| DNA polymerase eta (Poleta) functions in error-free bypass of ultraviolet light-induced DNA lesions, and inactivation of human Poleta causes the cancer prone syndrome, xeroderma pigmentosum variant form. In Schizosaccharomyces pombe, the eso1+ encoded protein is comprised of two domains, wherein the N-terminus is highly homologous to Polil, and the C-terminus is highly homologous to the S. cerevisiae Ctf7 protein which is essential for establishing sister chromatid cohesion during S phase.;In order to address the function of this unusual fusion and to characterize the biochemical properties of the Poleta domain, I characterized the DNA polymerase activity of S. pombe Eso1 protein and a truncated version containing only the Poleta domain. Both proteins exhibit similar DNA polymerase activity and low processivity. Steady-state kinetic analyses show that on undamaged DNA both proteins misincorporate nucleotides with frequencies of ∼10-2 to 10-4. Both proteins similarly replicate a CPD-containing DNA template, preferentially inserting adenine opposite the affected thymines. Thus, fusion with CO has no significant effect on the DNA replication or damage bypass properties of S. pombe Poleta.;Next, I deleted translesion synthesis genes from S. pombe strains to assess an in vivo role for Poleta. UV-induced reversion studies confirm that S. pombe Poleta is involved in error-free bypass of UV-induced DNA damage, and additionally suggest a role for Poleta in error-prone bypass. To explain these genetic findings, I examined the ability of Poleta to extend from mismatched primer-termini. Steady-state kinetics revealed that Poleta extends mismatched primer-termini on nondamaged templates more efficiently than it incorporates mismatches. At a CPD, Poleta extends as efficiently from G-T mismatches as from A-T matches. Thus, incorporation of guanine opposite either thymine of a CPD would be extended by Poleta, resulting in error-prone bypass. Therefore, Poleta is indeed capable of both error-free and error-prone bypass of UV-induced DNA damage.;Another S. pombe protein containing sequence homology to Poleta is the DinB1 protein, shown in other species to be a DNA polymerase. I performed initial characterization of the S. pombe DinB1 protein and showed that it is a DNA polymerase, but requires an accessory protein for optimal activity. |
