Effects of Accessory Proteins on the Bypass of a cis-syn Thymine−Thymine Dimer by Saccharomyces cerevisiae DNA Polymerase η †

Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Biochemistry (Impact Factor: 3.02). 08/2007; 46(30):8888-96. DOI: 10.1021/bi700234t
Source: PubMed


Among several hypotheses to explain how translesion synthesis (TLS) by DNA polymerase eta (pol eta) suppresses ultraviolet light-induced mutagenesis in vivo despite the fact that pol eta copies DNA with low fidelity, here we test whether replication accessory proteins enhance the fidelity of TLS by pol eta. We first show that the single-stranded DNA binding protein RPA, the sliding clamp PCNA, and the clamp loader RFC slightly increase the processivity of yeast pol eta and its ability to recycle to new template primers. However, these increases are small, and they are similar when copying an undamaged template and a template containing a cis-syn TT dimer. Consequently, the accessory proteins do not strongly stimulate the already robust TT dimer bypass efficiency of pol eta. We then perform a comprehensive analysis of yeast pol eta fidelity. We show that it is much less accurate than other yeast DNA polymerases and that the accessory proteins have little effect on fidelity when copying undamaged templates or when bypassing a TT dimer. Thus, although accessory proteins clearly participate in pol eta functions in vivo, they do not appear to help suppress UV mutagenesis by improving pol eta bypass fidelity per se.

14 Reads
  • Source
    • "Samples were analyzed in 12% denaturing polyacrylamide gels. Products were quantified and polymerization parameters were calculated as previously described (48). "
    [Show abstract] [Hide abstract]
    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.
    Nucleic Acids Research 05/2009; 37(11):3774-87. DOI:10.1093/nar/gkp238 · 9.11 Impact Factor
  • Source
    • "One possibility is that POLH-1 degradation is part of the mechanism that allows exchange of POLH-1 for the replicative polymerase after TLS. Current models for TLS suggest that post-TLS exchange occurs because pol eta, which exhibits low processivity in vitro (Washington et al., 1999; McCulloch et al., 2004, 2007), simply disassociates from the template after TLS, and this allows the replicative polymerase to regain access to the primed site. We propose that early embryos may employ a more active mechanism to remove POLH-1 from the template after TLS, and that this involves the CRL4-Cdt2 mediated proteolysis step that we have identified. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Both the POLH-1 (pol eta) translesion synthesis (TLS) DNA polymerase and the GEI-17 SUMO E3 ligase are essential for the efficient replication of damaged chromosomes in Caenorhabditis elegans embryos. Here we study how POLH-1 is regulated during a DNA-damage response in these embryos. We report that DNA damage triggers the degradation of POLH-1 and that degradation is mediated by the Cul4-Ddb1-Cdt2 (CRL4-Cdt2) pathway that has previously been shown to degrade the replication factor Cdt1 during S phase. We also show that GEI-17 protects POLH-1 from CRL4-Cdt2-mediated destruction until after it has performed its function in TLS, and this is likely via SUMOylation of POLH-1. These studies reveal that POLH-1 undergoes DNA-damage-induced proteolysis and that GEI-17 regulates the timing of this proteolysis. Implications for how this system may control the removal of POLH-1 from replication forks after TLS are discussed.
    Molecular cell 01/2009; 32(6):757-66. DOI:10.1016/j.molcel.2008.11.016 · 14.02 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Not Available
    Aerospace Conference, 2003. Proceedings. 2003 IEEE; 02/2003
Show more

Preview (2 Sources)

14 Reads
Available from