The results described in this report show that MutS and MutSΔ800 proteins, produced from multicopy plasmids, impart different properties to cells. Although cells with the MutSΔ800 or MutS protein are not mutators ( and ), the MutSΔ800-containing cells are deficient in antirecombination () and not sensitized to cisplatin cytotoxicity (). This is the first demonstration of a separation of function phenotype for an E.coli mutS mutation. We propose that the C-terminal domain of MutS is required for efficient antirecombination and cisplatin sensitization, but not mutation avoidance or MNNG sensitization. Given that MutSΔ800 can only form monomers and dimers, we postulate that the inability to form tetrameric MutS could be responsible for this phenotype. The data reported here also suggests that although MutS and MutSΔ800 may bind base mismatches as dimers, conversion to the terameric form may be required for subsequent reactions in mismatch repair.
The results we have obtained in this report are best explained by the findings of Bjornson et al
) who have argued that the MutS tetramer is likely the active form of MutS in mutation avoidance. To support this conclusion, they found that MutSΔ800 protein bound to mismatched DNA less efficiently than MutS. Our results also indicate that MutSΔ800 does have reduced affinity for GG–CT base mismatches () but not with the one base IDL () or O6
-meG mismatches (). Given that MutSΔ800 has reduced affinity for some base mismatches, we propose that the lack of a mutator phenotype in cells over-expressing MutSΔ800 ( and ) can be explained by the MutS dimer being able to cope with the few mismatches generated behind the replication fork. The atomic structure of MutSΔ800 indicates that mismatch binding does occur. In interspecies crosses, however, the much larger number of mismatches overwhelms the MutSΔ800 protein's reduced mismatch binding and processing ability, thereby leading to reduced antirecombination as shown in . An essential part of our proposal, therefore, is that the cell's response with dimeric MutS depends on the number of mismatches involved; if there are few, overproduced dimeric MutS can substitute for tetrameric MutS at its normal cellular concentration. The data in and , showing a greater amount of M13-fd heteroduplex formation by MutSΔ800 compared to MutS, is consistent with this model. The combination of MutL and MutSΔ800 is as effective as MutS and MutL where the level of mismatching is 3%. We predict that at higher levels of mismatching, such as the 17% in E.coli
crosses, MutSΔ800 would be much less effective than MutS, even in the presence of MutL.
Bjornson et al
) also showed that MutH-induced incision of heteroduplex DNA, in the presence of MutL, was reduced by MutSΔ800. This observation can also help to explain the response of dam
mutants with the over-expressed MutSΔ800 protein to cisplatin and MNNG. For cells exposed to MNNG, there is sufficient binding of MutSΔ800 to O6
-meG mismatches to provoke a sensitization response even though there is reduced MutH-induced incision (). For dam
cells expressing MutSΔ800 and exposed to cisplatin, however, the reduced binding of MutSΔ800 to intrastrand diguanyl-cisplatin cross-links and the reduced MutH-induced incision activity allow the cell to repair, by recombination, the few DSBs that might be formed ().
The model proposed above regarding the number of mismatched base pairs and the ability of over-expressed MutSΔ800 to deal with them makes the strong prediction that when MutSΔ800 is expressed from a single-copy gene, the dam cells containing it would have different responses for spontaneous mutagenesis and resistance to cisplatin and MNNG compared to expression from a multicopy plasmid. Experiments to test this prediction are in progress.
Surprisingly, MutSΔ800 when expressed in a wild-type background does not display a dominant negative phenotype with regard to cisplatin sensitivity (). This result appears to rule out the possibility that MutS, which is at a low cellular concentration, could be titrated out by the plasmid expressed MutSΔ800, leading to a dominant negative phenotype. We have not yet quantitated the protein levels of the plasmid expressed MutSΔ800 versus the chromosomally expressed copy of MutS. If the MutSΔ800 protein is less stable than MutS, then the ratio of the two proteins may be such that active mixed multimers are formed allowing for near-full functionality of the protein (), which is absent in a population containing only the MutSΔ800.
The results in indicate that the C-terminal end of MutS is required for binding to platinated GG–CC cross-links, which could also indicate a requirement for MutS tertamerization, in that the dimeric MutSΔ800 cannot recognize the lesion, whereas the MutS does. At present, it is not known how this cross-link opposite CC differs in structure from that opposite CT to allow efficient recognition by MutS, but it is possible that binding to a platinated lesion requires critical contacts with residues residing in the C-terminus of MutS, offering an alternative to the tetramerization requirement. Furthermore, deletion of the C-terminus may disrupt the binding of MutS to low-affinity lesions, such as the intrastrand GG cross-links (10- to 40-fold lower than a G–T mismatch (20
), but not to a high affinity one base IDL (). Either explanation is supported by the survival results for cells with the mutSΔ800
plasmid exposed to cisplatin (). In addition to these alternatives to tetramerization, we cannot rule out a critical loss of interaction(s) between MutSΔ800 and other proteins, such as the beta-clamp (32
) which can ultimately affect the efficiency of recognition or repair processes.
That MutS can bind to platinated GG–CC cross-links suggests that MMR-induced DSBs can occur in unreplicated DNA of dam
mutants at these sequences, as well as cross-links opposite CT bases, which can be formed by translesion polymerases (33
). Platinated GG–CC cross-links can also be formed during recombinational repair of DSBs and these are bound by MutS which can block further branch migration (17
), thereby preventing DSB repair. The ability of MutS to bind these cross-links can explain the observed cisplatin sensitivity of the dam mutS+
strain in . In contrast, the decreased binding of MutSΔ800 to such lesions reduces the number of mismatch repair-induced DSBs and thereby promotes survival of the dam mutSΔ800
strain to cisplatin ().
Finally, we note that mammalian cell lines also show sensitivity to cisplatin and MNNG and MMR-deficient lines derived from them are resistant to both compounds (35
), although whether cisplatin resistance is due specifically to MMR-deficiency has recently been challenged (37
). Cisplatin-resistant cells isolated from patients treated with this drug have also been shown to be deficient in MMR (39
). Like the bacterial MutS protein, the human MutS-alpha counterpart also binds to cisplatin intrastrand cross-links and O6
-meG mismatches (40
). The various models proposed to explain mismatch repair-dependent drug-resistance in mammalian cells have been reviewed in (41
). The data reported here with E.coli
MutSΔ800 have no counterpart in eukaryotic systems, thereby preventing selection of one model over another.