Synthetic Lethality between DNA Polymerase Epsilon and RTEL1 in Metazoan DNA Replication.

Genome stability requires coordination of DNA replication origin activation and replication fork progression. RTEL1 is a regulator of homologous recombination (HR) implicated in meiotic cross-over control and DNA repair in C. elegans. Through a genome-wide synthetic lethal screen, we uncovered an essential genetic interaction between RTEL1 and DNA polymerase (Pol) epsilon. Loss of POLE4, an accessory subunit of Pol epsilon, has no overt phenotype in worms. In contrast, the combined loss of POLE-4 and RTEL-1 results in embryonic lethality, accumulation of HR intermediates, genome instability, and cessation of DNA replication. Similarly, loss of Rtel1 in Pole4-/- mouse cells inhibits cellular proliferation, which is associated with persistent HR intermediates and incomplete DNA replication. We propose that RTEL1 facilitates genome-wide fork progression through its ability to metabolize DNA secondary structures that form during DNA replication. Loss of this function becomes incompatible with cell survival under conditions of reduced origin activation, such as Pol epsilon hypomorphy.

RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity.

T loops and telomeric G-quadruplex (G4) DNA structures pose a potential threat to genome stability and must be dismantled to permit efficient telomere replication. Here we implicate the helicase RTEL1 in the removal of telomeric DNA secondary structures, which is essential for preventing telomere fragility and loss. In the absence of RTEL1, T loops are inappropriately resolved by the SLX4 nuclease complex, resulting in loss of the telomere as a circle. Depleting SLX4 or blocking DNA replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(-/-) cells but failed to suppress telomere fragility. Conversely, stabilization of telomeric G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient cells but had no impact on TC formation or telomere loss. We propose that RTEL1 performs two distinct functions at telomeres: it disassembles T loops and also counteracts telomeric G4-DNA structures, which together ensure the dynamics and stability of the telomere.

Cooperative assembly of CYK-4/MgcRacGAP and ZEN-4/MKLP1 to form the centralspindlin complex.

Cytokinesis in metazoan cells requires a set of antiparallel microtubules that become bundled upon anaphase onset to form a structure known as the central spindle. Bundling of these microtubules requires a protein complex, centralspindlin, that consists of the CYK-4/MgcRacGAP Rho-family GTPase-activating protein and the ZEN-4/MKLP1 kinesin-6 motor protein. Centralspindlin, but not its individual subunits, is sufficient to bundle microtubules in vitro. Here, we present a biochemical and genetic dissection of centralspindlin. We show that each of the two subunits of centralspindlin dimerize via a parallel coiled coil. The two homodimers assemble into a high-affinity heterotetrameric complex by virtue of two low-affinity interactions. Conditional mutations in the regions that mediate complex assembly can be readily suppressed by numerous second site mutations in the interacting regions. This unexpected plasticity explains the lack of primary sequence conservation of the regions critical for this essential protein-protein interaction.