The Role Of RAD18 In DNA Double Strand Break Repair

In the DNA postreplication repair (PRR) RAD6 epitasis pathway, the error-free group comprises RAD 18, RAD5 and the MMS2-UBC13 heterodimer factors. Both Rad6 and UbclS are ubiquitin-conjugating enzymes, and Mms2 is a UBC-like protein. RadlS and Rad5 are both single strand binding RING linger proteins. RAD6 binds to RadlS and Mms2-UbclS binds to Rad5. The interaction between RadlS and Rad5 mediates the cooperation between the Ubcs, and also localizes the Ubcs on DNA. However, the ubiquitylated substrate protein of these Ubcs remains unknown, and the nature of the DNA to which the repair factors bind is still unclear. So the main objective of my PhD work is to investigate the role played by the ubiquitin system during DNA repair. We intended to use RadlS as a key element to start the work, as RadlS interacts with both Ubc and DNA. We intended to investigate what types of DNA structures RadlS is associated with in vivo.The DNA double strand break (DSB) can be rejoined by at least two discrete pathways, i.e., the homologous recombination and the nonhomologous end-joining. At least 10 genes were found involved in NHEJ. Among them, Yku70 (Hdfl) and YkuSO (Hdf2) are break ends binding proteins, and RAD52, MRE11, XRS2 are found both in HR and NHEJ. In Saccharomyces cerevisiae, switching of mating-type is associated with a DSB, created by the site-specific endonuclease HO. It is reported that RAD 18 is found to be involved in Ho degradation via the ubiquitin 26S proteasome system. RAD5 is found involved in the avoidance of non-homologous end-joining of DSB in S. cerevisiae. Therefore, our hypothesis is that RadlS may directly associate with DSB. And also for the establishment of the technique, we were interested in examining whether RAD18 plays a role in this HO-induced DSB repair in S. cerevisiae.To measure the association of RadlS with DSB, we used the chromatin immunoprecipitation (CHIP). In this technique, first the specific protein-DNA interaction is crosslinked by formaldehyde, and the proteins bound with the specific DNA are immunoprecipitated by antibodies. Then the precipitated DNA is purified by reversing theprotein-DNA with 1% SDS and heat In our experiment, RAD 18 was tagged with 9myc in vivo. We introduced the DSB by the induction of HO expression, which is under the control of the Gal 4 Promoter. Shortly after DSB induction, CHIP was performed with anti-myc antibodies. For multiplex PCR, primers specific for Z and Y a, which are involved in the DSB, were employed. And also, a pair of primers of ACT1 was used as a negative control.In this experiment, we also used Yku70 as a positive control, since it has been well defined as a DSB end binding protein. We found RadlS directly binds to the two ends of DSB. The results suggested that RadlS plays a role in DSB repair, as well as in PRR. This work is also suggestive of the discovery of a novel DSB repair factor.