Lesions in DNA: hurdles for polymerases.

Translesion synthesis (TLS) is one of the DNA damage tolerance strategies, which have evolved to enable organisms to replicate their genome despite the presence of unrepaired damage. The process of TLS has the propensity to produce mutations, a potential origin of cancer, and is therefore of medical interest. Significant progress in our understanding of TLS has come primarily from studies of the bacterium Escherichia coli, the budding yeast Saccharomyces cerevisiae and, more recently, human cells. Results from these analyses indicate that the fundamental mechanism of TLS and the proteins involved have been conserved throughout evolution from bacteria to humans.

Distinct roles for Rev1p and Rev7p during translesion synthesis in Saccharomyces cerevisiae.

Translesion synthesis (TLS) in Saccharomyces cerevisiae requires at least Rev1p and polymerase zeta (Pol zeta), a complex of the Rev3 polymerase and its accessory factor Rev7p. Although their precise role(s) are poorly characterized, in vitro studies suggest that each protein contributes to TLS in a manner dependent on the particular lesion and surrounding DNA sequence. In the present study, strand segregation analysis is used to attempt to identify the role(s) of the Rev1 and Rev7 proteins during TLS. This assay uses double-stranded plasmids containing a genetic marker opposite to a replication blocking lesion (N-2-acetylaminofluorene; AAF) to measure TLS quantitatively and qualitatively in vivo. The AAF adduct is localized within a repetitive sequence in a manner that allows the formation of misaligned primer-template replication intermediates. Elongation from a misaligned intermediate fixes a frameshift mutation (slipped TLS), while extension of the correctly aligned lesion terminus yields error-free (non-slipped) TLS. The results indicate that there is a strong requirement for Rev7p during Pol zeta-mediated TLS measured in vivo. Furthermore, Rev1p is needed only for non-slipped TLS; slipped TLS remains efficient in its absence, revealing a previously uncharacterized Rev1p activity similar to Escherichia coli UmuDC function. Specifically, this activity is required for elongation from a correctly aligned lesion terminus.

Analysis of damage tolerance pathways in Saccharomyces cerevisiae: a requirement for Rev3 DNA polymerase in translesion synthesis.

The replication of double-stranded plasmids containing a single N-2-acetylaminofluorene (AAF) adduct located in a short, heteroduplex sequence was analyzed in Saccharomyces cerevisiae. The strains used were proficient or deficient for the activity of DNA polymerase zeta (REV3 and rev3delta, respectively) in a mismatch and nucleotide excision repair-defective background (msh2delta rad10delta). The plasmid design enabled the determination of the frequency with which translesion synthesis (TLS) and mechanisms avoiding the adduct by using the undamaged, complementary strand (damage avoidance mechanisms) are invoked to complete replication. To this end, a hybridization technique was implemented to probe plasmid DNA isolated from individual yeast transformants by using short, 32P-end-labeled oligonucleotides specific to each strand of the heteroduplex. In both the REV3 and rev3delta strains, the two strands of an unmodified heteroduplex plasmid were replicated in approximately 80% of the transformants, with the remaining 20% having possibly undergone prereplicative MSH2-independent mismatch repair. However, in the presence of the AAF adduct, TLS occurred in only 8% of the REV3 transformants, among which 97% was mostly error free and only 3% resulted in a mutation. All TLS observed in the REV3 strain was abolished in the rev3delta mutant, providing for the first time in vivo biochemical evidence of a requirement for the Rev3 protein in TLS.

Inactivation of horseradish peroxidase by phenol and hydrogen peroxide: a kinetic investigation.

Inactivation of horseradish peroxidase (HRP) was examined in the presence of hydrogen peroxide alone and in the presence of hydrogen peroxide plus phenol. HRP is inactivated upon exposure to hydrogen peroxide (H2O2) by the combination of two possible pathways, dependent upon hydrogen peroxide concentration. At low H2O2 concentrations (below 1.0 mM in the absence of phenol), inactivation is predominantly reversible, resulting from the formation and accumulation of catalytically inert intermediate compound III. As H2O2 concentrations increase, an irreversible mechanism-based inactivation process becomes predominant. The overall inactivation comprised of both processes exhibits a second-order inactivation rate constant (kapp) of 0.023 +/- 0.005 M-1 s-1 at pH 7.4 and 25 degrees C. In the presence of both hydrogen peroxide fixed at 0.5 mM and phenol, HRP was inactivated in an irreversible, time- and phenol concentration-dependent process, also mechanism-based, with a kapp of 0.019 +/- 0.004 M-1 s-1.