Weaver, T. M. | Search bioRxiv

bioRxiv2021

REPROGRAMMING CBX8-PRC1 FUNCTION WITH A POSITIVE ALLOSTERIC MODULATOR

ABSTRACTCanonical targeting of Polycomb Repressive Complex 1 (PRC1) to repress developmental genes is mediated by cell type-specific, paralogous chromobox (CBX) proteins (CBX2, 4, 6, 7 and 8). Based on their central role in silencing and their misregulation associated with human disease including cancer, CBX proteins are attractive targets for small molecule chemical probe development. Here, we have used a quantitative and target-specific cellular assay to discover a potent positive allosteric modulator (PAM) of CBX8. The PAM activity of UNC7040 antagonizes H3K27me3 binding by CBX8 while increasing interactions with nucleic acids and participation in variant PRC1. We show that treatment with UNC7040 leads to efficient PRC1 chromatin eviction, loss of silencing and reduced proliferation across different cancer cell lines. Our discovery and characterization of UNC7040 not only revealed the most cellularly potent CBX8-specific chemical probe to date, but also corroborates a mechanism of polycomb regulation by non-histone lysine methylated interaction partners.

synthetic biology↗

bioRxiv2020

Visualizing Rev1 Catalyze Protein-template DNA Synthesis

During DNA replication, replicative DNA polymerases may encounter DNA lesions, which can stall replication forks. One way to prevent replication fork stalling is through the recruitment of specialized translesion synthesis (TLS) polymerases that have evolved to incorporate nucleotides opposite DNA lesions. Rev1 is a specialized TLS polymerase that bypasses abasic sites as well as minor-groove and exocyclic guanine adducts. It does this by using a unique protein-template mechanism in which the template base is flipped out of the DNA helix and the incoming dCTP hydrogen bonds with an arginine side chain. To observe Rev1 catalysis at the atomic level, we employed time-lapse X-ray crystallography. We found that Rev1 flips out the template base prior to binding the incoming nucleotide. Binding the incoming nucleotide changes the conformation of the DNA substrate to orient it for nucleotidyl transfer, and this is not coupled to large structural changes in the protein like those observed with other DNA polymerases. Moreover, we found that following nucleotide incorporation, Rev1 converts the pyrophosphate product to two mono-phosphates, which drives the reaction in the forward direction. Following nucleotide incorporation, the hydrogen bonds between the incorporated nucleotide and the arginine side chain are broken, but the templating base remains extrahelical. These post-catalytic changes prevent potentially mutagenic processive synthesis by Rev1 and facilitate dissociation of the DNA product from the enzyme.

biochemistry↗