymatic function that breaks and reseals double stranded DNA. This novel activity may explain the metastasis function of NM23 in terms of structural modifications introduced into DNA that may, in metastasis, be different from primary tumor formation. Chormatin modification catalyzed by topoisomerase reaction may also be a significant component of the decision making process that leads the c-myc oncogene to program cells toward proliferation or differentiation.
The aim of the present proposal is to understand the biochemical and structural basis of this novel activity, its relationship to c-myc transcription, and its potential biological cancer relevance.
The specific aims are as follows: (1) to elucidate the biochemistry of this enzyme reaction by studying the importance of the architecture and topology of the DNA substrate to DNA binding, catalytic activity, and in vitro transcription; to determine (a) the role of effectors such as ATP (binding vs. Hydrolysis), (b) whether NM23 contains a DNA-dependent ATPase activity, and (c) the structure of the cleavage site, including the DNA ends and amino acids involved in the reaction; (2) to identify, via structure determinations of cocrystals of NM23 with c-myc DNA, amino acid contacts and conformational changes induced by DNA and effectors, and other structural features associated with this novel mechanism; (3) to elucidate, through mutational analyses including naturally occurring mutations between the two enzymatic activities of NM23 and its relevance to biological function.
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