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Sukhyun Kang Laboratory
Institute for Basic Science - Center for Genomic Integrity
Research Interests
We aim to understand the molecular dynamics of DNA replication and repair machineries. Chromosomal DNA replication and repair are essential cellular processes to maintain genetic information from one generation to the next. Eukaryotic DNA replication is a complex process in which many multi-subunit protein complexes participate. Although various kinds of replication factors have been identified, it has been remained largely unknown how those factors work together to faithfully duplicate the chromosome. We investigate the interplay between replication proteins and nucleic acids during DNA synthesis using in vitro reconstitution systems. We especially focus on the regulatory mechanism for the assembly and disassembly of replication/repair machineries on DNA. Our studies will provide a detailed understanding of how replication and repair processes are controlled to maintain genomic integrity.
Research Background
Function of AAA+ Clamp Loader Complex
AAA+ clamp-loader complexes load or unload ring-shaped DNA clamps to the chromatin during DNA replication and repair. RFC is a canonical DNA clamp-loader complex that loads PCNA (Proliferating Cell Nuclear Antigen) to the primer-template junctions before DNA synthesis. PCNA is a homo-trimeric ring that tethers DNA polymerases and increases their processivity. RFC is a pentameric AAA+ ATPase complex composed of RFC1, RFC2, RFC3, RFC4 and RFC5. Eukaryotic cells have three alternative RFC-like complexes (RLCs; CTF18-RLC, ATAD5-RLC and Rad17-RLC) that share four subunits (RFC2, 3, 4 and 5) with RFC. CTF18-RLC is another PCNA clamp loader and participates in DNA repair and sister chromatid cohesion establishment. ATAD5-RLC is a putative PCNA unloader that removes PCNA after DNA replication. Rad17-RLC loads the 9-1-1 complex to damaged sites during the DNA repair process. Although RLCs are essential to maintain genomic integrity, the molecular mechanisms of clamp loading/unloading by each complex remain to be elucidated. We aim to uncover the function of RLCs in DNA replication using biochemical approaches
Chromatin and DNA replication
In eukaryotic cells genomic DNA is highly compacted by chromatin structures. DNA replication and nucleosome disassembly and re-deposition have to be tightly controlled. However, we do not fully understand how DNA replication and chromatin assembly are coordinated. We are investigating the relationship between DNA replication and chromatin at the point of PCNA cycling. It was reported that PCNA interacts with histone chaperones and facilitates replication-coupled chromatin assembly. On the other hand, PCNA has to be removed from DNA after DNA replication to facilitate proper chromatin formation. Therefore, it is important to understand the connection between chromatin assembly with PCNA-loading and unloading. We found several chromatin factors, such as BRD4, that interact with ATAD5. We are currently investigating how those factors regulate the activity of PCNA unloader.
Architecture of Replication Fork
Replication proteins at the eukaryotic replication fork form a protein-DNA mega-complex referred to as replisome. The architecture of the replisome is important for coordinated synthesis of leading strand and lagging strand. Furthermore, the dynamic association and dissociation of replication proteins is critical for the regulation of replication initiation and elongation. We aim to understand the dynamic change of replisome architecture during chromosomal replication. The interactome of DNA replication proteins will be monitored throughout the cell cycle using Mass-Spec Analysis. Furthermore, the dynamic association of each replication protein will be analyzed using an in vitro replication system.
