A novel Rad24 checkpoint protein complex closely related to replication factor C

Rad24 functions in the DNA damage checkpoint pathway of Saccharomyces cerev isiae [1,2], Here, analysis of Rad24 in whole cell extracts demonstrated th at its mass was considerably greater than its predicted molecular weight, s uggesting that Rad24 is a component of a protein complex. The Rad24 complex was purified to homogeneity, In addition to Rad24, the complex included po lypeptides of 40 kDa and 35 kDa. The 40 kDa species was found by mass spect rometry to contain Rfc2 and Rfc3, subunits of replication factor C (RFC), a five subunit protein that is required for the loading of polymerases onto DNA during replication and repair [], We hypothesised that other RFC subuni ts, all of which share sequence homologies with Rad24 [], might also be com ponents of the Rad24 complex. Reciprocal co-immunoprecipitation studies wer e performed using extracts prepared from strains containing epitope-tagged RFC proteins. These experiments showed that the small RFC proteins, Rfc2, R fc3, Rfc4 and Rfc5, interacted with Rad24, whereas the Rfc1 subunit did not . We suggest that this RFC-like Rad24 complex may function as a structure-s pecific sensor in the DNA damage checkpoint pathway.