How Chromosome Structure Influences Nuclear Processes
The overall objective of our research is to determine how chromosome structure influences nuclear processes and to identify and characterize the cellular machines that contend with this structure. Over the years, our general strategy has been to employ yeast molecular genetics to develop detailed models that describe complex nuclear events that can then be directly tested and expanded by subsequent biophysical and biochemical approaches. Much of our efforts over the past few years have focused on ATP-dependent chromatin remodeling enzymes (e.g. SWI/SNF and INO80) that hydrolyze ~1,000 ATPs per minute to alter chromatin structure and thereby regulate transcription, DNA repair, or replication. Our studies are centered on both the regulation and mechanism of this chromatin "remodeling" reaction. Since most of these enzymes are enormous (>1 MDa), multi-subunit enzymes, we are also interested in understanding how these enzymes are assembled and what roles are played by individual subunits. To address these goals we use a broad spectrum of methodologies, including yeast molecular and classical genetics, modern analytical ultracentrifugation, molecular biology, and traditional biochemistry. Notably, these remodeling enzymes are conserved from yeast to mammals, play key roles in gene expression and the maintenance of genome integrity, and loss of their function leads to various disease states.
In addition to our studies on chromatin remodeling enzymes, we also wish to understand the dynamics of chromatin fibers and how fiber condensation influences DNA repair, transcription, and DNA replication. These projects involve the biochemical reconstitution of defined nucleosomal arrays from recombinant histones and DNA templates that contain head-to-tail repeats of nucleosome positioning sequences. Typically, we perform sedimentation velocity experiments in the analytical ultracentrifuge to investigate how histone modifications (e.g. H4 K16 acetylation), histone variants, or heterochromatin proteins (e.g. HP1, Sir3) influence the folding dynamics of these reconstituted chromatin fibers. These biophysical studies are complemented by powerful biochemical assays where we assess how the structure of a chromatin fiber regulates various steps of DNA double strand break repair or DNA replication.
As we learn more about the dynamics of chromatin fibers and the basic mechanics of DNA repair and DNA replication, we have initiated in vivo studies that probe how these processes are coordinated and regulated within cells. For instance, we have recently, found that a DNA double strand break can induce the re-localization of a chromosomal domain to the nuclear envelope and that this compartmentalization inhibits recombinational repair. Interestingly, localization to the nuclear periphery requires components of the telomerase complex and seems to be due to an attempt to heal the chromosome by formation of a new telomere. Similar types of chromosome healing events may also occur at stalled replication forks. We are currently using a variety of cell biological and molecular genetic approaches to dissect the complex decision-making processes that a cell employs in its attempts to maintain genome integrity.