Mutations in Pol eta are responsible for the inherited disorder, the variant form of xeroderma pigmentosum (XP-V). XP-V patients are extremely prone to sunlight induced skin cancers. The high incidence of skin cancers in these patients is due to the absence of functional Pol eta, which has the remarkable ability to replicate through UV lesions such as a cis-syn cyclobutane thymine-thymine (T-T) dimer in an error-free way. Unlike classical DNA polymerases that become stalled at the UV induced T-T dimer, Pol eta can efficiently and accurately replicate past this common sunlight induced lesion. Besides a T-T dimer, Pol eta can also efficiently and accurately replicate DNA containing 7,8-dihydro-8-oxoguanine (8-oxoG) adducts formed by oxidative damage. The discovery of Pol eta has led to the description of a whole new class of translesion synthesis (TLS) DNA polymerases within the last two years, including the DinB homolog Polk in humans. However, despite similarities in sequence, TLS polymerases differ in the type of DNA damage they bypass, consistent with specialized cellular roles.
The specific aims are as follows: 1. Determine the structure of Pol eta. 2. Determine the structures of Pol eta bound to damaged and undamaged DNA. 3. Structure based mutational analysis of Pol eta. 4. Determine the structure of Pol eta in complex with PCNA. 5. Determine the structure of Dpo4 (a Polk homolog) in the mismatch extension mode. These structures will be tested throughout by extensive mutagenesis experiments aimed at defining the molecular mechanisms of these new DNA polyrnerases and the origins of their distinct activities.
