Single-molecule dissection of a tumor- and virus-suppressing Smc complex involved in genome maintenance

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Project Summary/Abstract The structural maintenance of chromosomes (Smc) 5/6 complex plays a critical role in tumor suppression and the repression of tumor-causing viruses, such as the hepatitis B virus. Smc5/6 exerts these clinical functions by promoting faithful genome replication, coordinating DNA repair, and silencing extra-chromosomal DNA. However, there is little understanding of how Smc5/6 operates as a molecular machine, hindering our ability to intervene in Smc5/6’s health-related functions. Our central hypothesis is that Smc5/6 operates as a dynamic molecular machine that compacts DNA, intrinsically binds to DNA fork junctions, and co-localizes with replication factors. The long-term goal of this research is to understand how defects in Smc5/6 promote genome instability and malignant transformation. This project’s immediate objective is to elucidate the biophysical properties of the Smc5/6 complex by utilizing correlative single-molecule fluorescence and force microscopy, which combines optical tweezers, automated microfluidics, and multi-color confocal microscopy. In Specific Aim 1, Smc5/6’s DNA compaction abilities will be assayed on individual DNA tethers. The effect of the subunits of Smc5/6 and ATP will be systematically tested. The outcome of this work will define the role of each of these components on Smc5/6’s DNA compaction behavior. In Specific Aim 2, the binding behavior and dynamic movement of fluorescently-labeled Smc5/6 will be monitored on double-stranded DNA, single-stranded DNA, and fork junctions in real time. In Specific Aim 3, Smc5/6’s interactions with replication factors will be defined by a first-of-its-kind in vitro reconstitution of the eukaryotic replisome. Overall, this project will: (1) consolidate our understanding of Smc5/6 at the molecular level; (2) yield important insights into how eukaryotes maintain genome integrity and suppress tumors; and (3) potentiate new strategies to modulate Smc5/6’s physiological functions as a tumor suppressor and host restriction factor. Dr. Xiaolan Zhao, an expert on Smc5/6 biology who has a proven track-record for training successful scientists, and Dr. Shixin Liu, an expert on single-molecule technology who practices active mentorship, are co-sponsoring this proposal. The research efforts will take place at the Rockefeller University within the deeply supportive Tri-Institutional MD- PhD Program. This proposal and fellowship is an important career milestone for dual-degree students seeking to become independent investigators.