Structural and functional characterization of human regulator of telomere elongation helicase 1 (RTEL1)

Abstract

RTEL1 is an ATP-dependent DNA helicase that translocates on the single-stranded DNA (ssDNA) from 5′ to 3′ direction. It is an iron-sulfur (Fe-S) cluster and DEAH motif-containing helicase. RTEL1 has been shown to play essential roles in telomere maintenance, DNA repair, meiotic recombination, and genome-wide replication. The 1300 amino acids long isoform of human RTEL1 (Uniprot identifier Q9NZ71-6) has the following identified domains and motifs: an N-terminal helicase domain, two tandem harmonin homology domains 1 & 2 (HHD1 and HHD2), a PCNA-interacting protein-box (PIP-box) motif, and a C-terminal C4C4 type RING domain. RTEL1 disassembles telomere T/D-loop and unwinds G-quadruplexes via its helicase activity. The helicase domain is followed by a nuclear localization signal (NLS) motif, which is essential for the localization of RTEL1 to the nucleus. The central part of RTEL1 consists of tandem harmonin homology domains HHD1 and HHD2. HHD1 and HHD2 are predicted to mediate protein-protein interactions. The PIP-box motif of RTEL1 interacts with the replisome's proliferating cell nuclear antigen (PCNA) and helps in genome-wide replication. The C-terminal Zn2+-binding C4C4 type RING domain interacts with the TRF2 of the shelterin complex, which facilitates the recruitment of RTEL1 to the telomere. Several mutations in RTEL1 are associated with genetic diseases such as Hoyeraal-Hreidarsson syndrome (HHS) and familial pulmonary fibrosis (FPF). These diseases are associated with short telomere length in patients, resulting in premature aging, bone marrow failure, and predisposition to cancer. Despite the importance of RTEL1 in telomere length maintenance and DNA metabolism, the details of its molecular mechanism of action are missing. No structure was available of either full length or any of its domains. Also, the molecular basis of various mutations in RTEL1 is unknown. Therefore, characterizing the structure and function of RTEL1 will help to understand its role in telomere DNA remodeling and the molecular basis of various disease mutations. In this thesis, we have identified RPA as a novel interacting partner of RTEL1. 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. We have biophysically characterized the HHD1, HHD2, and C4C4 RING domain of RTEL1 and studied its interaction with partner proteins and nucleic acids through NMR spectroscopy and ITC. We also determined the X-ray crystal structure of the HHD2 domain of RTEL1. This study establishes HHD2 as a novel accessory domain of RTEL1 that mediates protein – protein, and protein – DNA interactions. Interestingly, we also found that ssDNA competitively displaces the RPA 32C from the RTEL1 HHD2–RPA 32C complex. Thus, we uncovered an interplay among RTEL1, RPA, and DNA, suggesting a possible mechanism for RPA-mediated recruitment of RTEL1 at D-loops at the DNA repair and recombination sites.