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.
College of Pharmacy,University of Arizona, Tucson, Arizona 85721, USA.
Thomas S
TS
Dexheimer
Steven S
SS
Carey
Song
S
Zuohe
Vijay M
VM
Gokhale
Xiaohui
X
Hu
Lauren B
LB
Murata
Estelle M
EM
Maes
Andrzej
A
Weichsel
Daekyu
D
Sun
Emmanuelle J
EJ
Meuillet
William R
WR
Montfort
Laurence H
LH
Hurley
D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D045744:Cell Line, Tumor; D054856:G-Quadruplexes / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D023061:Gene Order; D016259:Genes, myc / Q000235:genetics*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D054778:NM23 Nucleoside Diphosphate Kinases / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D034622:RNA Interference; D015533:Transcriptional Activation / Q000187:drug effects* / Q000235:genetics*
D054778:NM23 Nucleoside Diphosphate Kinases