Kumar, N and Taneja, A and Ghosh, M and Rothweiler, U and Sundaresan, NR and Singh, M (2024) Harmonin homology domain-mediated interaction of RTEL1 helicase with RPA and DNA provides insights into its recruitment to DNA repair sites. In: Nucleic Acids Research, 52 (3). pp. 1450-1470.
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Abstract
The regulator of telomere elongation helicase 1 (RTEL1) plays roles in telomere DNA maintenance, DNA repair, and genome stability by dismantling D-loops and unwinding G-quadruplex structures. RTEL1 comprises a helicase domain, two tandem harmonin homology domains 1&2 (HHD1 and HHD2), and a Zn2+-binding RING domain. In vitro D-loop disassembly by RTEL1 is enhanced in the presence of replication protein A (RPA). However, the mechanism of RTEL1 recruitment at non-telomeric D-loops remains unknown. In this study, we have unravelled a direct physical interaction between RTEL1 and RPA. Under DNA damage conditions, we showed that RTEL1 and RPA colocalise in the cell. Coimmunoprecipitation showed that RTEL1 and RPA interact, and the deletion of HHDs of RTEL1 significantly reduced this interaction. NMR chemical shift perturbations (CSPs) showed that RPA uses its 32C domain to interact with the HHD2 of RTEL1. Interestingly, HHD2 also interacted with DNA in the in vitro experiments. HHD2 structure was determined using X-ray crystallography, and NMR CSPs mapping revealed that both RPA 32C and DNA competitively bind to HHD2 on an overlapping surface. These results establish novel roles of accessory HHDs in RTEL1�s functions and provide mechanistic insights into the RPA-mediated recruitment of RTEL1 to DNA repair sites. © 2024 Oxford University Press. All rights reserved.
| Item Type: | Journal Article |
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| Publication: | Nucleic Acids Research |
| Publisher: | Oxford University Press |
| Additional Information: | The copyright for this article belongs to the Author. |
| Keywords: | helicase; K ras protein; meprobamate; small interfering RNA, affinity chromatography; amino acid sequence; Article; base pairing; binding affinity; binding site; carcinogenesis; cell culture; cell proliferation; circular dichroism; coimmunoprecipitation; conformational transition; crystal structure; crystallization; DNA damage; DNA repair; DNA replication; DNA sequence; entropy; Escherichia coli; expression vector; gene; genetic transfection; genomic instability; heteronuclear single quantum coherence; HHD2 gene; immunoblotting; immunofluorescence; immunofluorescence microscopy; immunoprecipitation; ion exchange chromatography; isothermal titration calorimetry; mass spectrometry; meiotic recombination; molecular cloning; nonhuman; nuclear magnetic resonance spectroscopy; protein conformation; protein expression; protein interaction; protein purification; protein secondary structure; proton nuclear magnetic resonance; recombinase polymerase amplification; RING finger motif; RTEL1 gene; Sanger sequencing; sequence alignment; site directed mutagenesis; size exclusion chromatography; spectroscopy; stoichiometry; surface plasmon resonance; telomere; telomere length; ultraviolet spectrophotometry; Western blotting; X ray crystallography; X ray diffraction |
| Department/Centre: | Division of Biological Sciences > Molecular Biophysics Unit Division of Biological Sciences > Microbiology & Cell Biology |
| Date Deposited: | 09 Apr 2024 10:47 |
| Last Modified: | 09 Apr 2024 10:47 |
| URI: | https://eprints.iisc.ac.in/id/eprint/84689 |
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