Human DNA polymerase h (hPolh) is one of the newly identi®ed Y-family of DNA polymerases. These polymerases synthesize past template lesions that are postulated to block replication fork progression. hPolh accurately bypasses UV-associated cis±syn cyclobutane thymine dimers in vitro and contributes to normal resistance to sunlight-induced skin cancer. We describe here mutational analysis of motif II, a highly conserved sequence, recently reported to reside in the ®ngers domain and to form part of the active site in Y-family DNA polymerases. We used a yeast-based complementation system to isolate biologically active mutants created by random sequence mutagenesis, synthesized the mutant proteins in vitro and assessed their ability to bypass thymine dimers. The mutability of motif II in 210 active mutants has parallels with natural evolution and identi®es Tyr52 and Ala54 as prime candidates for involvement in catalytic activity or bypass. We describe the ability of hPolh S62G, a mutant polymerase with enhanced activity, to bypass ®ve other site-speci®c lesions. Our results may serve as a prototype for studying other members of the Y-family DNA polymerases.
DNA polymerase (Pol ) is a member of a new class of DNA polymerases that is able to copy DNA containing damaged nucleotides. These polymerases are highly error-prone during copying of unaltered DNA templates. We analyzed the relationship between bypass efficiency and fidelity of DNA synthesis by introducing substitutions for Tyr-52, a highly conserved amino acid, within the human DNA polymerase (hPol ) finger domain. Most substitutions for Tyr-52 caused reduction in bypass of UV-associated damage, measured by the ability to rescue the viability of UV-sensitive yeast cells at a high UV dose. For most mutants, the reduction in bypass ability paralleled the reduction in polymerization activity. Interestingly, the hPol Y52E mutant exhibited a greater reduction in bypass efficiency than polymerization activity. The reduction in bypass efficiency was accompanied by an up to 11-fold increase in the incorporation of complementary nucleotides relative to noncomplementary nucleotides. The fidelity of DNA synthesis, measured by copying a gapped M13 DNA template in vitro, was also enhanced as much as 15-fold; the enhancement resulted from a decrease in transitions, which were relatively frequent, and a large decrease in transversions. Our demonstration that an amino acid substitution within the active site enhances the fidelity of DNA synthesis by hPol , one of the most inaccurate of DNA polymerases, supports the hypothesis that even error-prone DNA polymerases function in base selection.Accurate DNA replication is required for the maintenance of a species. The accuracy of cellular DNA replication results from the exceptionally high fidelity of DNA synthesis by replicative DNA polymerases and multiple mechanisms for the repair of DNA damage. As a result, the mutation rate in human cells has been estimated to be as low as 1 ϫ 10 Ϫ10 mutations/nucleotide/ cell division (1). Replicational accuracy is maintained despite the large number of lesions caused by both exogenous and endogenous DNA damaging agents. For example, evidence suggests that spontaneous depurination of DNA can result in 10,000 abasic sites per cell per day (2), and an even larger number of lesions are produced as a result of DNA damage by reactive oxygen species (3). Many of the lesions are likely to escape DNA repair mechanisms and therefore are present in DNA templates during copying by replicative DNA polymerases. Bulky lesions can block the progression of DNA replication, forcing the cell to employ additional mechanisms to complete replication in the presence of unrepaired lesions (4, 5).Recently, a new family of DNA polymerases, referred to as the Y-family, has been identified and categorized as translesion synthesis DNA polymerases. The Y-family of DNA polymerases is highly conserved through evolution. Thus far, four Y-family DNA polymerases have been identified in human cells: DNA polymerase (hPol ) 1 (6, 7), DNA polymerase (hPol ) (8), DNA polymerase (hPol ) (9), and the DNA-dependent dCMP transferase Rev1 (10). In contrast to replicative polymerases, ...
Photoreceptor light responses are encoded by changes in synaptic vesicle release. Release from rods is triggered by the opening of calcium channels beneath plate-like synaptic ribbons. Maintained depolarization can activate CICR and enhance release. Using total internal reflection fluorescence microscopy (TIRFM) to visualize release of single synaptic vesicles, we tested whether CICR enhances release from rods by stimulating fusion at nonribbon sites. Rods from salamander retina were loaded with activity-dependent dyes, FM1-43 or dextran-conjugated pHrodo, and visualized by TIRFM. Rods were depolarized with steps to À10 mV under voltage-clamp or by puff application of 50 mM KCl. CICR was activated with 10 mM ryanodine and inhibited with 100 mM ryanodine. Ribbon locations were identified with a fluorescent ribbonbinding peptide or from hot spots of depolarization-evoked calcium entry visualized with Fluo5F. In terminals loaded with FM1-43 or pHrodo, depolarization stimulated rapid disappearance of vesicles with kinetics similar to that measured electrophysiologically. Additionally, stimulation-evoked vesicle disappearance was blocked by Cd 2þ , indicating that it was due to calcium-dependent exocytosis. Vesicles docked for about 200 ms before fusion. Most release events occurred close to ribbons, but some also occurred further away. Activation of CICR with 10 mM ryanodine stimulated intracellular calcium increases and vesicle release. Ryanodine-evoked release events were less clustered than release evoked by depolarization, consistent with greater non-ribbon release. The spread of calcium evoked by 500 ms steps (but not 50 ms steps) was inhibited by blocking CICR with 100 mM ryanodine in the patch pipette. Release evoked by 500 ms steps also involved sites further from the ribbon than release evoked by 50 ms steps. These results indicate that the stimulation of CICR by maintained depolarization enhances release from rods by triggering fusion of vesicles at nonribbon sites.
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