The integrity of the genome is under continuous attack from external insults as well as a result of the normal cellular metabolism or errors that take place during DNA replication or repair. Cells have therefore evolved a large arsenal of mechanisms that help them cope with various forms of DNA damage. The FA pathway has been shown to play an important role in repairing inter-strand cross links (ICLs).4
This pathway, composed of at least 15 proteins in mammalian cells, appears to be at least partially conserved in yeast. Two recent publications indeed have shown that the yeast orthologs of human FA proteins participate in ICL repair.45,46
Interestingly, a connection was found between the yeast FA pathway and portions of the post-replication repair (PRR) pathway as well as with PCNA modifications.45
The yeast Elg1 protein has been suggested to act as an unloader of SUMOylated PCNA, and it also genetically interacts with the PRR pathway: for example, mutations in ELG1
suppress the sensitivity of rad5
mutants to DNA damaging agents.33
Here, we have analyzed the physical and genetic interactions between Elg1 and members of the FA pathway in yeast.
We have uncovered a physical interaction between Elg1 and the Mhf1 protein (). Mhf1 and Mhf2 are small conserved proteins.9
Their crystal structure has recently been solved, and it shows that the two proteins form a tetramer, that resembles the histone (H3-H4)2
Deletion of any of the two proteins, or of both, does not result in sensitivity to any DNA damaging agent tested in S. pombe9,41
or in budding yeast (this work). Indeed, in previous publications, the effect of mhf1
Δ and mhf2
Δ could be discerned only on the background of an srs2
Here, we show an additional phenotype for these mutants: they suppress the sensitivity to hydroxyurea of elg1
Δ mutants ().
The Mhf proteins have been shown to form a complex with the FANCM/Mph1 helicase.9,45
Consistent with this proposal, Elg1 showed physical interactions with both Mhf1 and Mph1 (). However, our genetic results suggest that the Mhf proteins do not always act in a complex with Mph1. The phenotypes of mph1
Δ mutants were very different from those observed in strains mutated for the MHF
genes. For example, although mutations in MPH1
do not affect the sensitivity of elg1
Δ mutants to HU, this sensitivity was rescued by mutations in the MHF genes. Furthermore, the rescue was independent of Mph1. These findings suggest that the Mhf proteins can function independently of Mph1 to modulate HU resistance. Moreover, mutations in MPH1
increase the sensitivity of elg1
mutants to MMS, whereas mutations in MHF1, MHF2
or both have no additional effect ().
The results of our analysis show complex genetic interactions between the components of the FA pathway (), which depend on the DNA damaging agent tested. Below, we discuss each of the damaging agents separately.
Methyl methanesulfonate (MMS) methylates DNA on N7-deoxyguanine and N3-deoxyadenine, and is believed to stall the replication fork. Mutations in ELG1, MPH1
cause a mild sensitivity to this agent, similar for all single mutants. However, when combined, a synergistic effect can be observed: elg1
Δ and chl1
Δ double mutants are sensitive to 0.010% MMS (). In contrast, elg1
Δ mutants shows an even stronger sensitivity, being unable to form colonies on plates containing as little as 0.003% MMS. Deletion of MPH1
in this strain does not confer further sensitivity. Our results thus point to a hierarchical order between these DNA repair proteins. It is clear from our results that Chl1 is a major player and constitutes a strong alternative to Elg1: in the absence of both Chl1 and Elg1, cells are very MMS-sensitive. Moreover, under these circumstances mutations in Mph1 have no further effect (). These results can be explained () by assuming that there are two alternative pathways to bypass the replication stalling, one Chl1-dependent and another Elg1- (and Mph1-) dependent. The helicase activity of Chl1 could be involved in fork reversal, whereas Elg1 is necessary to remove SUMOylated PCNA from the stalled fork38
. Mph1 has been implicated in D-loop formation and may contribute to a pathway of homologous recombination involving the recently synthesized sister chromatid35
(). It should also be noted that both Elg1 and Chl1 have known roles in sister chromatid cohesion.47,48
Cohesion between the sisters, or some kind of interaction with the cohesin complexes, could constitute a pre-requisite for the activity of Mph1; without the activity provided either by Chl1 or by Elg1, mutations in MPH1
have no effect. Interestingly, as noted above, the Mhf proteins do not seem to play any role in the repair of MMS-caused lesions.
The picture is slightly different for hydroxyurea (HU). This drug causes inactivation of the ribonucleotide reductase complex, effectively depleting the pool of dNTPs and possibly causing fork stalling. Despite the fact that elg1
mutants have increased levels of dNTPs,49
these cells are sensitive to HU at high concentrations. Interestingly, this sensitivity depends on the presence of the Mhf proteins, as deletion of any of them or both, suppresses the HU sensitivity of elg1
strains (). As explained above, it has been proposed that the Mhf proteins and Mph1 form a complex. However, in contrast to the expectation from a single protein complex, a double mutant elg1 mph1
is as sensitive to HU as the single elg1
mutant (), whereas mutations in the MHF
genes suppress the sensitivity of elg1
, suggesting that only the Mhf proteins, and not Mph1, play roles in HU resistance. We thus suggest that Mhf1 and Mhf2 can form a complex with Elg1, which may control their loading or activity. In the absence of Elg1, the Mhf1/2 activity becomes toxic, and deletion of any of these two proteins alleviates the sensitivity of elg1
mutants to HU (). The toxicity of the Mhf proteins could be related to their resemblance to the histone (H3-H4)2
which may be required as a molecular decoy during DNA repair but could impede normal genomic activity if left unchecked. Interestingly, recent work has suggested that the FA pathway in mammals may play also a role in controlling histone deposition and its regulation during DNA repair.16
Mutations in CHL1 confer sensitivity to HU similar to that of the elg1Δ mutant. The two mutations showed an additive phenotype, which was not further affected by mutations in MPH1. This again suggests the existence of two parallel pathways, one ruled by Chl1 and the other by Elg1. The role of Mph1 in resistance to HU could be seen only in the absence of Chl1: the double mutant chl1Δ mph1Δ was more sensitive than the single chl1Δ strain (). The triple elg1Δ chl1Δ mph1Δ was not more sensitive than the elg1Δ chl1Δ double mutant, which supports the idea that Mph1 plays a role in the Chl1-independent Elg1 pathway (). Fork reversal by the Chl1 helicase seems to be the preferred mechanism of replication fork re-initiation in the presence of HU, with Elg1 serving as a backup by controlling the activity of Mhf1/2. The Mph1-dependent homologous recombination sub-pathway, however, is not used much in the presence of HU, if the Chl1 pathway is active ().
Our results thus show that Chl1 and Elg1 play alternative roles with respect to survival of both MMS and HU. The need for either Elg1 or Chl1 is seen not only in the sensitivity to DNA damaging agents: the elg1
Δ and chl1
Δ mutations exhibit a synthetic fitness defect ( and ). We have investigated what region in Elg1 is responsible for the essential function in the absence of Chl1. Our results () show that neither the N terminus, which has been implicated in the interactions between Elg1 and SUMOylated proteins,38,48
nor the C terminus, which is important for its repair function ( and ref. 42
) are necessary. The region of Elg1 defined by our studies (between aas 517 and 731) has been shown to be important for the incorporation of Elg1 into an RFC-like complex,42
suggesting that its interactions with the small Rfc subunits (Rfc2–5) are important here.
We have explored the interactions between the yeast FA pathway members and PCNA. Interestingly, the two PCNA mutations analyzed behaved in very different fashion: pol30–104 mutants, carrying the A251V substitution, showed epistatic interactions with elg1Δ with respect to the sensitivity to MMS and HU, and was able to slightly suppress the synthetic sick phenotype of elg1Δ chl1Δ double mutants. This PCNA mutant indeed shares with elg1Δ a number of synthetic genetic interactions, suggesting that either the region of PCNA affected (the inter-domain loop) is responsible for the attachment of Elg1, or, alternatively, that binding of a still unknown factor to this region is essential to carry out Elg1’s function. The suppression of the synthetic sickness of an elg1Δ chl1Δ mutant supports the second model: binding of the unknown factor may be toxic in a strain devoid of both Elg1 and Chl1; a mutation that prevents its binding alleviates the synthetic sickness.
In striking contrast, mutations in the lysines 127 and 164 of PCNA, which abrogate post-translational modifications of the clamp, exhibited increased toxicity and sensitivity to MMS. Lysine 164 can undergo both mono- and poly-ubiquitination, which direct lesion bypass by trans-lesion synthesis (mono-ubiquitination) or template-switch synthesis (poly-ubiquitination). In addition, lysine 164 (and lysine 127 as well) can also undergo SUMOylation. This modification is believed to prevent homologous recombination events at the fork (reviewed in ref. 50
). Thus, in a pol30-RR
mutant, the bypass pathways are abolished and recombination is unchecked. The additional synthetic sickness and sensitivity to MMS conferred to the elg1
Δ mutant by the pol30-RR
mutation suggests that the bypass pathways abolished play a central role when both Chl1 and Elg1 are inactivated; alternatively, the unchecked recombination may be toxic. Deletion of RAD52
in a elg1
Δ background enhanced the synthetic sickness (data not shown), indicating that the low fitness is not caused by increased recombination. Surprisingly, the epistasis observed in HU suggests that Elg1 might cooperate with one of the bypass mechanisms to deal with the effects of HU.
Two papers have very recently characterized the role of the yeast FA orthologs in the repair of ICLs. Daee and coworkers found that the pathway is required for the repair of nitrogen mustard-induced ICLs by a mechanism that is independent of the Pso2 protein, previously identified as essential for ICL repair,51
but relies on the Rad5 component of the post-replication repair pathway. Moreover, they also found evidence for a role of Mph1 in preventing ICL-stalled replication intermediates from collapsing into double-strand breaks.45
Ward and coworkers also identified the yeast FA pathway as an alternative to Pso2 in the repair of ICLs, and suggested that this pathway includes the yeast mismatch repair system (Mutsα).46
In both papers, the yeast FA genes appear genetically to work as a single pathway. We have shown here that this is not true when dealing with other forms of DNA damage.