DNA -protein conjugates for structural studies of polymerase eta and for nucleic acid triggered prodrug activation

The ability to conjugate proteins to DNA has facilitated structural studies of protein-DNA complexes and the development of hybrid enzymes with new catalytic activities. Polymerase eta replicates past cis-syn thymidine dimers in a highly non-mutagenic manner and mutations in the human polymerase results in a condition known as xeroderma pigmentosum variant. The detailed structure of the ternary polymerase complex with a dimer-containing template-primer and a dNTP is of great interest in understanding how this enzyme is able to bypass dimers in such an error free manner. Whereas a crystal structure of the enzyme was obtained five years ago, there still has been no report of a ternary complex with a cis-syn dimer, presumably because of the difficulty in obtaining the required co-crystals. To address this potential problem we have constructed pol eta template conjugates to form a more robust binary complex with a primer for crystallographic studies by a site-specific disulfide bond forming strategy. In this strategy, primer-templates containing a convertible nucleotide are used to introduce a disulfide bearing appendage that can form a disulfide with a cysteine engineered to anchor the template at the correct position on the protein. Cysteines were introduced into sites expected to be near the DNA bases of the template in the major groove by site-directed mutagenesis. We found that S394C, M396C, and R402C mutants can selectively form stable disulfide crosslinks with an ethylene sulfhydryl on C9 on the template and effectively stop the movement of the polymerase and extension of the primer. Initial attempts at crystallization have yielded co-crystals, but not with sufficient order for high resolution crystallographic studies and will require further study.;A new approach to nucleic acid triggered prodrug activation was also developed based on protein-DNA conjugates. The idea was to use a disease-specific mRNA transcript to template the assembly of two catalytically inactive halves of a protein to form an active prodrug activating enzyme. To this end, we selected to make use of the well studied assembly of catalytically inactive S-protein with S-peptide to form RNase S, which has an identical ribonuclease activity to RNase A. The S peptide-DNA conjugate was prepared by disulfide conjugation of a mutant S peptide with a 15-mer 5'-thiol-modified DNA. The S protein-DNA conjugate was prepared from a C-terminal acylhydrazide His-tagged S protein prepared via intein mediated protein splicing and a 5'-aldehydo 15-mer DNA. The protein-DNA conjugate was found to be active only in the presence of wild type S peptide and its DNA conjugate, or a high concentration of mutant S peptide and its DNA conjugate. Initial attempts to demonstrate DNA-templated activation of RNase S were not successful and will require further study and optimization.