Positive coactivator of transcription (PC4) is a human single-stranded DNA (ssDNA) binding protein involved in multiple nucleic acid metabolism processes that has been identified as a novel G4 interactor which binds G4 structures with a low nM Kd value (~2 nM), similar to that observed for ssDNA. Based on the structural differences between G4 and ssDNA, we surmised that the mode of binding by PC4 may be substantially different for G4DNA. While many proteins have been identified as G4- binding proteins, much less is known about the structural mechanisms utilized to mediate G4 binding.
The work described herein set out to detail the molecular interactions which allow the binding of G4DNA by PC4. A combination of biophysical, biochemical, and in silico techniques confirmed PC4 can bind non-discriminatively to multiple G4 topologies and utilizes a similar binding interface to that observed for ssDNA. Further evidence to support a binding model in which the G4DNA is grasped by PC4 and nested within the shallow PC4 DNA binding groove is described.
The impact of the PC4-G4DNA interaction was also investigated, and it was determined that PC4 functions to stabilize G4 structures as no apparent G4 unfolding capacity could be observed with PC4 alone. This unfolding activity was also not observed for less stable G4 structures. Additionally, helicase-catalyzed unfolding of G4DNA was severely perturbed in the presence of PC4 supporting a G4 stabilizing function.
Lastly, the binding of PC4 to G4DNA was considered in the context of duplex substrate that has been termed the G4-multiplex. Based on the canonical ssDNA binding model, an expanded binding hypothesis which included a G4 structure was investigated. The conclusions drawn from this investigation provide evidence that further support a stabilizing effect on G4DNA by PC4 and an expanded model of DNA binding.
|Advisor:||Raney, Kevin D.|
|Commitee:||Diekman, Alan B., Eoff, Robert L., Peterson, Eric C., Tackett, Alan J.|
|School:||University of Arkansas for Medical Sciences|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 79/04(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry, Biophysics|
|Keywords:||G-quadruplex DNA, Nucleic acid chemistry, Nucleic acid structure, Positive coactivator 4, ssDNA binding protein|
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