The Activation of ATR in Response to Double-Stranded DNA Breaks

Citation

Ramírez-Lugo, Juan S. (2010) The Activation of ATR in Response to Double-Stranded DNA Breaks. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/EG9Z-0H60. https://resolver.caltech.edu/CaltechTHESIS:05262010-155732939

Abstract

The cellular response to the presence of double-stranded DNA breaks (DSBs) is primarily mediated by the ATM protein kinase. A related kinase, ATR, regulates the responses to dysfunctional DNA replication and is also activated, in an ATM-dependent manner, when breaks occur during S-phase. The latter is achieved by the ability of ATM to interact with TopBP1, an inducer of ATR activity. Additionally, in Xenopus egg extracts, the Mre11-Rad50-Nbs1 (MRN) complex is required to bridge ATM and TopBP1 together. With our current work, we show that CtIP, a known MRN-interacting protein, is recruited to DSB-containing chromatin and interacts with TopBP1 in a damage-dependent manner. A region containing the first two BRCT repeats of TopBP1 is essential for this interaction. Furthermore, two distinct regions of CtIP participate in mediating the association between CtIP and TopBP1. The first region includes two putative ATM/ATR phosphorylation sites. Secondly, an MRN-binding region in the N-terminal region of CtIP is involved. In addition, the binding between CtIP and TopBP1 is diminished in Nbs1-depleted extracts and, reciprocally, the binding of Nbs1 to TopBP1 decreases in the absence of CtIP. This suggests the formation of a complex containing CtIP, TopBP1 and the MRN complex. When CtIP is removed from egg extracts, the levels of TopBP1 and Nbs1 in damaged nuclei are reduced, thereby compromising the activation of the damage response. Thus, CtIP interacts with TopBP1 in a damage-stimulated, MRN-dependent manner to mediate the activation of ATR in response to DSBs.

We additionally explore the involvement of the chromatin remodeling ATPase ISWI in the responses to DNA damage. We find that ISWI associates with ATR, ATRIP, and TopBP1 on DNA in the presence of damage. In addition, ISWI is a substrate of both ATM and ATR in vitro. Furthermore, the activities of ATM and ATR stimulate an increase in the levels of ISWI on chromatin that contains DSBs. Finally, we assessed the role of ISWI in the activation of multiple damage responses in Xenopus egg extracts. Taken together, our work describes several previously uncharacterized features of ISWI with implications in the response to damaged and incompletely replicated DNA.

Thesis Files