Towards a better understanding of translesion synthesis and polymerase specificity through directed evolution.

Abstract

When the canonical replicative polymerase in E. coli, pol III, encounters DNA damage it stalls and is unable to proceed until the DNA damage is either repaired or a translesion synthesis (TLS) polymerase bypasses the DNA damage. How various polymerases evolved to overcome DNA damage via TLS and acquired their specificity for different kinds of DNA damage are open questions in chemical biology. TLS polymerases are implicated in cancer and antibiotic resistance due to their ability to be utilized in transient hypermutation and to overcome the types of inter- and intra-strand crosslinks caused by chemotherapy drugs, such as cisplatin. In E. coli, pol IV is normally responsible for TLS bypass of N2-dG damage, whereas pol V is the polymerase whose cognate damage is caused by UV light. By either (1) giving pol III the novel ability to perform TLS-like replication, or (2) changing/adding specificity of pol IV or pol V to bypass non-cognate DNA damage, we can better understand how nature designs enzymes, evolves novel functions, and performs translesion synthesis with specificity and efficiency. Utilizing directed evolution, we hope to elucidate these evolutionary mechanisms. Libraries of random variant polymerases were created by hydroxylamine mutagenesis, and then used to transform cells deficient in UV damage repair proteins, including pol V. After exposure to UV light, survivors that showed potential to exceed survival of wild-type were purified and assayed.