J Gong, W Siede
J Gong, W Siede. Influence Of Deubiquitinating Enzymes On Mutagenesis In Saccharomyces Cerevisiae. The Internet Journal of Microbiology. 2010 Volume 9 Number 2.
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
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 (
Pathways leading to mutation are best understood for DNA damage by UV-C radiation, resulting in pyrimidine dimers. The prototype of an
In contrast to this error-prone bypass, a mostly
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).
Given the importance of Ub for translesion synthesis, it is perhaps surprising that the impact of cellular Ub concentration on mutagenesis has not been studied in detail. For example, whereas a deletion of the polyubiquitin gene
Posttranslational protein modification by Ub and specifically by K48-linked polyUb has been traditionally linked with promoting degradation through the 26S proteasome. Ub is a long lived-protein and, consequently, deubiquitination enzymes (DUBs) that recycle Ub from proteins marked for degradation play an important role in Ub homoeostasis. DUBs are also needed to reverse ubiquitination from target proteins whose activity or localization was altered by Ub and from those that were accidentally ubiquitinated. This is a topic of considerable complexity, given the identification of more than 100 DUBs in humans (Amerik and Hochstrasser, 2004; Reyes-Turcu et al., 2009).
Our interest in exploring the relationship between DUBs and induced mutagenesis stems from the isolation of a mutant of Doa4, a DUB important for Ub homoeostasis, in a screen for mutants defective in UV mutagenesis (Gong and Siede, 2009). Doa1 may represent a similar case, studied by other investigators (Lis and Romesberg, 2006). Here, we present a characterization and discussion of the role of Doa4 in DNA-damage-induced mutagenesis.
Material and Methods
The initially used strains were all derivatives of BY4741 (
Cells were grown to logarithmic phase for 16 h or to saturation for 48 h in YPD (1% yeast extract, 2% peptone, 2% dextrose) at 30°C. For UV radiation assays, cells were washed and resuspended in sterile water, appropriate dilutions were plated on synthetic media plates with or without canavanine or tryptophane (Amberg et al., 2005) and irradiated (germicidal UV-C). For MMS assays, cells were incubated in YPD at a titer of 2x107 cells/ml for 1 h at 30°C, washed and plated. Colonies were counted after 3-5 days of incubation. Fractions of surviving cells and mutants per surviving cells were calculated.
The key data that initiated this project originated from a screen of the haploid
To prove the general validity of our observation, we transferred the
We noted a small fraction of distint fast growing mutants of the
However, this defect in mutagenesis appeared to be damage-specific. In response to the alkylating agent MMS,
We detected a partial defect in UV mutagenesis in deletion mutants of
Such a role of a DUB in DNA-damage responses is not without precedence. As described by Lis and Romesberg, Doa1 appears to represent a similar case since it is required for normal resistance to MMS and hydroxyurea (Lis and Romesberg, 2006) as well as UV (our data, Table 1). The protein is required for normal Ub levels (Ghislain et al., 1996), it binds Ub and prevents the accumulation of K-48 linked Ub trimers suggesting that Doa1 (or a co-purifying protein) acts as a Ub protease (Lis and Romesberg, 2006). MMS-induced histone H2B- and PCNA monoubiquitination are not detected in
It is important to distinguish between a general influence on bypassing DNA damage in a mutagenic fashion and effects restricted to this specific selection system (Gong and Siede, 2009; Lemontt, 1977; Lis et al., 2008). Preferably, mutation data need to be confirmed with mutational systems of different nature. Canavanine sensitivity is quite common in mutants of the Ub/proteasome system and has indeed been described for mutants of
Consequently, we constructed a
Two hypotheses can be invoked to explain these observations. First, the DUB in question may be required for Ub recycling and the
The first hypothesis is preferred because of the characterized Doa4 function that has now been primarily implicated in recycling Ub from vacuolar membrane-bound proteins (Amerik et al., 2000; Swaminathan et al., 1999). A direct interaction with DNA repair/mutagenesis proteins appears therefore somewhat unlikely.
An obvious candidate for an indirect DUB target that influences mutability is PCNA. A defect in PCNA ubiquitination following MMS treatment has already been detected for
However, three observations do not fit with this concept. First, UV sensitivity of the DUB mutants (Tab. 1) does not approach that of mutants of the Rad5/Mms2/Ubc13 complex, the E3/E2 enzymes required for polyubiquitination of PCNA and error-free damage tolerance (Fig. 1). If Ub homoeostasis is disturbed, it is not obvious why
Future studies will address these discrepancies and may reveal other, relevant targets. For example, H2B represents another Ub modified protein that is important for DNA-damage transactions (Lis and Romesberg, 2006). Additional candidate targets that may need to be degraded through the proteasome pathway include the checkpoint clamp loader Rad24 (if it behaves similar to the human homolog) (Zhang et al., 2010).
In the future, it should also be explored why only certain DUBs and not others may have an influence on mutagenesis although their effect on free Ub levels might be quite comparable. The concept of Ub channeling to certain targets has been brought up in conjunction with the
Protein degradation and Ub-metabolizing proteins were found to be conserved in evolution and thus, the suggested studies may very well be relevant for higher eukaryotes. Interestingly, Usp6 as a potential human ortholog of Doa4 is an oncogene critically important for the development of aneurysmal bone cysts (Oliveira et al., 2006). The described chromosome translocations in this disease lead predominantly to the overexpression of Usp6.
Many additional implications can be listed. For example, manipulation of cellular Ub levels may be used to modify the outcome of cancer chemotherapy and to reduce mutagenic side effects.
These studies were supported by a grant from the National Institutes of Health (NIEHS grant ES011163).