Molecular and evolutionary characterization of short-patch double illegitimate recombination, a recently discovered mutation mechanism

One of the definitions of "life" is that living beings can reproduce. In the simplest case, cell division, a single cell splits up into two daughter cells, and before the split, the genome (the DNA) is processed into two identical copies. DNA duplication normally is very faithful, but occasionally errors (mutations) occur. The consequence of a mutation can range from neutral (not affecting the organism) to lethal (e.g., when an essential gene is destroyed by that mutation); in some cases though, mutations are even beneficial. In the long run, rare beneficial mutations accumulate and change the gene content of the organism and its community. This effect is called evolution. Prior to this project, we have discovered and initially described a new, very rare class of mutation: small sections of DNA getting duplicated and inserted elsewhere in the DNA (we termed that mechanism "SPDIR", for "short-patch double illegitimate recombination"), and a typical outcome is a cluster of single base changes (up to 80). Such clustered changes have not been observed before. They are supposedly more detrimental than conventional mutations but can potentially confer complex genomic changes and thus speed up evolution. We discovered SPDIR mutations in a bacterium (Acinetobacter baylyi, a model laboratory strain) and found supporting evidence (by comparing DNA sequences) that SPDIR is widespread: Other bacterial and even human genomes show typical outcomes (clustered base changes by transferred, duplicated DNA sections). Remarkably, SPDIR frequency is elevated in cancer tissue. This project highlights in-depth characterization of SPDIR: How is SPDIR controlled, what factors affect the frequency and variability of SPDIR mutations, how widespread is SPDIR in the bacterial world? A second approach focuses on experimental evolution: Can SPDIR indeed speed up evolution of new traits in Acinetobacter baylyi, and if so, what are the requirements and limits?

The results from this project increase our understanding about microindel mutations caused by short-patch double illegitimate recombination as a new and previously unrecognized mutation mechanism. We have identified a new factor increasing microindel mutations in bacteria (specific plasmid carriage).

Mutations and genomic rearrangements result in remarkable genomic plasticity in all domains of life. These processes allow bacteria to develop of antibiotic resistance, enable pathogens to evade immune response, and, in higher organisms, can lead to cancer. We recently discovered and initially characterized a previously unrecognized mutation mechanism named "short-patch double illegitimate recombination" (SPDIR) that leads to the generation of highly variable clustered polymorphisms, microindels, and mosaic genes in a single generation. SPDIR mutations occur by two joined illegitimate recombinations with fully unrelated DNA molecules of intragenomic or extracellular origin at microhomologies (short DNA stretches of identical nucleotides). The outcome is genetic diversity within a single generation too complex to occur through point mutations. SPDIR has been experimentally demonstrated in the Gram-negative bacterial model organism Acinetobacter baylyi, and analyses of whole genome sequences showed that SPDIR is a mutation mechanism in the Gram-positive bacterial pathogen Streptococcus pneumoniae as well as in humans. In this project, we will further characterize genetic factors that modulate the frequency and the resulting sequence variability of SPDIR mutations. Using A. baylyi as model, we will investigate SPDIR in wildtype and mutant strains, as well as the general role of genotoxins, DNA damages and of added foreign DNA for horizontal gene transfer. SPDIR will be further characterized with newly developed detection constructs in A. baylyi and in different bacterial organisms. Finally, we will assess the potential of SPDIR to adapt gene properties and to shape bacterial evolution in an experimental evolution setup.