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Uncovering the roles of RNF8 ubiquitin signaling networks and BRCA1 in recruiting Fanconi Anemia proteins to DNA damage

Bick, Gregory P.
http://orcid.org/0000-0002-0643-4040
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1467987940

Abstract Details

2016, PhD, University of Cincinnati, Medicine: Molecular and Developmental Biology.
Fanconi Anemia is a multigenic DNA damage repair disorder and cancer predisposition syndrome. The 20 proteins in the FA pathway are all linked by their mutual necessity in responding to DNA interstrand crosslinks, such as those which arise from mitomycin C; however, there are clear distinctions between the proteins as well. For instance, loss of some proteins, such as FANCA or FANCD2 does not dramatically impact the cellular restistance to ionizing radiation, while loss of others such as BRCA1, PALB2 or BRCA2 leaves cells exquisitely sensitive. This distinction is echoed by differences in clinical phenotypes as well as biochemical properties. FANC A, B, C, E, F, G, L, and M, interact in a large complex, called the FA core complex, which monoubiquitinates FANCD2 and FANCI. BRCA1, PALB2, BRCA2, FANCJ, RAD51C, XRCC2, and RAD51 also form complexes which are important for homologous recombination. In this dissertation, we examine how these groups of proteins are recruited to DNA damage through protein-protein interactions and ubiquitin signaling. First, we examine the role of the PALB2 coiled coil domain in recruitment of itself and RAD51 to DNA damage. We show that PALB2 coiled coil domain interacts with BRCA1 and PALB2 coiled coils, it is dispensible for PALB2 mediated DNA repair, when PALB2 is fused to the BRCA1 BRCT-repeat domain. This domain is essential for BRCA1’s recruitment to DNA damage foci through the MDC1, RNF8, RAP80 and ABRAXAS ubiquitin signaling cascade. We show that both endogenous PALB2 and BRCT fusion PALB2 are also dependent on this pathway. This indicates the PALB2 is recruited via a coiled coil mediated interaction with BRCA1, allowing it to recruit other downstream complex proteins. Concommitant work showed that RNF8 plays a role in recruiting the upstream FA protein FANCD2. Our work shows links have been identified which may coordinate the different FA proteins to facilitate the specific steps of DNA repair. As such, we tested whether FANCD2 and PALB2 could be coordinately recruited to crosslinks via shared RNF8 mediated regulation. We showed that FANCD2 and PALB2 colocalize after crosslink induction and that both also colocalize with RNF8 dependent ubiquitin chains. Interestingly, while we show that RNF8 is indeed important for the regulation of both FANCD2 and PALB2, other proteins such as MDC1, RAP80, and the ubiquitin binding component of the FA-Core complex, FAAP20, differentially regulate PALB2 or FANCD2, respectively. This indicates that although RNF8 is a shared regulator of FANCD2 and PALB2 the mechanism through which it acts is distinct. This is interesting given that FANCD2 and PALB2 are involved in different steps in the repair of DNA crosslinks. More work must be done to understand what these mechanisms are and why they are distinct. Because of the continued use of DNA damaging agents such as crosslinkers and radiotherapy in the treatment of cancer, as well as to better help FA patients themselves, this work sheds light on important and possibly targetable regulatory pathways.
Paul Andreassen, Ph.D. (Committee Chair)
Ruhikanta Meetei, Ph.D. (Committee Member)
Satoshi Namekawa, Ph.D. (Committee Member)
Peter Stambrook, Ph.D. (Committee Member)
Yi Zheng, Ph.D. (Committee Member)
138 p.
Cellular Biology
PALB2; RNF8; DNA damage; FANCD2; Fanconi Anemia; ubiquitin

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Citations

  • Bick, G. P. (2016). Uncovering the roles of RNF8 ubiquitin signaling networks and BRCA1 in recruiting Fanconi Anemia proteins to DNA damage [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1467987940

    APA Style (7th edition)

  • Bick, Gregory. Uncovering the roles of RNF8 ubiquitin signaling networks and BRCA1 in recruiting Fanconi Anemia proteins to DNA damage. 2016. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1467987940.

    MLA Style (8th edition)

  • Bick, Gregory. "Uncovering the roles of RNF8 ubiquitin signaling networks and BRCA1 in recruiting Fanconi Anemia proteins to DNA damage." Doctoral dissertation, University of Cincinnati, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1467987940

    Chicago Manual of Style (17th edition)

ucin1467987940
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This open access ETD is published by University of Cincinnati and OhioLINK.