The storage of genetic information within DNA sequences is the underlying code that defines an organism and it’s function. Genomic integrity is dependent upon the faithful replication and maintenance of DNA. Not only must the cell coordinate standard processes essential for gene transcription, DNA replication, and division, but it must also deliver rescue mechanisms in response to DNA damaging agents.
Histone posttranslational modifications (PTMs) play an essential regulatory role in all aspects of chromatin function. Each of the core histones is vastly decorated with different PTMs, altering the side-chain chemistry and influencing changes in histone-DNA and histone-protein interactions. The chromatin landscape is shaped by an immense field of histone PTM writers, readers, and erasers. It is the concise balance of each of these factors that conserves chromatin plasticity and permits the fluid chromatin dynamics essential for proper function.
There remains an insufficient amount of data regarding the complicated molecular interactions that occur on the nucleosomal surface, particularly in the context of the living cell. My interests are focused on the subtleties of histone proteins and how their modifications influence chromatin architecture and molecular contacts that occur on the nucleosome. With the techniques we have established, we can now investigate the impact of histone PTMs in all aspects of chromatin biology as well as begin to illuminate the substantial interactome required for proper genome stability and integrity.
To address chromatin function and structure in living cells we exploit a synthetic biology technique that allows for the expansion of the genetic code in Saccharomyces cerevisiae. This system requires an evolved tRNA/aminoacyl-tRNA synthetase pair that act as orthogonal cellular components to encode for unnatural amino acids in response to an amber stop codon. The recombinant expression of these orthogonal pieces allow for a wide assortment of non-native chemistries to be introduced at the genetic level.
In particular we are interested in the amino acid, p-benzoylphenylalanine. It contains a photo-inducible chemical crosslinking side chain that allows us to capture and monitor protein-protein interactions in vivo. We genetically encode this UAA into histone proteins and scan the nucleosomal surface for binding partners. We aim to expose nucleosomal protein-protein interactions and the mechanistic details of chromatin dynamics in yeast.