Academic journal article Genetics

Roles of RAD6 Epistasis Group Members in Spontaneous Pol[zeta]-Dependent Translesion Synthesis in Saccharomyces Cerevisiae

Academic journal article Genetics

Roles of RAD6 Epistasis Group Members in Spontaneous Pol[zeta]-Dependent Translesion Synthesis in Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

DNA lesions that arise during normal cellular metabolism can block the progress of replicative DNA polymerases, leading to cell cycle arrest and, in higher eukaryotes, apoptosis. Alternatively, such blocking lesions can be temporarily tolerated using either a recombination- or a translesion synthesis-based bypass mechanism. In Saccharomyces cerevisiae, members of the RAD6 epistasis group are key players in the regulation of lesion bypass by the translesion DNA polymerase Polζ. In this study, changes in the reversion rate and spectrum of the lys2ΔA746 - 1 frameshift allele have been used to evaluate how the loss of members of the RAD6 epistasis group affects Polζ-dependent mutagenesis in response to spontaneous damage. Our data are consistent with a model in which Polζ-dependent mutagenesis relies on the presence of either Rad5 or Rad18, which promote two distinct error-prone pathways that partially overlap with respect to lesion specificity. The smallest subunit of Polδ, Pol32, is also required for Polζ-dependent spontaneous mutagenesis, suggesting a cooperative role between Polδ and Polζ for the bypass of spontaneous lesions. A third error-free pathway relies on the presence of Mms2, but may not require PCNA.

THE integrity of the genome is threatened not only by environmental agents such as UV and gamma rays, but also by DNA lesions generated spontaneously during normal cellular metabolism. Such spontaneous lesions include oxidative-based damage, base alkylation, loss of DNA bases, and chromosome breaks. Not only can endogenous DNA damage change the base-pairing properties of nucleotides, but also it creates noncoding lesions (FRIEDBERG et al. 1995). Such noncoding lesions can block the forward progression of the replication fork, resulting in cell cycle arrest and/or cell death. Normally, DNA repair processes such as nucleotide excision repair and base excision repair remove blocking lesions before the replication machinery encounters them (HOEIJMAKERS 2001; KUNKEL 2003). If, however, the damage escapes removal by a repair pathway, mechanisms exist to bypass the lesion in order to allow continued DNA replication. Such bypass can be accomplished in a relatively error-free manner via homologous recombination or template switching involving the sister chromatid. Alternatively, a blocking lesion can be bypassed by translesion synthesis, which employs a low-fidelity polymerase to replicate across the damage, often at the cost of increased mutations (KUNKEL 2003).

Three translesion polymerases have been described in Saccharomyces cerevisiae: Poln, Revl, and Polζ. Both Poln and Revl belong to the Y family of DNA polymerases, whose founding members include polymerases involved in the SOS response of Escherichia coli (OHMORI et al 1999). In vitro, Polζ (encoded by RAD30) can bypass thymine-thymine dimers and 8-oxo-G lesions in an error-free manner (JOHNSON et al. 1999; HARACSKA et al. 2000b; YUAN et al. 2000), while bypassing other lesions in an error-prone manner (YuAN et al. 2000). In vivo, lesion bypass by Polζ has a variable effect on spontaneous mutagenesis; rad30 mutants can exhibit an increase, a decrease, or no change in mutation rate, depending upon the assay system used (McDoNALD et al 1997; ROUSH et al 1998). REVl encodes a protein with limited polymerase activity in vitro, specifically incorporating cytosine across from both damaged and undamaged bases (LAWRENCE 2002). This polymerase activity, however, does not appear to be required for the role(s) of Revl in in vivo mutagenesis (HARACSKA et al. 2001; S. WILEY and S. JINKS-ROBERTSON, unpublished observations). Revl interacts genetically with the third translesion polymerase in yeast, ??ιζ (HARFE and JINKS-ROBERTSON 2000; LAWRENCE 2002). ??ιζ is composed of two subunits: a catalytic subunit encoded by KEV3 and an accessory factor encoded by KEV7 (NELSON et al. 1996). Like the other translesion polymerases, ??ιζ has no 3'-5' proofreading activity and demonstrates low processivity, with half of the molecules dissociating from a DNA template after the addition of only three to four nucleotides (NELSON et al. …

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