Related Experiment Video
Updated: Mar 29, 2026
Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging
Published on: April 28, 2021
Rtt107 BRCT domains act as a targeting module in the DNA damage response
Grace P Leung1, Joshua A R Brown1, J N Mark Glover2
1Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada.
Abstract:
Cells are constantly exposed to assaults that cause DNA damage, which must be detected and repaired to prevent genome instability. The DNA damage response is mediated by key kinases that activate various signaling pathways. In Saccharomyces cerevisiae, one of these kinases is Mec1, which phosphorylates numerous targets, including H2A and the DNA damage protein Rtt107. In addition to being phosphorylated, Rtt107 contains six BRCA1 C-terminal (BRCT) domains, which typically recognize phospho-peptides. Thus Rtt107 represented an opportunity to study complementary aspects of the phosphorylation cascades within one protein. Here we sought to describe the functional roles of the multiple BRCT domains in Rtt107. Rtt107 BRCT5/6 facilitated recruitment to sites of DNA lesions via its interaction with phosphorylated H2A. Rtt107 BRCT3/4 also contributed to Rtt107 recruitment, but BRCT3/4 was not sufficient for recruitment when BRCT5/6 was absent. Intriguingly, both mutations that affected Rtt107 recruitment also abrogated its phosphorylation. Pointing to its modular nature, replacing Rtt107 BRCT5/6 with the BRCT domains from the checkpoint protein Rad9 was able to sustain Rtt107 function. Although Rtt107 physically interacts with both the endonuclease Slx4 and the DNA replication and repair protein Dpb11, only Slx4 was dependent on Rtt107 for its recruitment to DNA lesions. Fusing Rtt107 BRCT5/6 to Slx4, which presumably allows artificial recruitment of Slx4 to DNA lesions, alleviated some phenotypes of rtt107Δ mutants, indicating the functional importance of Slx4 recruitment. Together this data revealed a key function of the Rtt107 BRCT domains for targeting of both itself and its interaction partners to DNA lesions.
Insights
The Rtt107 protein
Area of Science:
- Molecular Biology
- Cellular Biology
- DNA Repair Mechanisms
Background:
- Cells face constant DNA damage, necessitating robust detection and repair to maintain genome stability.
- The DNA damage response involves kinases like Mec1 in Saccharomyces cerevisiae, which phosphorylates key proteins such as H2A and Rtt107.
- Rtt107 possesses multiple BRCA1 C-terminal (BRCT) domains, crucial for recognizing phosphorylation events and mediating DNA repair.
Purpose of the Study:
- To elucidate the functional roles of the distinct BRCT domains within the Rtt107 protein.
- To investigate how Rtt107's BRCT domains contribute to its recruitment to DNA damage sites and its interactions with other repair factors.
- To understand the interplay between Rtt107 phosphorylation and its recruitment capabilities.
Main Methods:
- Site-directed mutagenesis was used to disrupt specific BRCT domains in Rtt107.
- Assays were performed to assess Rtt107 recruitment to DNA lesions and its phosphorylation status.
- Functional complementation studies involved replacing Rtt107's BRCT domains with those from Rad9 and assessing rescue of mutant phenotypes.
Main Results:
- Rtt107's BRCT5/6 domains were essential for recruitment to DNA lesions through interaction with phosphorylated H2A.
- BRCT3/4 domains also contributed to recruitment but were insufficient without BRCT5/6.
- Mutations affecting recruitment also abrogated Rtt107 phosphorylation, and functional replacement of BRCT5/6 with Rad9's domains restored Rtt107 function.
Conclusions:
- The BRCT domains of Rtt107 play a critical, modular role in targeting both Rtt107 and its interacting partners, like Slx4, to DNA damage sites.
- Rtt107's recruitment to lesions is intrinsically linked to its phosphorylation status.
- Understanding Rtt107's BRCT domain functions provides insights into the precise mechanisms of DNA damage response pathways.
Related Concept Videos
02:36DNA Damage can Stall the Cell Cycle
02:36DNA Damage Can Stall the Cell Cycle
01:18Other Unique Bacteria
02:31Homologous Recombination
01:26Receptor Tyrosine Kinases
02:10Translesion DNA Polymerases
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...