Table 2. Phosphorylation sites and genes selected for further characterization.
| Gene symbol | Target sequence | Modification position | Observed phenotype(s)a | Function |
|---|---|---|---|---|
| APN1 | ATAEPS(ph)DNDILSQMTK | S350 | MMS-sensitive | Base excision repair |
| APN1 | ATAEPSDNDILS(ph)QMTK | S356 | ||
| CTF4 | LFSDIT(ph)QEANAEDVFT(ph)QTHDGPSGLSEK | T401, T411 | MMS-sensitive | Checkpoint; Sister-chromatid cohesion |
| TOF1 | LTVSGS(ph)QALVDEK | S379 | MMS-sensitive; Interact with rad9Δ and dia2Δ. | |
| TOF1 | FNIS(ph)EGDITK | S626 | ||
| MPH1 | T(ph)GSSEEAQISGMNQK | T540 | MMS-sensitive | HR intermediate resolution |
| MPH1 | TGS(ph)S(ph)EEAQISGMNQK | S542 or S543b | ||
| RAD50 | QVFPLT(ph)QEFQR | T568 | MMS-sensitive | MRX |
| XRS2 | APEVEAS(ph)PVVSK | S349 | MMS-sensitive; Telomere maintenance; Interact with exo1Δ, yku80Δ, and sae2Δ | |
| XRS2 | NAAFLIT(ph)R | T675 | ||
| RAD18 | INFTSMT(ph)QS(ph)QIK | T282 or S284b | MMS-sensitive | PRR |
| RAD18 | SMT(ph)DILPLSSKPSK | T155 |
Since PRR genes play an essential role in the replication stress response, RAD18 was included even though the induction of phosphorylation by MMS for these proteins was detected in only one of the forward or label-swap experiments. The same is true for the phosphorylation of Mph1T540 and Apn1S356. The mass spectra of the detected phosphopeptides from these gene products (in either the forward or label-swap experiment) were manually inspected and confirmed.
The observed phenotypes reflect phenotypes of the phospho-mutants in which all detected MMS-inducible phosphosites are mutated.
The mass spectra were not able to distinguish between phosphorylation of Mph1 on S542 vs. S543 or phosphorylation of Rad18 on T282 vs. S284; in all ambiguous cases, both sites were mutated.