Endonuclease V (EndoV) initiates a major base repair pathway for nitrosative deamination resulting from endogenous processes and increased by oxidative stress from mitochondrial dysfunction or inflammatory responses. We solved the crystal structures of Thermotoga maritima EndoV in complex with a hypoxanthine lesion substrate and with product DNA. The PYIP wedge motif acts as a minor-groove damage sensor for helical distortions and base mismatches and separates DNA strands at the lesion. EndoV incises DNA with an unusual offset nick one nucleotide 3′ of the lesion, as the deaminated adenine is rotated ~90° into a recognition pocket ~8Å from the catalytic site. Tight binding by the lesion recognition pocket in addition to Mg2+ ion and hydrogen-bond interactions to the DNA ends stabilize the product complex, suggesting orderly recruitment of downstream proteins in this base repair pathway.

Preventing the formation of dysfunctional telomeres is essential for genomic stability. In most organisms, the ribo-nucleoprotein reverse transcriptase telomerase is responsible for telomere GT-strand elongation. However, in telomerase-negative cells, low-frequency recombination mechanisms can avert lethality by elongating critically short telomeres. This study focuses on the involvement of the budding yeast Mre11 in telomere recombination and homeostasis. We have identified a novel allele of MRE11, mre11-A470T, that, in telomerase-positive cells, confers a semidominant decrease in telomere size and a recessive defect in telomere healing. In addition, mutant cells lack normal telomere size homeostasis. Telomerase-negative mre11-A470T cells display a Rad51-dependent bypass of replicative senescence via induction of a highly efficient type I-related recombination pathway termed type IA. The type IA pathway involves an amplification of subtelomeric Y9 elements, coupled with elongated and more heterogeneous telomere tracts relative to the short telomere size of type I survivors. The data have led us to propose the involvement of break-induced replication in telomere expansion. The differing phenotypes elicited by the mre11-A470T mutants in telomerase-positive and telomerase-negative cells have also led us to speculate that the telomere end structure may be modified differentially in mre11-A470T cells, directing the telomere into specific pathways.

The function of the replication clamp loaders in the semi-conservative telomere replication and their relationship to telomerase- and recombination mechanisms of telomere addition remains ambiguous. We have investigated the variant clamp loader Ctf18 RFC (Replication Factor C). To understand the role of Ctf18 at the telomere, we first investigated genetic interactions after loss of Ctf18 and TLC1 (the yeast telomerase RNA). We find that the tlc1▵ ctf18▵ double mutant confers a rapid >1000-fold decrease in viability. The rate of loss was similar to the kinetics of cell death in rad52▵ tlc1▵ cells. However, the Ctf18 pathway is distinct from Rad52, required for the repair of DSBs, as demonstrated by the synthetic lethality of rad52▵ tlc1▵ ctf18▵ triple mutants. These data suggest that each mutant elicits non-redundant defects acting on the same substrate. Second, interactions of the yeast hyper-recombinational mutant, mre11A470T, with ctf18▵ confer a synergistic cold sensitivity. The phenotype of these double mutants ultimately results in telomere loss and the generation of recombinational survivors. We observed a similar synergism between single mutants that led to hypersensitivity to the DNA alkylating agent, methane methyl sulphonate (MMS), the replication fork inhibitor hydroxyurea (HU), and to a failure to separate telomeres of sister chromatids. Hence, ctf18▵ and mre11A470T act in different pathways on telomere substrates for multiple phenotypes. The mre11A470T cells also displayed a DNA damage response (DDR) at 15°C but not at 30°C while ctf18▵ mutants conferred a constitutive DDR activity. Both the 15°C DDR pattern and growth rate were reversible at 30°C and displayed telomerase activity in vivo. We hypothesize that Ctf18 confers protection against stalling and/or breaks at the replication fork in cells that either lack, or are compromised for, telomerase activity. This Ctf18-based function is likely to contribute another level to telomere size homeostasis.