Influence Of Deubiquitinating Enzymes On Mutagenesis In Saccharomyces Cerevisiae

摘要

In recent years, it became clear that the mutagenic effect of base alterations in a DNA template is dependent on bypass synthesis, carried out by one or more translesion polymerases. A critical role for proliferating cell nuclear antigen (PCNA) and its ubiquitination following DNA damage has been established. Among Saccharomyces cerevisiae deletion mutants in which UV mutagenesis is compromised, we identified and characterized a mutant of the ubiquitin recycling protein Doa4. Similar cases may be represented by Doa1, previously described by others, as well as Bro1 and Ubi4. We discuss overall altered ubiquitin levels or failure to deubiquitinate specific target proteins as likely explanations. This study is of relevance for understanding and possibly modifying the mutagenic effect of DNA-damaging agents in environmental toxicology, cancer treatment and cancer prevention. Introduction The cellular responses to various kinds of DNA damage have by now been elucidated to a significant degree in pro- and eukaryotes (Friedberg et al., 2006). However, concepts explaining one of the most severe consequences of DNA damaging treatments – the enhancement of mutagenesis – have emerged only during the last few years. With the discovery of translesion polymerases, however, the frequently postulated replicative bypass of altered bases of reduced coding capacity gained a mechanistic foundation. Data from the eukaryotic model budding yeast (Saccharomyces cerevisiae) were essential for this progress and the basic mechanisms appear to be evolutionarily conserved. Pathways leading to mutation are best understood for DNA damage by UV-C radiation, resulting in pyrimidine dimers. The prototype of an error-prone polymerase is polymerase ?eta, the complex of proteins Rev3 and Rev7 (Nelson et al., 1996b). This polymerase is responsible for most induced and spontaneous mutagenic events. Of equal importance is Rev1, a deoxycytidyl transferase with an independent structural role in mutagenesis, possibly as a platform for several bypass polymerases (Acharya et al., 2005; Acharya et al., 2007; Acharya et al., 2006; Guo et al., 2006a; Nelson et al., 1996a). In contrast to this error-prone bypass, a mostly error-free bypass of the most frequent UV photoproducts, dipyrimidine cyclobutane-type dimers is catalyzed by polymerase eta (Johnson et al., 1999; Yu et al., 2001). Consequently, the enzyme’s action lowers overall mutability since it competes with error-prone bypass mechanisms. A second major error-free tolerance pathway of different mechanism is the Rad5 pathway that appears to involve template switching (Li et al., 2002; Zhang and Lawrence, 2005). The current concept of bypass pathway choice is based upon PCNA and its modifications by ubiquitin (Ub) and SUMO (Fig. 1). Monoubiquitination of PCNA (at K164 in yeast) is found after methylmethane sulfonate (MMS) or UV treatment (Hoege et al., 2002; Kannouche et al., 2004) and a requirement of ubiquitinated PCNA for UV mutagenesis has indeed been demonstrated (Stelter and Ulrich, 2003; Zhang et al., 2006). The Rad6-Rad18 complex can bind to single-stranded DNA (Bailly et al., 1994; Bailly et al., 1997) which emerges as a consequence of blocked replication. The Ub conjugating activity of Rad6 catalyzes monoubiquitination of PCNA at K164 which increases its affinity for bypass polymerases (Guo et al., 2006b; Kannouche et al., 2004). Monoubiquitination of PCNA attracts error-prone polymerases mediating a mutagenic bypass, presumably through the ubiquitin-binding domain of Rev1. On the other hand, the signal for the error-free bypass by template-switching is subsequent K63-linked polyubiquitination, resulting from the action of the Ubc13-Mms2-Rad5 complex, with the Ubc13-Mms2 heterodimer functioning as the ubiquitin-conjugating (E2) enzyme in conjunction with the Rad5 (E3) ubiquitin ligase (Hoege et al., 2002; Ulrich and Jentsch, 2000).