Abstract
Telomere DNA ends with a single-stranded 3′ overhang. Long 3′ overhangs may cause aberrant DNA damage responses and accelerate telomere attrition, which is associated with cancer and aging, respectively. Genetic studies have indicated several important players in preventing 5′ end hyper-resection, yet detailed knowledge about the molecular mechanism in which they act is still lacking. Here, we use an in vitro DNA 5′ end protection assay, to study how N. castellii Cdc13 and Rap1 protect against 5′ exonucleolytic degradation by λ-exonuclease. The homogeneous telomeric repeat sequence of N. castellii allows us to study their protection ability at exact binding sites relative to the 5′ end. We find efficient protection by both Cdc13 and Rap1 when bound close to the 5′ end. Notably, Rap1 provides protection when binding dsDNA at a distance from the 5′ end. The DNA binding domain of Rap1 is sufficient for 5′ end protection, and its wrapping loop region is essential. Intriguingly, Rap1 facilitates protection also when its binding site contains 2 nt of ssDNA, thus spanning across the ds-ss junction. These results highlight a role of Rap1 in 5′ end protection and indicate that Cdc13 and Rap1 have complementary roles in maintaining proper 3′ overhang length.
Highlights
Telomeres are the nucleoprotein structures that protect the end of eukaryotic chromosomes
We find that Cdc[13] and Rap[1] have complementary roles in 5′ end protection, both being able to protect the 5′ end from exonucleolytic degradation when bound immediately adjacent to the ds-ss junction and further away on the ssDNA and dsDNA, respectively
We show that the Rap[1] DNA binding domain (DBD) is sufficient for mediating 5′ end protection, and that the wrapping loop of the DBD is essential for this function
Summary
Telomeres are the nucleoprotein structures that protect the end of eukaryotic chromosomes. Mutant strains carrying the temperature sensitive Cdc[] allele experience hyper resection of the 5′ end by Exo[1], resulting in long overhangs that are recognized by the DDR6, 21. This indicates a role for Cdc[13] in protection from 5′ resection, and it was recently shown that Cdc[13] inhibits resection by λ-exonuclease in a biochemical assay[26]. It was proposed that cell-cycle-specific processing may occur, where Rap[1] would act in preventing 5′ end resection in G0/G1-phase, while Cdc[13] would be crucial for preventing excessive 5′ end resection in S-phase[27]
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