Investigation of protein-protein interaction for DNA replication and transcription; Fanconi anemia group J and Replication protein A, and Forkhead box O4 and p53

Abstract

DNA replication and transcription are necessary biological processes to maintain living organisms. Replication is the process of forming complementary DNA strands using two strands of double-stranded DNA (dsDNA) as respective templates. DNA polymerases are mainly responsible for this process, but in order to proceed with replication, cooperation of various proteins is required. Representatively, DNA helicases are responsible for the release of dsDNA and single-stranded DNA (ssDNA) binding proteins (SSBs) contribute to maintenance of ssDNA. They physically bind to each other and participate in the replication process. Transcription is the process of transferring genetic information from DNA to mRNA. RNA polymerases play central role in this process, however, other factors also participate in the initiation or regulation of transcription through various protein-protein and protein-DNA interactions. Among them, Transcription factors (TFs) are key regulators that modulate gene expression by recognizing specific sequences of DNA. Here, over three chapters, we investigate the interaction between the human DNA helicase Fanconi anemia group J (FANCJ) and the SSB Replication protein A (RPA), the selective target DNA recognition of the TF Forkhead box O4 (FOXO4), and the dual binding surface between FOXO4 and another TF p53. The first chapter describes the interaction between the peptide sequence of FANCJ C-terminal region and the RPA70N domain. FANCJ is a human DNA helicase belonging to the superfamily 2 Fe-S helicase and is involved in various processes required for genome maintenance. After dsDNA is released by FANCJ, RPA binds to ssDNA and protects it from damage. Thus, these two proteins have a cooperative role in replication and chemo resistance processes in tumor cells. Nevertheless, it is only known that they interact with each other, and the structural understanding of the precise binding surface and properties is still lacking. Here, we observed the core binding regions of FANCJ and RPA and measured their binding affinity by using biophysical experiments such as NMR analyses and fluorescence polarization assays. Our results showed that an unstructured acidic rich peptide sequence within the C-terminal of FANCJ binds to the RPA70N domain with micromolar binding affinity and this peptide sits on the basic cleft of RPA70N. Also, we observed that this helicase-RPA binding can be mediated by electrostatic interactions with precisely tuned hydrophobic interactions using alanine mutants of aromatic residues of FANCJ peptide. This work is expected to provide fundamental structural information on helicase-RPA interaction that may inform inhibitory strategies with therapeutic applications. The second chapter shows that the recognition of its target DNA by the forkhead domain (FHD) of FOXO4 is regulated by another intramolecular domain, the transactivation domain (CR3). FOXO4 is a TF that participates in cell homeostasis. While the structure and DNA binding properties of the conserved FHD have been thoroughly investigated, how the CR3 regulates the DNA binding properties of the protein remains elusive. Here, we investigated the role of CR3 in modulating the DNA binding properties of FOXO4 using solution NMR. CR3 and DNA share the same surface of FHD for binding. While FHD did not differentiate binding to target and non-target DNA, the FHD-CR3 complex showed different behaviors depending on the DNA sequence. In the presence of CR3, free and DNA-bound FHD exhibited a slow exchange with target DNA and a fast exchange with non-target DNA. The interaction of the two domains affected the kinetic function of FHD depending on the type of DNA. Based on these findings, we suggested a transcription initiation model by which CR3 modulates FOXO4 recognition of its target promoter DNA sequences. This study describes the function of CR3 in FOXO4 and provides a new kinetic perspective on target sequence selection by TFs. In the last chapter, it is suggested that dual binding surfaces are involved in the interaction between the TFs of FOXO4 and p53, and that these bindings are mediated by DNA. Cellular senescence protects cells against external carcinogenic stress, but it causes aging-related diseases when it accumulates. Human TFs FOXO4 and p53 cooperate with each other to activate the transcription of p21 and accelerate senescence. Inhibition of the FOXO4-p53 interaction is attracting attention as a strategy to prevent the accumulation of senescent cells, but researches on the precise binding surfaces mediating this interaction is still lacking. Here, we investigated the dual binding sites involved in the interaction between FOXO4 and p53 and their binding affinities using NMR spectroscopy and isothermal calorimetry (ITC) experiments. The interaction between the FHD of FOXO4 and the TAD of p53, and the CR3 of FOXO4 and the DBD of p53, was detected using NMR chemical shift perturbation analysis. According to ITC data, both interactions have micromolar Kd values and the FOXO4 FHD-p53 TAD interaction has a greater binding affinity. We also discovered that the intramolecular CR3 and p53 TAD2 binding sites of FOXO4 FHD and the intramolecular p53 TAD and FOXO4 CR3 binding sites of p53 DBD overlap, respectively. This suggests that their intermolecular and intramolecular interactions can properly compete and/or cooperate to modulate transcription process. Based on these findings, we proposed a model for how the dual binding surfaces of FOXO4 and p53 contributes to the efficient recognition of the p21 promoter site. We expect this study to provide structural and functional information for understanding the mutual cooperation of the two TFs that induce cellular senescence.