Our work on RTEL1 has provided insights into its role of RTEL1 at telomeres and the consequences of RTEL1 mutations in the human telomere dysfunction disorder, Hoyeraal-Hreidarsson syndrome.
Eukaryotic genomes are organized into linear chromosomes, which require the presence of telomeres to protect and maintain the chromosome ends. Telomeres are repetitive sequences (TTAGGG in vertebrates) that form complex nucleoprotein structures that protect chromosome ends from promiscuous DNA repair activities and nucleolytic degradation. Telomeres also shorten with each cell division due to processing by nucleases, genotoxic stress and the “end-replication problem”. To counteract telomere shortening, telomeric repeats can be extended by means of telomerase, a reverse transcriptase that uses an RNA moiety (TERC) as a template to extend the 3’ end of the chromosome.
Telomerase is normally repressed in somatic tissues, but is frequently re-expressed in cancer cells as a means of maintaining telomere length during disease progression. Cancer cells can also employ Alternative Lengthening of Telomeres (ALT) to maintain telomeres by means of recombination between telomeres on different chromosomes. The function and integrity of the telomere is also critically dependent on a six-subunit protein complex called Shelterin, which associates specifically with telomere repeats and acts by i) suppressing ATM and ATR checkpoint signaling, ii) inhibiting DSB repair pathways, and iii) facilitating semi-conservative replication through the telomere. In addition to Shelterin, numerous other factors have been reported to engage with telomeres but their function in telomere metabolism remains largely unexplored.
RTEL1 is a helicase that was originally identified by mapping of loci that control telomere length differences between M. musculus and M. spretus.RTEL1 also plays a critical role in genome stability as knockout mice are embryonic lethal and cells derived from these mice exhibit chromosomal aberrations and dramatic telomere dysfunction (Fig. 1). The clinical importance of RTEL1’s role at telomeres was recently highlighted with the discovery that it is frequently mutated in Hoyeraal-Hreidarsson syndrome (HHS), a severe form of Dyskeratosis congenita (DKC) caused by critically short telomeres. HHS is characterized by its similarity to DKC as well as additional complications from bone marrow failure, microcephaly and immunodeficiency. An explaination as to why HHS patients present with this spectrum of phenotypes is currently lacking.
We previously identified RTEL1 as a key regulator of homologous recombination (HR) in a genetic screen for anti-recombinases . Our biochemical studies revealed that RTEL1 promotes the disassembly of D loop recombination intermediates in vitro, which we proposed could act to counteract toxic recombination events (Barber et al., Cell 2008). Based on these findings we speculated that RTEL1’s role at telomeres might reflect a need to constrain or regulate HR. Indeed, we proceeded to show that RTEL1 performs two distinct functions at vertebrate telomeres: i) it removes telomeric DNA structures, such as the t loops, to prevent the catastrophic processing of the telomere by the SLX1/4 nuclease complex, and ii) it counteracts the formation of telomeric G4-DNA structures to facilitate replication through the telomere (Vannier et al., Cell 2012).
Our subsequent work established that the targeting of RTEL1 to replication forks and telomeres is mediated by PIP-box (binds PCNA) and C4C4 (binds TRF2) motifs in RTEL1, respectively (Vannier et al., Science 2013; Sarek et al. Molecular Cell 2015). Most recently we discovered that telomerase is the source of telomere dysfunction in RTEL1 deficient cells, as loss of telomerase function is sufficient to rescue the RTEL1 telomere phenotype. We proceeded to show that telomerase inappropriately binds to reversed replication forks that accumulate at RTEL1 deficident as a consequence of the inability to efficiently unwind T-loops. In this context, telomerase inhibits telomere replication and fork restart, which triggers the catastrophic processing of telomeres by the SLX1/4 nuclease complex (Margalef et al., Cell 2017).
We are continuing to explore the regulation of RTEL1 at telomeres and during DNA replication.