Saccharomyces cerevisiae Rev7 Regulates Double-Strand Break Repair Pathway Choice by Binding and Inhibiting Mre11 Nuclease and Rad50 ATPase Activities

Core Concepts

Saccharomyces cerevisiae Rev7 directly interacts with the Mre11-Rad50-Xrs2 (MRX) complex, inhibits Mre11 nuclease and Rad50 ATPase activities, and thereby facilitates non-homologous end-joining (NHEJ) while antagonizing homologous recombination (HR) repair of double-strand breaks.

Abstract

The study explores the role of Saccharomyces cerevisiae Rev7 (ScRev7) in regulating the choice between non-homologous end-joining (NHEJ) and homologous recombination (HR) pathways for the repair of double-strand breaks (DSBs) in DNA.

Key highlights:

1. ScRev7 directly interacts with the subunits of the Mre11-Rad50-Xrs2 (MRX) complex through its C-terminal 42-amino acid fragment.
2. The interaction between ScRev7 and MRX inhibits the nuclease activity of Mre11 and the ATPase activity of Rad50, without affecting Rad50's ability to bind ATP.
3. The 42-amino acid C-terminal fragment of ScRev7 is critical for its binding to MRX and for protecting cells from the synergistic toxicity of G-quadruplex DNA and hydroxyurea (HU).
4. Deletion of REV7 in S. cerevisiae leads to a decrease in NHEJ efficiency and an increase in HR frequency, suggesting that ScRev7 promotes NHEJ while suppressing HR.
5. The C-terminal 42-amino acid fragment of ScRev7 is sufficient to restore NHEJ efficiency in rev7Δ cells to wild-type levels.

These findings uncover a novel mechanism by which ScRev7 regulates the choice between NHEJ and HR pathways for DSB repair in S. cerevisiae, by directly interacting with and modulating the activities of the MRX complex.

Stats

Apparent equilibrium dissociation constants (Kd) for the binding of ScRev7 to various G-quadruplex DNA substrates:

TP-G4: 0.12 ± 0.03 μM
6G3-G4: 0.18 ± 0.04 μM
4G3-G4: 0.25 ± 0.06 μM

Quotes

"Saccharomyces cerevisiae Rev7 robustly interacts with the Mre11-Rad50-Xrs2 (MRX) subunits, impedes G-quadruplex DNA synergised, HU-induced toxicity and facilitates NHEJ, while antagonizing HR."
"We further demonstrate that the full-length Rev7 impedes Mre11 nuclease and Rad50's ATPase activities, without affecting the latter's ATP-binding ability."
"Notably, we found that Rev7 binds with high affinity and specificity to G-quadruplex structures, as opposed to no binding to mixed-sequence single- and double-stranded DNA."

Key Insights Distilled From

Saccharomyces cerevisiae Rev7 regulates DSB repair pathway choice through binding and blocking Mre11 nuclease and Rad50 ATPase activities

by Badugu,S., D... at www.biorxiv.org 02-21-2024

https://www.biorxiv.org/content/10.1101/2024.02.20.581217v1

Deeper Inquiries

How do the interactions between Rev7 and the MRX complex differ in other eukaryotic species that lack Shieldin complex orthologs?

In other eukaryotic species that lack Shieldin complex orthologs, the interactions between Rev7 and the MRX complex may differ due to the absence of Shieldin components like SHLD1, SHLD2, and SHLD3. These species may have alternative mechanisms for regulating the double-strand break (DSB) repair pathway choice. For example, in Saccharomyces cerevisiae, Rev7 interacts directly with the Mre11-Rad50-Xrs2 (MRX) subunits, inhibits G-quadruplex DNA-induced toxicity, and promotes non-homologous end-joining (NHEJ) while antagonizing homologous recombination (HR). The absence of Shieldin orthologs in other species suggests that Rev7 may recruit different factors or utilize distinct mechanisms to regulate DSB repair pathway choice.

What are the potential implications of Rev7's ability to bind and inhibit G-quadruplex DNA structures in the context of genome stability and cellular processes?

The ability of Rev7 to bind and inhibit G-quadruplex DNA structures has significant implications for genome stability and cellular processes. G-quadruplex structures are known to form in the genome and play regulatory roles in various cellular processes. By binding to G-quadruplex DNA structures, Rev7 may prevent their adverse effects, such as replication stress and genomic instability. This interaction could help maintain genome integrity by protecting against G-quadruplex-induced DNA damage. Additionally, the inhibition of G-quadruplex DNA structures by Rev7 may influence processes like DNA replication, transcription, and DNA repair, ultimately contributing to the overall stability of the genome.

Could the insights gained from this study on the role of Rev7 in regulating DSB repair pathway choice be leveraged to develop novel therapeutic strategies for diseases associated with Rev7 dysfunction, such as Fanconi anemia?

The insights gained from studying the role of Rev7 in regulating the DSB repair pathway choice between HR and NHEJ could indeed be valuable for developing novel therapeutic strategies for diseases associated with Rev7 dysfunction, such as Fanconi anemia. Understanding how Rev7 interacts with the MRX complex and influences the repair pathway choice provides a basis for targeted interventions. By modulating the activity of Rev7 or its interactions with other repair factors, it may be possible to restore the balance between HR and NHEJ in cells with Rev7 mutations. This could potentially help in mitigating the DNA repair defects and genomic instability associated with Fanconi anemia and other diseases linked to Rev7 dysfunction. Developing therapies that target the Rev7-mediated DSB repair pathway choice could offer new avenues for treating these conditions.