ELG1, a regulator of genome stability, has a role in telomere length regulation and in silencing

Abstract

Telomeres, the natural ends of eukaryotic chromosomes, prevent the loss of chromosomal sequences and preclude their recognition as broken DNA. Telomere length is kept under strict boundaries by the action of various proteins, some with negative and others with positive effects on telomere length. Recently, data have been accumulating to support a role for DNA replication in the control of telomere length, although through a currently poorly understood mechanism. Elg1p, a replication factor C (RFC)-like protein of yeast, contributes to genome stability through a putative replication-associated function. Here, we show that Elg1p participates in negative control of telomere length and in telomeric silencing through a replication-mediated pathway. We show that the telomeric function of Elg1 is independent of recombination and completely dependent on an active telomerase. Additionally, this function depends on yKu and DNA polymerase. We discuss alternative models to explain how Elg1p affects telomere length.

Fig. 1.

Elongation of telomeres in Δelg1 depends on telomerase, but not on recombination. Shown is Southern blot analysis with an S. cerevisiae-specific telomeric probe. (A) Genomic DNA was prepared from spore derivatives dissected from ELG1/Δelg1 RAD52/Δrad52 tetrads. Lane 1, ELG1 RAD52; lane 2, Δelg1 RAD52; lane 3, ELG1 Δrad52 single mutants; lane 4, Δelg1 Δrad52 double mutant. (B) Genomic DNA was prepared from spore derivatives dissected from ELG1/Δelg1 EST2/Δest2 tetrads. Lane 1, Δelg1 Δest2 double mutant; lane 2, Δelg1 EST2; lane 3, ELG1 EST2; lane 4, ELG1 Δest2.

Fig. 2.

Elongation of telomeres in Δelg1 depends on KU70, but not on RIF2, RIF1,or TEL1. Southern blot analysis with an S. cerevisiae-specific telomeric probe was done. (A) Genomic DNA was prepared from spore derivatives dissected from ELG1/Δelg1 RIF2/Δrif2 tetrads. Lane 1, Δelg1RIF2; lane 2, ELG1 Δrif2; lane 3, ELG1 RIF2; lane 4, Δelg1 Δrif2 double mutant. (B) Genomic DNA was prepared from spore derivatives dissected from ELG1/Δelg1 RIF1/Δrif1 tetrads. Lane 1, ELG1 RIF1; lane 2, Δelg1RIF1; lane 3, ELG1 Δrif1 double mutant; lane 4, Δelg1 Δrif1.(C) Genomic DNA was prepared from spore derivatives dissected from ELG1/Δelg1 TEL1/Δtel1 tetrads. Lane 1, ELG1 TEL1; lane 2, ELG1 Δtel1; lane 3, Δelg1TEL1; lane 4, Δelg1 Δte1l double mutant. (D) Genomic DNA was prepared from spore derivatives dissected from ELG1/Δelg1 YKU70/Δyku70 tetrads. Lane 1, ELG1 YKU70; lane 2, Δelg1 YKU70; lane 3, Δelg1 Δyku70 double mutant; lane 4, ELG1 Δyku70.

Fig. 3.

ELG1 operates in telomere elongation in the same genetic pathway as POLα, but in a distinct genetic pathway from CTF18 and RFC1. DNA was analyzed by Southern blot hybridized with an S. cerevisiae-specific telomeric probe. (A) Genomic DNA was prepared from spore derivatives dissected from ELG1/Δelg1 CDC44/cdc44-1 tetrads. Lane 1, ELG1 CDC44; lane 2, ELG1 cdc44-1; lane 3, Δelg1 CDC44; lane 4, Δelg1 cdc44-1 double mutant. (B) Genomic DNA was prepared from spore derivatives dissected from ELG1/Δelg1 CTF18/Δctf18 tetrads. Lane 1, ELG1 CTF18; lane 2, ELG1/Δctf18; lane 3, Δelg1/Δctf18 double mutant; lane 4, Δelg1 CTF18. (C) Genomic DNA was prepared from spore derivatives dissected from ELG1/Δelg1 CDC17/cdc17-1 tetrads. Spores 1–4 were grown at 25°C, and spores 5–8 were grown at 30°C. Lane 1, ELG1 CDC17; lane 2, Δelg1 cdc17-1 double mutant; lane 3, Δelg1 CDC17; lane 4, ELG1 cdc17-1; lane 5, ELG1 CDC17; lane 6, ELG1 cdc17-1; lane 7, Δelg1 cdc17-1 double mutant; lane 8, Δelg1 CDC17.

Fig. 4.

Deletion of ELG1 elevates silencing at an ADE2 gene placed near the telomere in S. cerevisiae. (A) Complementation of the elevated telomeric silencing phenotype was tested in the indicated strains. Strains containing the Δelg::KanMX deletion allele or the ELG1 allele were transformed with control (pCM189) or pELG1 plasmids. The strains were grown on YPD media supplemented with adenine and plated on SD-Ura plates. (B) The uncoupling of silencing and length phenotype of Δelg1 was tested by picking separately Ura⁺ (lane 4) and Ura^- (lane 3) colonies for extraction of genomic DNA. DNA was analyzed by Southern blot hybridized with an S. cerevisiae-specific telomeric probe. Telomeric length was compared with Δelg::KanMX strain (lane 1) and ELG1 (lane 2). (C) Silencing was tested in a strain containing the Δelg::KanMX deletion allele and pELG1 plasmid. This strain was transferred from selective media (SD-Ura) to nonselective media (YPD). Colonies were replica-plated on SD-Ura plates. (D) Genomic DNA was prepared from tetrad created by the dissection of an ELG1/Δelg1 TEL1/Δtel1 diploid with an ADE2 telomeric gene. Spores with all gene combinations (ELG1, Δelg1, Δtel1, and Δelg1 Δtel1) and a telomeric ADE2 gene were grown on YPD liquid media supplemented with adenine and plated on a YPD plate.

Fig. 5.

Increased silencing at an ADE2 gene placed near the telomere in aΔelg1 background depends on YKU70 and SIR2. (A) ELG1/Δelg1 SIR2/Δsir2 diploid with an ADE2 telomeric gene was dissected. Spores with all gene combinations (ELG1, Δelg1, Δsir2, and Δelg1 Δsir2) and a telomeric ADE2 gene were grown on YPD liquid media supplemented with adenine and plated on a YPD plate. (B) ELG1/Δelg1 YKU70/Δyku70 diploid with an ADE2 telomeric gene was dissected. Spores with all gene combinations (ELG1, Δelg1, Δyku70, and Δelg1 Δyku70) and a telomeric ADE2 gene were grown on YPD liquid media supplemented with adenine and plated on a YPD plate.