The role of nonhomologous end-joining components in telomere metabolism in Kluyveromyces lactis

Abstract

The relationship between telomeres and nonhomologous end-joining (NHEJ) is paradoxical, as NHEJ proteins are part of the telomere cap, which serves to differentiate telomeres from DNA double-strand breaks. We explored these contradictory functions for NHEJ proteins by investigating their role in Kluyveromyces lactis telomere metabolism. The ter1-4LBsr allele of the TER1 gene resulted in the introduction of sequence altered telomeric repeats and subsequent telomere-telomere fusions (T-TFs). In this background, Lig4 and Ku80 were necessary for T-TFs to form. Nej1, essential for NHEJ at internal positions, was not. Hence, T-TF formation was mediated by an unusual NHEJ mechanism. Rad50 and mre11 strains exhibited stable short telomeres, suggesting that Rad50 and Mre11 were required for telomerase recruitment. Introduction of the ter1-4LBsr allele into these strains failed to result in telomere elongation as normally observed with the ter1-4LBsr allele. Thus, the role of Rad50 and Mre11 in the formation of T-TFs was unclear. Furthermore, rad50 and mre11 mutants had highly increased subtelomeric recombination rates, while ku80 and lig4 mutants displayed moderate increases. Ku80 mutant strains also contained extended single-stranded 3' telomeric overhangs. We concluded that NHEJ proteins have multiple roles at telomeres, mediating fusions of mutant telomeres and ensuring end protection of normal telomeres.

Figure 1.—

Telomere length in strains compromised for NHEJ. DNA blot of EcoRI-digested chromosomal DNA hybridized with a probe specific to K. lactis telomeric repeats. (A) Telomere lengths from strains CK213-4C (wt, or wild type), AKY124 (lig4), SAY572 (nej1), SAY573 (ku80), SAY559 (mre11), and SAY557 (rad50). (B) Telomere lengths of strains SAY100 (sir4), AKY116 (sir4 ku80), SAY703 (sir4 lig4), SAY701 (sir4 nej1), SAY704 (sir4 mre11), and SAY559 (mre11). Molecular weight markers are in kilobases.

Figure 2.—

Long 3′ overhangs in ku80 cells. Nondenaturing in-gel hybridization of genomic DNA digested with PstI from wt (wild type), ku80, lig4, mre11, and rad50 strains. (A) Shows hybridization to a G-strand telomeric probe. (B) Shows the EtBr-stained gel prior to hybridization. Molecular weight markers are in kilobases.

Figure 3.—

T–TFs in NHEJ mutant strains. (A) LIG4, but not NEJ1, was required for formation of T–TFs. Shown is a DNA blot using two independent isolates of ter1-4LBsr (ter1-4L), ter1-4L lig4, and ter1-4L nej1 and one ter1-4L lig4 nej1 strain. Chromosomal DNA was prepared from the strains following 10 streaks. (B) KU80 was required for formation of T–TFs. The blot is divided into early and late lanes. Chromosomal DNA from early lanes was isolated immediately following the generation of the mutant strains. Late lanes are of the same mutant strains following 24 serial restreaks. Two independent isolates of ter1-4L ku80 and one isolate of a ter1-4L single mutant are shown. (C) ter1-4L mre11 double-mutant strains had short, stable telomeres. The blot is divided into early and late lanes as described above. Shown are ter1-4L, mre11, and ter1-4L mre11 strains. Wild types for all blots are TER1 strains recovered from the same tetrads used in the generation of the respective mutant strains. (D) A ter1-4L rad50 double-mutant strain incorporated mutant repeats. EcoRI and EcoRI/BsrGI digestions of genomic DNA isolated from a ter1-4L rad50 mutant strain following 24 serial restreaks. Molecular weight markers are in kilobases.

Figure 4.—

Molecular characterization of T–TFs. (A) Agarose gel electrophoresis of PCR amplifications using genomic DNA from pre- and postfusion strains, as indicated above the lanes, and subtelomeric specific primers. Results were from strains SAY561 and SAY605 following 5 and 25 serial restreaks, respectively. Control lanes result from PCR of genomic DNA isolated from the parental strains used in the generation of each mutant strain prior to the TER1 loop-out procedure. Size markers indicated on the left. (B) K. lactis T–TFs analyzed by restriction digests and DNA sequencing. Seven different T–TFs (first column) were analyzed, one to three originating from strain SAY605 and four to seven from strain SAY561. The second column shows an estimate of the number of mutant repeats present as determined by BsrGI digestion of each cloned fragment followed by agarose gel electrophoresis. The third column is a schematic of the sequencing results. Sequenced wild-type repeats (black arrows) and mutant repeats (blue arrows) are indicated. Red and yellow boxes represent subtelomeric sequences. The numbers within brackets indicate the estimated difference, in base pairs, between the fragment submitted for sequencing and the actual sequence recovered from the analysis.

Figure 5.—

Multiple T–TFs are detrimental to the ter1-AccSna strain undergoing meiosis. Shown is a DNA blot of EcoRI-digested genomic DNA from 16 haploid strains (lanes 1–16) derived from a cross between a ter1-AccSna strain with stable T–TFs and the wild-type strain GG1958. Lanes marked as 7B520, GG1958, and ter1-AccSna represent the parental haploid controls used to set up the crosses. The sharp bands visible in lanes 1–16, which are identical in size to bands present in the ter1-AccSna parent, represent T–TFs passed unaltered through meiosis. The genomic DNA was hybridized to a G-strand telomere probe (Klac1-25). Molecular weight markers are in kilobases. Above the blot is a schematic of the ter1-AccSna allele, indicating the position of the introduced mutations.