To identify proteins that are required for induced mutagenesis, we carried out a screen for non-essential genes in S. cerevisiae
that when absent, render cells less able to mutate. The screen identified all of the key non-essential PRR genes previously known to be involved in mutation, RAD5
, and SRS2
. The screen also identified FYV6
, which appear to cooperate in a second, distinct pathway of induced mutation. Fyv6 has no sequence homology to a known protein and its identification in high-throughput screens for mutants sensitive to the antifungal K1 killertoxin [46
] and mutants with altered telomere length [47
] suggest no obvious function. While it has been suggested that Fyv6 plays a role in regulation of non-homologous end joining in stationary cells [34
], this activity does not appear to contribute to the protein’s role in induced mutation. However, FYV6
has also been isolated in a high-throughput screen for genes required for resistance to DTT [48
]. This screen also identified the genes encoding thioredoxin, which is an essential electron donor for RNR. Given the role of RNR in this pathway, one plausible role for Fyv6 is that it interacts in some manner with thioredoxin to help regulate RNR activity. Nonetheless, Fyv6 appears to play a role in only a subset of the mutations induced by Rnr4.
encodes one of the four subunits of RNR. We find that deletion of RNR4
results in an up to 10-fold reduction in UV-induced mutation. Previously, Rnr3, another subunit of RNR has been implicated in MMS-induced mutation. Specifically, the inhibition of the TORC1 pathway, which controls transcription of RNR3
via Rad53, also results in a reduction of mutation in response to MMS [49
]. These data suggest that the effect on induced mutation may be generalized to other subunits of the RNR complex. We also demonstrated that RNR4
is required for UV- and MMS-induced increases in dNTP levels, consistent with the ability of the protein to stimulate TLS as proposed previously [38
]. Moreover, our data suggest that RNR4 functions in a previously unknown second pathway of induced mutation that involves TLS by the replicative polymerase Polδ. While this pathway contributes significantly to UV and MMS-induced mutation, it appears to be the major pathway responsible for EMS-induced mutation.
Interestingly, Polδ has been previously implicated in TLS across an abasic site in vivo
, at least when the abasic site is located within a single-stranded region of a plasmid [50
]. Moreover, the hypermutability associated with rendering Polδ exonuclease deficient has been shown to depend on the activity of the S-phase checkpoint [51
]. The hypermutability is significantly diminished by deletion of DUN1
, which encodes a substrate of Rad53 that regulates RNR activity, and this hypermutability is not affected by deletion of REV3
. These data are consistent with an S-phase checkpoint-mediated upregulation of dNTP levels for Polδ mediated TLS.
A model consistent with all of the data is presented in . In the case of UV-damage, the majority of photo-lesions are repaired by NER, but unrepaired photolesions, or lesions induced by other types of mutagens (i.e. MMS or EMS) that are not repaired prior to S-phase, cause replication forks to stall and the induction of the intra-S checkpoint response. As part of this response, Rnr4 is upregulated and the resulting increase in dNTPs facilitates error-prone TLS by Polδ, perhaps specifically during lagging strand synthesis where Polδ is thought to predominantly function [52
]. This pathway is also favored by disabling Polδ exonuclease proofreading activity, presumably because this activity competes with TLS. If TLS does not occur, genome replication is completed by error-free or mutagenic PRR, perhaps involving gap filling after re-initiation of synthesis downstream of the blocking lesion. This explains why factors that favor Polδ TLS, including disabling its exonuclease activity or the presence of RNR4
, strongly reduce the damage sensitivity of PRR deficient cells.
Figure 6 Model of induced mutation. The X in the template represents a lesion and the box in the newly synthesized DNA represents a mutation. See discussion for details.
The data does not rule out a contribution of Rnr4 to Polζ-mediated mutagenic PRR, and examining this issue will require additional studies. What is clear from the data is that the Polζ and Polδ pathways are distinct and that the Polδ pathway relies on Rnr4, but not on Polζ. Both pathways, Rnr4/Polδ and mutagenic PRR, appear to contribute to mutation induced by many types of DNA damage, including UV and MMS, but the Rnr4/Polδ pathway alone appears to play an important role in EMS-induced mutagenesis. It seems likely that UV- and MMS-induced damage are more potent blocks of replication, and thus are more likely to require the specialized activities of the conventional TLS polymerases, while EMS induces damage that may be more easily replicated through by Polδ, at least in the presence of elevated levels of dNTPs. The effects of other mutagens may similarly partition between these two pathways depending on the ability of Polδ at elevated levels of dNTPs to synthesize through the associated lesion.
In addition to facilitating lesion bypass, the presence of elevated dNTP levels may also reduce the fidelity of Polδ acting on undamaged templates and may result in the induction of undirected mutations. In fact, elevated nucleotide pools have been associated previously with reduced fidelity of DNA replication possibly by making competitive the rate at which a mispair is extended with the rate at which it is excised [54
]. Additional work is required to deconvolute the contributions of lesion directed and undirected mutation mediated by the Rnr4/Polδ pathway.
The ability of HU to inhibit Rnr4/Polδ-mediated mutation is particularly interesting, as it suggests that if similar mechanisms are conserved in human cells, the mutations induced during chemotherapy might be inhibited. The potential therapeutic utility of such an approach depends on the proportion of mutations mediated by Rnr4/Polδ as opposed to PRR, which is currently under investigation. Whatever the result, the data suggest that there are a finite number of pathways, perhaps only two, that independently induce mutation. Thus, HU combined with an inhibitor of Polζ, might represent a therapeutic combination that efficiently eliminates all forms of chemotherapy-induced mutation. Studies directed towards reaching these goals promise to provide an unprecedented opportunity to understand genome instability in eukaryotic cells and also to intervene in mutation, and thus in the development and progression of cancer.