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Updated: Sep 5, 2025
Author Spotlight: Decoding DNA Repair by Extrachromosomal NHEJ Assay and HR Assays
Published on: February 2, 2024
Rad5 and Its Human Homologs, HLTF and SHPRH, Are Novel Interactors of Mismatch Repair
Anna K Miller1, Guogen Mao1, Breanna G Knicely1
1College of Medicine Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, KY, United States.
Abstract:
DNA mismatch repair (MMR) repairs replication errors, and MMR defects play a role in both inherited cancer predisposition syndromes and in sporadic cancers. MMR also recognizes mispairs caused by environmental and chemotherapeutic agents; however, in these cases mispair recognition leads to apoptosis and not repair. Although mutation avoidance by MMR is fairly well understood, MMR-associated proteins are still being identified. We performed a bioinformatic analysis that implicated Saccharomyces cerevisiae Rad5 as a candidate for interacting with the MMR proteins Msh2 and Mlh1. Rad5 is a DNA helicase and E3 ubiquitin ligase involved in post-replicative repair and damage tolerance. We confirmed both interactions and found that the Mlh1 interaction is mediated by a conserved Mlh1-interacting motif (MIP box). Despite this, we did not find a clear role for Rad5 in the canonical MMR mutation avoidance pathway. The interaction of Rad5 with Msh2 and Mlh1 is conserved in humans, although each of the Rad5 human homologs, HLTF and SHPRH, shared only one of the interactions: HLTF interacts with MSH2, and SHPRH interacts with MLH1. Moreover, depletion of SHPRH, but not HLTF, results in a mild increase in resistance to alkylating agents although not as strong as loss of MMR, suggesting gene duplication led to specialization of the MMR-protein associated roles of the human Rad5 homologs. These results provide insights into how MMR accessory factors involved in the MMR-dependent apoptotic response interact with the core MMR machinery and have important health implications into how human cells respond to environmental toxins, tumor development, and treatment choices of tumors with defects in Rad5 homologs.
Insights
DNA mismatch repair (MMR) proteins interact with Rad5 homologs, revealing new roles in DNA repair and apoptosis. This discovery impacts understanding of cancer development and treatment strategies for tumors with MMR defects.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA mismatch repair (MMR) corrects replication errors and is crucial in preventing cancer.
- MMR also triggers apoptosis in response to environmental or chemotherapeutic DNA damage.
- While MMR's role in mutation avoidance is known, its accessory proteins are still being discovered.
Purpose of the Study:
- To identify novel proteins interacting with the DNA mismatch repair (MMR) machinery.
- To investigate the functional significance of interactions between Rad5 and MMR proteins.
- To explore the conserved roles of Rad5 homologs in DNA repair and cellular responses to damage.
Main Methods:
- Bioinformatic analysis to predict protein interactions.
- Biochemical assays to confirm interactions between yeast Rad5 and human MMR proteins (Msh2, Mlh1).
- Functional studies involving depletion of human Rad5 homologs (HLTF, SHPRH) to assess cellular responses to DNA damage.
Main Results:
- Bioinformatic analysis implicated *Saccharomyces cerevisiae* Rad5 in interacting with Msh2 and Mlh1.
- Experimental validation confirmed Rad5 interaction with Msh2 and Mlh1, with the latter mediated by a MIP box.
- Human Rad5 homologs HLTF and SHPRH showed conserved, yet specialized, interactions with MSH2 and MLH1, respectively.
- Depletion of SHPRH, not HLTF, conferred mild resistance to alkylating agents, suggesting functional divergence after gene duplication.
Conclusions:
- Rad5 homologs interact with core MMR proteins, suggesting roles beyond canonical MMR mutation avoidance.
- These interactions are important for MMR-associated apoptotic responses to DNA damage.
- Understanding these interactions offers insights into cellular responses to toxins, cancer development, and therapeutic strategies.
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