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Low-fidelity DNA synthesis by the L979F mutator derivative of Saccharomyces cerevisiae DNA polymerase zeta.

Authors:
Stone JE, Kissling GE, Lujan SA, Rogozin IB, Stith CM, Burgers PM, Kunkel TA
Affiliation:
Journal:
Nucleic acids research

Abstract

To probe Pol zeta functions in vivo via its error signature, here we report the properties of Saccharomyces cerevisiae Pol zeta in which phenyalanine was substituted for the conserved Leu-979 in the catalytic (Rev3) subunit. We show that purified L979F Pol zeta is 30% as active as wild-type Pol zeta when replicating undamaged DNA. L979F Pol zeta shares with wild-type Pol zeta the ability to perform moderately processive DNA synthesis. When copying undamaged DNA, L979F Pol zeta is error-prone compared to wild-type Pol zeta, providing a biochemical rationale for the observed mutator phenotype of rev3-L979F yeast strains. Errors generated by L979F Pol zeta in vitro include single-base insertions, deletions and substitutions, with the highest error rates involving stable misincorporation of dAMP and dGMP. L979F Pol zeta also generates multiple errors in close proximity to each other. The frequency of these events far exceeds that expected for independent single changes, indicating that the first error increases the probability of additional errors within 10 nucleotides. Thus L979F Pol zeta, and perhaps wild-type Pol zeta, which also generates clustered mutations at a lower but significant rate, performs short patches of processive, error-prone DNA synthesis. This may explain the origin of some multiple clustered mutations observed in vivo.

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