Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans

Abstract

DNA lesions that block replication fork progression are drivers of cancer-associated genome alterations, but the error-prone DNA repair mechanisms acting on collapsed replication are incompletely understood, and their contribution to genome evolution largely unexplored. Here, through whole-genome sequencing of animal populations that were clonally propagated for more than 50 generations, we identify a distinct class of deletions that spontaneously accumulate in C. elegans strains lacking translesion synthesis (TLS) polymerases. Emerging DNA double-strand breaks are repaired via an error-prone mechanism in which the outermost nucleotide of one end serves to prime DNA synthesis on the other end. This pathway critically depends on the A-family polymerase theta, which protects the genome against gross chromosomal rearrangements. By comparing the genomes of isolates of C. elegans from different geographical regions, we found that in fact most spontaneously evolving structural variations match the signature of polymerase theta-mediated end joining (TMEJ), illustrating that this pathway is an important source of genetic diversification.

Figure 1.

Spontaneous mutagenesis in TLS-deficient strains. (A) Generation of mutation accumulation (MA) lines. For each genotype, multiple populations were started by cloning out single worms from a single hermaphrodite P0. Cultures were propagated by transferring animals to new plates each generation. At generation F_n, a single animal was grown to a full population, from which genomic DNA was isolated and subjected to whole-genome sequencing on an Illumina HiSeq. (B) Substitution and microsatellite mutation rates for the indicated genotypes. Mutation rates are expressed as the number of mutations per generation divided by the fraction of the genome covered >3 times in all samples. (C) Rates of structural variations for the indicated genotypes. (D) Size distribution of deletions in the different mutant backgrounds. The median sizes are indicated by the gray horizontal lines.

Figure 2.

Deletion footprints in TLS mutants indicate a priming-based end joining mechanism. (A) Distribution of deletion footprints in polh-1polk-1 mutants. (B) Schematic illustration of a deletion associated with a templated insertion. Deleted sequence in pink; newly inserted sequence in purple and its template boxed; nonaltered DNA in gray. (C) Sequence context of deletions with templated insertions derived from polh-1polk-1 animals. Matching sequences are underlined. (D) Schematic illustration of a deletion not accompanied by insertions. Deleted sequence in pink; nonaltered DNA in gray. The eight-nucleotide window, capturing neighboring flanking and deleted sequences and used for the generation of the heat maps, is underlined. (E) The strategy to score junction homology: For each simple deletion, matching bases between the 5′ and 3′ junction were scored 1, nonmatching bases were scored 0, thus creating one map per deletion. (F) A heat map representing the sum of all individual deletion maps derived from polh-1polk-1 animals (n = 102). A heat map for a simulated set of deletions (n = 6796) with random distribution is displayed on the right. (G) Base composition at the 5′ and 3′ junctions. The flanking sequences have positive numbers, the deleted sequences have negative; −1 being the first nucleotide within the deletion. Dotted lines indicate the relative abundance of a particular base for a simulated set of deletions (n = 6796).

Figure 3.

Pol theta mediates end joining of breaks in pol eta- and pol kappa-deficient animals. (A) Fecundity of single, double, and triple knockout mutants of pol theta and TLS polymerases pol eta and pol kappa. (B) Quantification and (C) representative pictures of RAD-51 immunostainings on germlines of the indicated genotype. Scale bar, 10 µm. (D) Schematic representation of the unc-22 reporter gene and spontaneous deletions (in red) isolated from either polh-1polk-1 or polh-1polk-1polq-1 mutant animals. Three of five deletions extended beyond the borders of the unc-22 locus.

Figure 4.

Signature of pol theta-mediated end joining in natural isolates of C. elegans. (A) Phylogenetic tree diagram of the different isolates of C. elegans used in this study. (B) Size distribution of deletions of evolutionary distinct C. elegans species compared to size distribution of polh-1polk-1-derived deletions. An exponential regression curve describes the size distribution of deletions in both natural isolates up to 20 bp; deletions up to 200 bp are overrepresented. (C) Deletions in natural isolates, especially in size class 50–200 bp, show templated insertions analogously to deletion footprints in polh-1polk-1 animals. (D) Microhomology for deletions in natural isolates as compared to deletions in polh-1polk-1 animals. (E) unc-93 mutagenesis in polq-1 worms and wild-type controls.

Figure 5.

A tentative model for TMEJ of breaks induced at replication fork barriers. DNA lesions from endogenous sources—with increased frequency in the absence of functional TLS—causes replication fork blocks, leading to double-stranded breaks. The broken ends are repaired by pol theta-mediated end joining (TMEJ), which is stimulated by minimal priming of 1 base pair, explaining deletions with single nucleotide homology (left). Iterative cycles of priming, extending, and dissociation will result in deletions with templated insertions (right). In pol theta-deficient cells, DNA breaks resulting from replication fork stalling are differently processed, eventually leading to larger size deletions.