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Structural and functional characterization of the yeast DUN1 FHA domain

Lee, Hyun
http://rave.ohiolink.edu/etdc/view?acc_num=osu1155669890

Abstract Details

2006, Doctor of Philosophy, Ohio State University, Biophysics.
The Dun1 protein of Saccharomyces cerevisiae plays a crucial role within cell signaling pathways responsible for coping with DNA damage. DNA damage activates Mec1 kinase which, in association with Rad9 kinase, phosphorylates Rad53. Activated Rad53 then activates Dun1 by phosphorylation. This Mec1/Rad53/Dun1 pathway regulates ribonucleotide reductase (RNR) activity by modulationg the concentration of the RNR inhibitor Sml1. The fork-head associated (FHA) domain of Dun1 has an essential role in regulating direct kinase-kinase interaction between Rad53 and Dun1 through the first SQ/TQ-rich cluster domain (SCD) of Rad53. However, exactly how Dun1 FHA functions in this mechanism is unknown: neither the structure of Dun1 FHA nor its phospho-protein recognition specificity have been determined. Once activated, Rad53 amplifies DNA damage signaling by oligomerization-promoted autophosphorylation. Importantly, the same SCD of Rad53 that regulates its oligomerization via its FHA domain prior to autophosphorylation is also responsible for interaction with Dun1 FHA. The work described here was undertaken to determine exactly how SCD1 of Rad53 interacts with both Rad53 FHA1 and Dun1 FHA. Our working hypothesis was as follows: 1) Rad53 FHA1 preferentially binds singly phosphorylated SCD1, thereby promoting oligomerization and autophosphorylation, 2) Rad53 FHA1 subsequently releases the multiply phosphorylated SCD1, 3) Dun1 FHA specifically recognizes the multiply phosphorylated SCD1. Consistent with this hypothesis, the data presented here shows that FHA1 (14-164) has higher affinity for SCD1 than does Dun1 FHA when there is a single phosphorylation of any of the four SCD1 threonines. In contrast, Dun1 FHA has higher affinity for multiply phosphorylated SCD1 than does FHA1. According to nuclear magnetic resonance (NMR) titration and surface plasmon resonance (SPR) studies, double phosphorylations of SCD1 are sufficient for recognition by Dun1FHA. Additionally, we solved the solution structure of free Dun1 FHA by NMR. Autophosphorylation of Dun1 in vitro was found to be concentration-dependent, and seven autophosphorylation sites were identified by mass spectrometry. Two of them (Ser4 and Ser10) could be key residues that initiate dimerization prior to autophosphorylation. In addition, new binding partners of Dun1 FHA were screened by pull down assays and confirmed by co-immunoprecipitation.
Ming-Daw Tsai (Advisor)

Recommended Citations

Citations

  • Lee, H. (2006). Structural and functional characterization of the yeast DUN1 FHA domain [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1155669890

    APA Style (7th edition)

  • Lee, Hyun. Structural and functional characterization of the yeast DUN1 FHA domain. 2006. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1155669890.

    MLA Style (8th edition)

  • Lee, Hyun. "Structural and functional characterization of the yeast DUN1 FHA domain." Doctoral dissertation, Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=osu1155669890

    Chicago Manual of Style (17th edition)

osu1155669890
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© 2006, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.