Retrotransposon overdose and genome integrity

Abstract

Yeast and mammalian genomes are replete with nearly identical copies of long dispersed repeats in the form of retrotransposons. Mechanisms clearly exist to maintain genome structure in the face of potential rearrangement between the dispersed repeats, but the nature of this machinery is poorly understood. Here we describe a series of distinct "retrotransposon overdose" (RO) lineages in which the number of Ty1 elements in the Saccharomyces cerevisiae genome has been increased by as much as 10 fold. Although these RO strains are remarkably normal in growth rate, they demonstrate an intrinsic supersensitivity to DNA-damaging agents. We describe the identification of mutants in the DNA replication pathway that enhance this RO-specific DNA damage supersensitivity by promoting ectopic recombination between Ty1 elements. Abrogation of normal DNA replication leads to rampant genome instability primarily in the form of chromosomal aberrations and confirms the central role of DNA replication accuracy in the stabilization of repetitive DNA.

Fig. 1.

DNA content of high-copy Ty1 strains. (A) Real-time PCR (hatched bars) and RT-PCR (solid bars) analysis of Ty1 copy number and gene expression in RO strains (L26–10C, L27–10C, L28–10C, L29–10C, L30–10C, and L31–10C), control strain (L48–10C), and plasmid-bearing strains (WT + pJEF2631 and WT + pJEF2631-Ty1) relative to the parental strain (GRF167). (B) Southern blot of Ty1 elements in RO strains, distinguishing endogenous Ty1 elements that contain 2 restriction sites (END) from introduced Ty1 elements that contain 1 site (INT; including some native Ty1 elements lacking an AvaI/XhoI site in one LTR) in RO strains. (C) Pulsed-field gel electrophoresis of yeast chromosomes, indicating the increased relative mobility of chromosomes of RO strains. The migration of chromosomes in the WT strain is indicated (Left). (D) Pulsed-field gel electrophoresis of strains from the L30 lineage after 3, 6, 7, and 10 cycles of Ty1 transposition.

Fig. 2.

Sensitivity of high-copy Ty1 strains to DNA-damaging agents. Serial dilutions of yeast strains were plated onto YPD for 2 d (A), YPD + 150 mM HU for 4 d (B), or YPD + 0.0375% MMS for 2 d (C). Serial dilutions of high-copy strains with and without deletion of the SPT3 gene were plated onto YPD for 2 d (D), YPD + 25 mM HU for 3 d (E), or YPD + 150 mM HU for 4 d (F). (G) Higher magnification of WT and L26–10C spt3Δ colonies grown on 150 mM HU reveals that the L26–10C strain forms smaller colonies even in the absence of ongoing retrotransposition.

Fig. 3.

RO synthetic fitness interactions. (A) Nodes in the network diagram represent all the DNA replication and repair genes we assayed for genetic interaction with the RO Ty1 state and are colored according to GO biological process as annotated in the legend. Light green and dark green lines represent synthetic growth defects and synthetic lethality interactions, respectively, between cellular genes (from Yeast GRID database); light blue lines represent synthetic fitness interactions generated by deletion of cellular genes from RO strains (present study) whereas dark blue lines represent synthetic fitness interactions generated by deletion of cellular genes from RO strains that further result in HU supersensitivity. (B–E) Sensitivity of RO strains to a mutant allele of POL1. WT and an RO strain each carrying a WT or mutant pol1–17 allele were serially diluted and plated onto YPD (B and C) or YPD + 150 mM HU (D and E) and incubated at 22 °C for 3 d (B and D) or 30 °C for 5 d (C and E). Strains designated WT and L26–10C contain the WT POL1 allele, those designated pol1–17 contain the mutant allele, and those labeled POL1 contain the mutant allele as well as the pRS413-POL1 plasmid.

Fig. 4.

Chromosomal alterations in RO pol1–17 strains. (A and B) WT and RO strain L26–10C containing WT or mutant alleles of POL1 were held at 22 °C or shifted to 30 °C for 2.5 h in the presence of 200 mM HU; recovered isolates were examined for changes in chromosome structure by pulsed-field gel electrophoresis. Strains are labeled as in Fig. 3. (C and D) Model for generation of chromosomal translocations in RO strains. DNA lesions within chromatids (gray and red bars) are more likely to occur within Ty1 elements (purple arrows) in RO Ty1 strains (D) relative to a WT strain (C) as they comprise a greater fraction of the RO strain genome. These lesions can either be repaired from an allelic element (Left), regenerating the original chromosome structure, or from an ectopic Ty1 sequence (Right) located intra- or inter-chromosomally and resulting in the formation of deletions, inversions, and translocations.