Spectrum of variations in dog-1/FANCJ and mdf-1/MAD1 defective Caenorhabditis elegans strains after long-term propagation

Abstract

Background: Whole and partial chromosome losses or gains and structural chromosome changes are hallmarks of human tumors. Guanine-rich DNA, which has a potential to form a G-quadruplex (G4) structure, is particularly vulnerable to changes. In Caenorhabditis elegans, faithful transmission of G-rich DNA is ensured by the DOG-1/FANCJ deadbox helicase.

Results: To identify a spectrum of mutations, after long-term propagation, we combined whole genome sequencing (WGS) and oligonucleotide array Comparative Genomic Hybridization (oaCGH) analysis of a C. elegans strain that was propagated, in the absence of DOG-1 and MDF-1/MAD1, for a total of 470 generations, with samples taken for long term storage (by freezing) in generations 170 and 270. We compared the genomes of F170 and F470 strains and identified 94 substitutions, 17 InDels, 3 duplications, and 139 deletions larger than 20 bp. These homozygous variants were predicted to impact 101 protein-coding genes. Phenotypic analysis of this strain revealed remarkable fitness recovery indicating that mutations, which have accumulated in the strain, are not only tolerated but also cooperate to achieve long-term population survival in the absence of DOG-1 and MDF-1. Furthermore, deletions larger than 20 bp were the only variants that frequently occurred in G-rich DNA. We showed that 126 of the possible 954 predicted monoG/C tracts, larger than 14 bp, were deleted in unc-46 mdf-1 such-4; dog-1 F470 (JNC170).

Conclusions: Here, we identified variants that accumulated in C. elegans' genome after long-term propagation in the absence of DOG-1 and MDF-1. We showed that DNA sequences, with G4-forming potential, are vulnerable to deletion-formation in this genetic background.

Figure 1

A schematic representation of the long-term propagation of unc-46 mdf-1 such-4; dog-1 homozygotes for 470 generations. We isolated a suppressor (such-4) in F₄ from the only plate that we set up that propagated in the absence of MDF-1 and DOG-1 for longer than three generations. We generated a strain (KR4233) by crossing away dog-1(gk10). A second line was propagated for the total of 470 generations. The worms were frozen at generations 170 (green), 270 (purple) and 470 (blue). Note that unc-46 (used as a visible marker) is tightly linked to mdf-1(gk2) in all of our strains.

Figure 2

Mutation rate estimates. The variants analyzed are the 94 SBSs that were identified between generations 170 and 470.

Figure 3

The majority of large deletions initiates at G-rich DNA. Figures (A) through (D) are Genome Browser snapshots of single deletions; x-axis represents genome location, while y-axis represents number of reads that cover the region; blue reads depict coverage of more than 10, while orange depicts 10 or less; the gaps depict no coverage, which is indicative of deletions. The reference sequence is depicted with deleted bases underlined. (A) A 99 bp deletion that initiates at G₂₀ homopolymer. (B) A 96 bp deletion that initiates at G₁₄TG₆AGAAG₃ sequence. (C) A 112 bp deletion that initiates at a G-rich sequence, G₂N₃G₂N₅G₂N₂G₂. (D) A 55 bp deletion that initiates at a non G-rich sequence. (E) Size distribution of homozygous deletions that occur at G-rich sequences (n = 123).