This training program will be carried out at the post-doctoral level in the laboratory of Dr. Wade Harper at Harvard Medical School. The research study, which is estimated to require 3 years to complete will focus on a recently discovered Holliday junction resolvase component, SLX4. This is an excellent system to study DNA recombinational repair. Numerous technologies exist both in Dr. Harper's lab and at Harvard Medical School which should greatly facilitate the proposed experimental studies such as proteomics and microscopy imaging facilities. In the first aim of the research study, the regulatory mechanisms controlling SLX4 localization and activation and as well as the role of SLX4 in telomere maintenance will be investigated. Since post-translational modifications are important elements in the localization of proteins involved in the damage response, SLX4 and its associated factors will be examined in the context of these processes. Mass spectrometric approaches will be used to identify residues on SLX4 that are phosphorylated in the absence and presence of DNA damage. These residues will be mutated to test if abrogation of SLX4 phosphorylation at specific residues affects its recruitment to sites of damage, or affects its HJ resolution activity. SLX4 contains two tandem ubiquitin interaction motifs and thus will be tested for binding to ubiquitin. If these motifs are found to be important for ubiquitin interaction, SLX4 mutant isoforms that do not bind ubiquitin will be tested for localization to damage sites, as well as HJ resolution activity in vitro and in vivo. SLX4 has been observed at telomeres in telomerase- negative (ALT) cells and a recent study highlights the importance of MUS81, a subunit of the SLX4 complex, in telomere recombination in ALT cells. In the final part of the first aim, the role of SLX4 in the recruitment of MUS81 to telomeres in ALT cells will be examined. This will be tested by depleting SLX4 by siRNA coupled with co-precipitation and fluorescence-based localization experiments of MUS81 with the telomere-binding protein, TRF2. In the second aim, the mechanistic details of SLX4 resolution of Holliday junctions will be elucidated. In SLX4 complexes, symmetrical cleavage across Holliday junctions is catalyzed by the SLX1 enzyme, while the MUS81-EME1 endonuclease is thought to cleave HJ-like structures through a "nick- counternick" mechanism. A plasmid with a cruciform extension that mimics a Holliday junction will be utilized to examine what favors one process over the other, and to establish for the first time that the SLX1-SLX4 complex cleaves both strands of the HJ by a synchronous mechanism, analogous to that displayed by baterial HJ resolvases such as RuvC. structure-based mutagenesis of SLX1 and SLX4 will also be performed to examine the molecular factors that control the selectivity and symmetry of SLX1-dependent HJ resolution. These in vitro results will be followed by experiments that test the role of asymmetric versus symmetric HJ resolvase activity in vivo.
This training program will be devoted to the study of the SLX4 Holliday junction resolvase component, which interacts with multiple elements of recombination-mediated genome maintenance and stability. This will include investigating the recruitment of SLX4 to sites of DNA damage and mechanistic aspects of its catalytic activity. Since SLX4 occupies a central node in homologous recombination, a process that is often deregulated in human cancers, this program can potentially make significant contributions to human health and disease.