Ubiquitin is a small polypeptide that is transferred to proteins via ubiquitin ligases. Often ubiquitylation of a protein will target it for degradation; other times it will alter its localization or function. Many aspects of cell cycle control and DNA repair are regulated by ubiquitylation. We are interested in defining how these regulatory mechanisms operate in normal and cancer cells.
In one project we focused on the Cul4-DDB1 ubiquitin ligase. Cul4 is a scaffold protein that assembles a large number of proteins required to catalyze the addition of ubiquitin onto substrates. DDB1 is one subunit of this complex that helps to determine what proteins serve as substrates. DDB1 was first identified as a component of a DNA damage binding protein complex that also contains DDB2. Defects in this damaged DNA binding activity cause the disease xeroderma pigmentosum in humans, which is characterized by ultraviolet light sensitivity and skin cancer. Mutations in DDB2 impair nucleotide excision repair, which is required to remove UV-induced DNA lesions.
The Cul4-DDB1 complex has many substrates. These include histones, DDB2, c-jun and Cdt1. We have recently discovered that defects in the Cul4-DDB1 complex cause genome instability even when cells are not challenged by UV radiation. In fact, silencing the DDB1 gene rapidly leads to the appearance of double strand breaks throughout the genome. This is in part due to defects in the regulation of DNA replication. The Cdt1 protein, one of the Cul4-DDB1 targets, is a critical regulator of replication. The degradation of this protein, which is initiated by ubiquitylation, ensures that only one round of DNA synthesis occurs per cell cycle. This is important to maintain the number of chromosomes in the cell. We found that cells lacking DDB1 do not regulate Cdt1 properly, leading to a partial re-replication of DNA within one cell cycle. This is extremely problematic for the cell. Our data also suggest that other, unknown substrates of Cul4-DDB1 are also important for genome stability. We are interested in identifying all of the genome maintenance pathways that are regulated by this ubiquitin ligase complex.
In a second project, we used a synthetic lethal approach in an RNAi screen to identify modifiers of cancer cell sensitivity to newly developed drugs targeting the PARP enzyme. PARP inhibitors are being developed to treat cancers that have defects in specific genome maintenance pathways. Our screen identified the ubiquitin specific protease USP11 as a new regulator of double-strand break repair.