The replication of chromosomal DNA is an essential process for all organisms and needs to be tightly regulated. A cell limits its DNA replication activity to once per cell division cycle to maintain its genomic integrity through multiple pathways. Key amongst these is the regulation of replication initiator proteins such as ORC, Cdc6, Cdt1 and MCM complex. Cdt1 is vital to the recruitment of MCM complex to pre-RC and regulation of Cdt1 activity is a crucial control point in the initiation of DNA replication. Higher eukaryotes have evolved redundant mechanism to restrain Cdt1 activity. An inhibitor called Geminin has been reported directly binds with Cdt1 and block the interaction between Cdt1 and MCM6, therefore offering additional safeguard to the control of the initiation of DNA repl...[ Read more ]

The replication of chromosomal DNA is an essential process for all organisms and needs to be tightly regulated. A cell limits its DNA replication activity to once per cell division cycle to maintain its genomic integrity through multiple pathways. Key amongst these is the regulation of replication initiator proteins such as ORC, Cdc6, Cdt1 and MCM complex. Cdt1 is vital to the recruitment of MCM complex to pre-RC and regulation of Cdt1 activity is a crucial control point in the initiation of DNA replication. Higher eukaryotes have evolved redundant mechanism to restrain Cdt1 activity. An inhibitor called Geminin has been reported directly binds with Cdt1 and block the interaction between Cdt1 and MCM6, therefore offering additional safeguard to the control of the initiation of DNA replication. So it is much meaningful to delineate the mechanism how Geminin sequesters Cdt1 and inhibits MCM complex loading onto chromatin. To understand the regulation mechanisms of Cdt1 and its interactions with Geminin, our collaborators from Prof. Liang Chun’s lab have mapped the interaction regions between Cdt1 and Geminin using the yeast two-hybrid and co-IP assays. Except for the binding interfaces published, they found a new Geminin interacting region on Cdt1. Here we further confirmed the new interaction region between Geminin coiled-coil domain and Cdt1 (residues 117-150) by in vitro GST pull-down assay and NMR titration method. In addition, we also found that the coiled coil domain of Geminin interacts with Cdt1 (residues 410-440) region, which overlaps with the binding region between Cdt1 and MCM6. This observation may be sufficient to explain the inhibition mechanism of MCM loading by Geminin. We also identified that both the interaction region with Cdt1 (117-150) and Cdt1 (410-440) on Geminin were located at the C-terminal of the coiled coil domain. Furthermore, only dimerized Geminin can bind with these two fragments and these two interactions are independent with each other. We crystallized the whole Geminin/Cdt1 complex including the three interaction sites and the work of structure determination has been carrying out. On the other hand, we are also trying to solve the structure of Geminin (110-160)/ Cdt1 (117-158 plus 410-440) by NMR technique.

Recently, interest in G-quadruplex (G4) structures has significantly increased. In the regulation of DNA replication, DNA G-quadruplex seems to have a double-faced role: as impediments to replication and as components of metazoan replication origins. Recently it is reported that 90% of human DNA replication origins contain G4 motifs. So G-rich DNA sequence in human genome may be involved in DNA replication initiation. To understand the mechanism, we started to work on G-rich DNA structure and its interaction with pre-RC. Initial effort is to determine structure of human G-quadruplex DNA. Conserved DNA sequence repeats of telomeres was first demonstrated to form G-quadruplex structures in vitro. Human telomeric DNA contains tandem repeats of the sequence 5' -GGGTTA- 3'. Under physiological ionic conditions, this G-rich strand can fold into a four-strand G-quadruplex structure involving multiple G-tetrads which are important for telomere biology. Many different G-quadruplex topologies are known and the four-repeat human telomeric G-rich sequences show a diverse range of intramolecular G-quadruplex structures. Previous studies have demonstrated the presence of multiple G-quadruplex conformations in K+ solution. ¹²⁵I-radioprobing indicated the antiparallel basket arrangement in Na+, and possibly a chair-type quadruplex coexisted with a mixed parallel/antiparallel G-quadruplex in K+ solution. Until now the structure of a chair type G-quadruplex of human telomeric G-rich sequences [GGGTTA]n remains elusive. Here, we present the first chair-type G-quadruplex fold of the human telomeric sequence d[(GGGTTA)₂GGGTTTGGG] (htel21T₁₈), substituting an adenine with thymine in K+ solution and solved its structure using X-ray crystallization. In this structure, loops are successively edgewise; glycosidic conformation of guanines is syn•anti•syn•anti around each tetrad, and each strand of the core has two antiparallel adjacent strands. The hydrogen-bond directionalities of the three G-tetrads are anti-clockwise, clockwise and clockwise, respectively. This novel structure highlights the conformational heterogeneity of human telomeric DNA and provides a unique target for structure-based anticancer drug design. Subsequently, we also found that it interacts with Cdc6, one component of pre-RC and solved the complex structure of truncated Cdc6 peptide and G-quadruplex DNA. We will investigate its interaction with other pre-RC and try to reveal the mechanism in DNA replication.