Table 2.
In vitro complementation of mismatch repair defects in 2APurR isolates
| 2APurR cell extract
|
||||||||
|---|---|---|---|---|---|---|---|---|
| None | VB101 | VB105 | VB201 | VB301 | VB305 | VB401 | VB701 | |
| Repair (fmol/hr per 0.1 mg extract) | ||||||||
| Addition | ||||||||
| None | — | <0.5 | <0.5 | <0.5 | <0.5 | <0.5 | <0.5 | <0.5 |
| mutS− extract | <0.5 | <0.5 | 13 | 12 | 5.2 | 12 | <0.5 | 11 |
| mutL− extract | <0.5 | 10 | 0.8 | <0.5 | 9.2 | <0.5 | 7.4 | <0.5 |
| mutH− extract | <0.5 | 13 | 6.6 | 4.9 | 13 | 8.8 | 13 | 6.7 |
| ExoI | — | <0.5 | <0.5 | <0.5 | <0.5 | <0.5 | <0.5 | <0.5 |
| ExoI + MutS | — | 6.7 | n.t. | <0.5 | 4.3 | n.t. | 1.3 | n.t. |
| ExoI + MutL | — | <0.5 | 20 | 7.2 | 1.0 | 21 | <0.5 | 20 |
| ExoI + MutH | — | <0.5 | <0.5 | <0.5 | 1.3 | <0.5 | <0.5 | <0.5 |
| ExoI + SSB | — | 2.5 | n.t. | <0.5 | 10 | n.t. | <0.5 | n.t. |
| ExoI + UvrD | — | <0.5 | <0.5 | <0.5 | 3.2 | <0.5 | <0.5 | <0.5 |
| Western blot | MutS | − | + | + | + | + | + | + |
| MutL | + | − | − | + | − | + | + | |
Extracts of 2APur resistant strains were assayed (Experimental Procedures) for repair of a 3′-G-T heteroduplex (9) in the presence of extracts derived from mutS−, mutL−, or mutH− derivatives of the parental strain BT199 or in the presence of purified proteins as indicated. For extract complementation, assays contained approximately 0.1 mg of each extract tested. For complementation with purified proteins, individual extracts (0.1–0.2 mg) were supplemented with ExoI (10 ng), together with MutS (35 ng), MutL (80 ng), MutH (1 ng), single-strand binding protein (400 ng), or UvrD (DNA helicase II, 20 ng) as shown. ExoI restores mismatch repair directed by a 3′ strand signal to E. coli extracts deficient in RecJ, ExoVII, ExoI, and ExoX (unpublished work). No detectable repair was observed in VB101 or VB401 extracts supplemented with MutS in the absence of ExoI, or in VB105, VB201, VB305, or VB701 extracts supplemented with MutL in the absence of the exonuclease. Despite the efficient complementation of VB401 extracts by extracts deficient in MutH or MutL, restoration of repair by addition of ExoI and purified MutS was consistently low, although other purified proteins did not increase repair to detectable levels. Immunological blot analysis was performed on extract samples using polyclonal serum against MutS, MutL, or DNA helicase II by a method described previously (44). Normal levels of the helicase II polypeptide were present in all extracts. n.t., not tested.