Proteasomal regulation of the mutagenic translesion DNA polymerase, Saccharomyces cerevisiae Rev1.

Translesion DNA synthesis (TLS) functions as a tolerance mechanism for DNA damage at a potentially mutagenic cost. Three TLS polymerases (Pols) function to bypass DNA damage in Saccharomyces cerevisiae: Rev1, Pol ζ, a heterodimer of the Rev3 and Rev7 proteins, and Pol η (Rad30). Our lab has shown that S. cerevisiae Rev1 protein levels are under striking cell cycle regulation, being ∼50-fold higher during G2/M than during G1 and much of S phase (Waters and Walker, 2006). REV1 transcript levels only vary ∼3-fold in a similar cell cycle pattern, suggesting a posttranscriptional mechanism controls protein levels. Here, we show that the S. cerevisiae Rev1 protein is unstable during both the G1 and the G2/M phases of the cell cycle, however, the protein's half-life is shorter in G1 arrested cells than in G2/M arrested cells, indicating that the rate of proteolysis strongly contributes to Rev1's cell cycle regulation. In the presence of the proteasome inhibitor, MG132, the steady-state levels and half-life of Rev1 increase during G1 and G2/M. Through the use of a viable proteasome mutant, we confirm that the levels of Rev1 protein are dependent on proteasome-mediated degradation. The accumulation of higher migrating forms of Rev1 under certain conditions shows that the degradation of Rev1 is possibly directed through the addition of a polyubiquitination signal or another modification. These results support a model that proteasomal degradation acts as a regulatory system of mutagenic TLS mediated by Rev1.

The DNA polymerase activity of Saccharomyces cerevisiae Rev1 is biologically significant.

A cell's ability to tolerate DNA damage is directly connected to the human development of diseases and cancer. To better understand the processes underlying mutagenesis, we studied the cell's reliance on the potentially error-prone translesion synthesis (TLS), and an error-free, template-switching pathway in Saccharomyces cerevisiae. The primary proteins mediating S. cerevisiae TLS are three DNA polymerases (Pols): Rev1, Pol ζ (Rev3/7), and Pol η (Rad30), all with human homologs. Rev1's noncatalytic role in recruiting other DNA polymerases is known to be important for TLS. However, the biological significance of Rev1's unusual conserved DNA polymerase activity, which inserts dC, is much less well understood. Here, we demonstrate that inactivating Rev1's DNA polymerase function sensitizes cells to both chronic and acute exposure to 4-nitroquinoline-1-oxide (4-NQO) but not to UV or cisplatin. Full Rev1-dependent resistance to 4-NQO, however, also requires the additional Rev1 functions. When error-free tolerance is disrupted through deletion of MMS2, Rev1's catalytic activity is more vital for 4-NQO resistance, possibly explaining why the biological significance of Rev1's catalytic activity has been elusive. In the presence or absence of Mms2-dependent error-free tolerance, the catalytic dead strain of Rev1 exhibits a lower 4-NQO-induced mutation frequency than wild type. Furthermore, Pol ζ, but not Pol η, also contributes to 4-NQO resistance. These results show that Rev1's catalytic activity is important in vivo when the cell has to cope with specific DNA lesions, such as N(2)-dG.

Eukaryotic translesion polymerases and their roles and regulation in DNA damage tolerance.

DNA repair and DNA damage tolerance machineries are crucial to overcome the vast array of DNA damage that a cell encounters during its lifetime. In this review, we summarize the current state of knowledge about the eukaryotic DNA damage tolerance pathway translesion synthesis (TLS), a process in which specialized DNA polymerases replicate across from DNA lesions. TLS aids in resistance to DNA damage, presumably by restarting stalled replication forks or filling in gaps that remain in the genome due to the presence of DNA lesions. One consequence of this process is the potential risk of introducing mutations. Given the role of these translesion polymerases in mutagenesis, we discuss the significant regulatory mechanisms that control the five known eukaryotic translesion polymerases: Rev1, Pol zeta, Pol kappa, Pol eta, and Pol iota.

A biophysical investigation of recombinant hemoglobins with aromatic B10 mutations in the distal heme pockets.

This study examines the structural and functional effects of amino acid substitutions in the distal side of both the alpha- and beta-chain heme pockets of human normal adult hemoglobin (Hb A). Using our Escherichia coli expression system, we have constructed four recombinant hemoglobins: rHb(alphaL29F), rHb(alphaL29W), rHb(betaL28F), and rHb(betaL28W). The alpha29 and beta28 residues are located in the B10 helix of the alpha- and beta-chains of Hb A, respectively. The B10 helix is significant because of its proximity to the ligand-binding site. Previous work showed the ability of the L29F mutation to inhibit oxidation. rHb(alphaL29W), rHb(betaL28F), and rHb(betaL28W) exhibit very low oxygen affinity and reduced cooperativity compared to those of Hb A, while the previously studied rHb(alphaL29F) exhibits high oxygen affinity. Proton nuclear magnetic resonance spectroscopy indicates that these mutations in the B10 helix do not significantly perturb the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces, while as expected, the tertiary structures near the heme pockets are affected. Experiments in which visible spectrophotometry was utilized reveal that rHb(alphaL29F) has equivalent or slower rates of autoxidation and azide-induced oxidation than does Hb A, while rHb(alphaL29W), rHb(betaL28F), and rHb(betaL28W) have increased rates. Bimolecular rate constants for NO-induced oxidation have been determined using a stopped-flow apparatus. These findings indicate that amino acid residues in the B10 helix of the alpha- and beta-chains can play different roles in regulating the functional properties and stability of the hemoglobin molecule. These results may provide new insights for designing a new generation of hemoglobin-based oxygen carriers.