Abstract
For more than half a century, genotoxic agents have been used to induce mutations in the genome of model organisms to establish genotype-phenotype relationships. While inaccurate replication across damaged bases can explain the formation of single nucleotide variants, it remained unknown how DNA damage induces more severe genomic alterations. Here, we demonstrate for two of the most widely used mutagens, i.e. ethyl methanesulfonate (EMS) and photo-activated trimethylpsoralen (UV/TMP), that deletion mutagenesis is the result of polymerase Theta (POLQ)-mediated end joining (TMEJ) of double strand breaks (DSBs). This discovery allowed us to survey many thousands of available C. elegans deletion alleles to address the biology of this alternative end-joining repair mechanism. Analysis of ~7,000 deletion breakpoints and their cognate junctions reveals a distinct order of events. We found that nascent strands blocked at sites of DNA damage can engage in one or more cycles of primer extension using a more downstream located break end as a template. Resolution is accomplished when 3’ overhangs have matching ends. Our study provides a step-wise and versatile model for the in vivo mechanism of POLQ action, which explains the molecular nature of mutagen-induced deletion alleles.
Highlights
DNA mutations fuel evolution of organisms giving rise to speciation, and of cells within an organisms giving rise to cancer
We demonstrate in C. elegans that exposure to mutagenic agents leads to replication-associated double strand breaks (DSBs) that require polymerase Theta (POLQ)-mediated end joining for their repair
We provide a mechanistic basis for understanding how mutagens that interfere with DNA replication induce the type of deletion mutations that have been generated in a great variety of organisms in order to establish genetic null alleles
Summary
DNA mutations fuel evolution of organisms giving rise to speciation, and of cells within an organisms giving rise to cancer. TLS polymerases, in contrast to the replicative polymerases, have the ability to extend nascent DNA strands across non- or poorly coding damaged bases, often leading to mutation. It is, less well understood which mechanisms are responsible for other types of genomic alterations, such as deletions that are larger than a few bases. A similar profile of mutagenesis was observed resulting from DNA double-strand break repair, which hinted towards DSBs as being a very prominent source of genome diversification during evolution, and towards errorprone DSB repair as the mechanism responsible for this type of genome alterations [1]
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