NM23-H2 may play an indirect role in transcriptional activation of c-myc gene expression but does not cleave the nuclease hypersensitive element III(1)

The formation of G-quadruplex structures within the Nuclease hypersensitive element (NHE) III(1) region of the c-Myc promoter and the ability of these structures to repress c-Myc transcription have been well established. However, just how these extremely stable DNA secondary structures are transformed to activate c-Myc transcription is still unknown. NM23-H2/nucleoside diphosphate kinase B has been recognized as an activator of c-Myc transcription via interactions with the NHE III(1) region of the c-Myc gene promoter. Through the use of RNA interference, we confirmed the transcriptional regulatory role of NM23-H2. In addition, we find that further purification of NM23-H2 results in loss of the previously identified DNA strand cleavage activity, but retention of its DNA binding activity. NM23-H2 binds to both single-stranded guanine- and cytosine-rich strands of the c-Myc NHE III(1) and, to a lesser extent, to a random single-stranded DNA template. However, it does not bind to or cleave the NHE III(1) in duplex form. Significantly, potassium ions and compounds that stabilize the G-quadruplex and i-motif structures have an inhibitory effect on NM23-H2 DNA-binding activity. Mutation of Arg(88) to Ala(88) (R88A) reduced both DNA and nucleotide binding but had minimal effect on the NM23-H2 crystal structure. On the basis of these data and molecular modeling studies, we have proposed a stepwise trapping-out of the NHE III(1) region in a single-stranded form, thus allowing single-stranded transcription factors to bind and activate c-Myc transcription. Furthermore, this model provides a rationale for how the stabilization of the G-quadruplex or i-motif structures formed within the c-Myc gene promoter region can inhibit NM23-H2 from activating c-Myc gene expression.