Title

Base pairing modes and replicative properties of site-specific urea residues in DNA

Date of Completion

January 2005

Keywords

Biology, Molecular

Degree

Ph.D.

Abstract

Urea (Ur) lesion is one of the significant pyrimidine ring fragmentation products generated by radical-mediated processes initiated by ionizing radiation or oxidation. Ur exists as a mixture of two conformational isomers, and, though they can be separated by HPLC, the conformers equilibrate at room temperature. The in vitro study revealed that urea lesion is poorly bypassed by the newly discovered translesion synthesis (TLS) polymerases, such as Dpo4 enzyme and its human homolog pol κ. The mutagenic and cytotoxic effects or Ur was investigated using an M13mp7L2 or pMS2 genome containing a mixture of the two conformers at a specific site. Replication of the lesion-containing M13mp7L2 genome in both uninduced and SOS-induced E. coli showed a significant reduction of the viability of the modified M13mp7L2 genome (∼97%) and possible mutations at the site of this lesion. Analysis of the progeny phage provided evidence that Ur was preferentially replaced by a T. Moreover, Ur-to-C point mutations (3% frequency) and targeted one-base deletions (4% frequency) were detected, as well as Ur-to-G (< 0.1% frequency) and Ur-to-A (< 0.1% frequency) point mutations. SOS induction caused the Ur-to-C mutations to increase by several fold, though frequency of deletions did not increase. These observations were in general agreement with the results obtained from the replication of the modified pMS2 genome in E. coli . A different trend was observed when the pMS2 genome containing the lesion was introduced in COS7 cells. Analysis of the progeny DNA indicated that the urea lesion promoted misincorporation of C, which leads to Ur-to-G (51% frequency) mutation. In addition, Ur-to-C (12% frequency), Ur-to-A (5% frequency), and targeted one-base deletion (2% frequency) mutations were detected. The thermal stability of DNA duplexes containing Ur lesion were determined and compared with the unmodified duplexes. Thermal melting studies showed that the presence of Ur destabilized the duplex DNA when it was paired with G, C, A, or T, except when the opposite base was deleted. This suggested that urea is an instructional lesion and that the thermodynamic stability may play a role in its replicative bypass in vivo. ^