Structural Studies Of The Cdt1 Binding Domain And The Mcm6 Binding Domain By NMR

This thesis contains two parts:1) In all eukaryotic cells, initiation of genomic DNA replication is tightly controlled to ensure that DNA replication occurs only once in each cell cycle. DNA replication initiation is the critical step during the whole DNA replication process, initiation step is strictly controlled by the formation of pre-replication complex (pre-RC) which. is composed of several important protein complexes and proteins including the. minichromosome maintenance MCM complex. The MCM complex is composed of six evolutionally conserved proteins Mcm2-Mcm7, and it is believed to be the replicative helicase in eukaryotic species, and it plays essential roles in the initiation and elongation phases of DNA replication. During late M and early G1, the Mcm2-7 complex is loaded onto chromatin to form pre-RC in a Cdtl-dependent manner.Mcm6 is one of the six subunit of the Mcm2-7 complex, and it contains the highly conserved MCM domain and one uncharacterized C-terminal domain.In this study we demonstrate that the previously uncharacterized C-terminal domain of human Mcm6 is the Cdtl binding domain (CBD).With the use of high-resolution nuclear magnetic resonance (NMR) technique, we solved the 3D structure of CBD. The structure of CBD exhibits a typical "winged-helix" fold that is generally involved in protein-nucleic acid interaction. Nevertheless, the CBD failed to interact with DNA in our studies indicating that it is specific for protein-protein interaction. Also, we mapped the Mcm6 binding domain (MBD) of Cdtl. Based on NMR titration experiment and GST Pull-down assay we identified E757 and L766 on CBD are the critical residues for the interaction with Cdt1.The fundamental role of these two residues in Mcm6-Cdtl interaction was further confirmed with subcellular co-localization assay. Meanwhile, we solved the 3D structure of MBD which contains a a helix in solution and mapped the residues involved in binding with CBD by NMR. Based on the observed intermolecular NOEs in 13C-edited (F1), 13C/15N-filtered (F3)3D NOESY experiment, we have determined the structure of the CBD-MBD complex and mapped the key residues on the interface in solution. The structure of the CBD-MBD complex elucidates the specific recognization through the HTH motif between CBD and MBD. We speculate that Cdtl directs Mcm2-7 complex chromatin loading to accomplish pre-RC formation through the direct interaction between the MBD of Cdtl and CBD of Mcm6. In conclusion, our structural and functional studies in Mcm6-Cdtl interaction will definitely help to better understand the process of Mcm2-7 complex chromatin loading and pre-RC assembly which are the vital steps in eukaryotic DNA replication.2) The evolutionarily conserved SWIRM (Swi3p, Rsc8p and Moira) domain is a module found in many chromatin modification or remodeling proteins, such as the Ada2 and LSD1 subunits of chromatin modification complexes. In this study, we present the solution NMR structure of the SWIRM domain from LSD1 and compare with the published crystal and NMR structure which contains the same sequence. We found that the N-terminal 10 amino acids show a random coil but not a short helix in the published structure. The additional 5 amino acids were added on the N terminus. Based on the 2D 1H-15N HSQC spectrum and heteronuclear 1H-15N NOE data, the additional 5 amino acids are very important for the formation and stabilization of the N-terminal helix which is not observed in our structure.