Change of Gene Structure and Function by Non-Homologous End-Joining, Homologous Recombination, and Transposition of DNA

and Transposition of DNA. PLoS Genet 5(6): e1000516. doi:10.1371/journal.pgen.1000516 Change of Gene Structure and Function by Non- Homologous End-Joining, Homologous Recombination, and Transposition of DNA Wolfgang Goettel 0 Joachim Messing 0 Mathilde Grelon, Institut Jean-Pierre Bourgin, INRA de Versailles, France 0 Waksman Institute of Microbiology, Rutgers University , Piscataway, New Jersey , United States of America An important objective in genome research is to relate genome structure to gene function. Sequence comparisons among orthologous and paralogous genes and their allelic variants can reveal sequences of functional significance. Here, we describe a 379-kb region on chromosome 1 of maize that enables us to reconstruct chromosome breakage, transposition, non-homologous end-joining, and homologous recombination events. Such a high-density composition of various mechanisms in a small chromosomal interval exemplifies the evolution of gene regulation and allelic diversity in general. It also illustrates the evolutionary pace of changes in plants, where many of the above mechanisms are of somatic origin. In contrast to animals, somatic alterations can easily be transmitted through meiosis because the germline in plants is contiguous to somatic tissue, permitting the recovery of such chromosomal rearrangements. The analyzed region contains the P1-wr allele, a variant of the genetically well-defined p1 gene, which encodes a Myb-like transcriptional activator in maize. The P1-wr allele consists of eleven nearly perfect P1-wr 12-kb repeats that are arranged in a tandem head-to-tail array. Although a technical challenge to sequence such a structure by shotgun sequencing, we overcame this problem by subcloning each repeat and ordering them based on nucleotide variations. These polymorphisms were also critical for recombination and expression analysis in presence and absence of the trans-acting epigenetic factor Ufo1. Interestingly, chimeras of the p1 and p2 genes, p2/p1 and p1/p2, are framing the P1-wr cluster. Reconstruction of sequence amplification steps at the p locus showed the evolution from a single Myb-homolog to the multi-gene P1-wr cluster. It also demonstrates how non-homologous end-joining can create novel gene fusions. Comparisons to orthologous regions in sorghum and rice also indicate a greater instability of the maize genome, probably due to diploidization following allotetraploidization. - Evolution is based on genome instability. Because genome instability can be detrimental to an individual organism, highly sophisticated mechanisms evolved to maintain genome integrity. Processes to prevent instability, such as DNA damage repair systems, however, are error-prone. Consequently, chromosomal changes are passed onto the next generation and will be tested in evolution on the individual and population level. Species as well as inter-species sequence comparisons reveal the dynamic structure of plant genomes as a consequence of genomic instability. It appears that just a few mechanisms are required to explain genomic instability. Minor or local changes that can cause mutations are associated with inaccurate DNA replication, or DNA repair, or recombination [1]. Replication errors, impairment of base excision and mismatch repair, or error-prone translesion synthesis can lead to base substitutions, micro-insertions and micro-deletions. Microand minisatellite instability that results in expansion or contraction of short, repetitive sequences is caused by unequal homologous recombination, replication slippage, or by repair impairment. More dramatic or global changes in chromosome structure occur when two DNA fragments that were previously unlinked are being joined. Such chromosomal rearrangements include deletions, insertions, duplications, inversions, and translocations, and they can occur by transposition, unequal homologous recombination, or illegitimate recombination [2]. All of these processes involve DNA Double-Strand-Breaks (DSBs) and ligations. Already McClintock demonstrated that chromosomal rearrangements such as translocations, deficiencies, ring chromosomes and end fusions could be consequences of chromosome breaks [3]. DSBs can arise in all tissues at all stages of development and are induced by excision of transposable elements, endonucleases, ionizing irradiation (UV, decay of naturally occurring radioisotopes), reactive oxygen species, and mechanical pulling of dicentric chromosomes. DSBs result in cell-cycle arrest and the recruitment of the DSBrepair machinery. An unrepaired DSB leads ultimately to cell death. Dependent on the phase of the cell cycle, availability of homologous sequences close to the break site, DSBs are repaired by illegitimate recombination (also known as non-homologous endjoining (NHEJ)), homologous recombination (HR), or even a combination of both mechanisms (reviewed in [46]). During meiosis DSBs are probably exclusively repaired by HR. NHEJ is a major pathway for DSB repair in somatic tissue. The rejoining of the broken ends via NHEJ is associated with deletions Plant genomes analyzed to date contain 15% or more genes that are arranged in tandem arrays. Tandem duplications are a source for allelic variability since their homologous sequences can serve in recombination events. For example, unequal crossing over between amplified genes can result in contraction and expansion of the array. Tandem gene multiplications are also subject to repeat induced gene silencing (RIGS). Most importantly, gene duplications create the evolutionary potential for genetic novelty (neo- or subfunctionalization). In addition to homologous recombination during meiosis, illegitimate recombination in somatic tissues of plants can create events that potentially can be transmitted through reproductive tissue to further enrich genetic diversity. Here we illustrate the evolution from a single Myb homolog to a multigene cluster that exemplifies the evolution of the maize genome. We used the p locus to demonstrate how plant genomes expand by polyploidization, gene duplication, and transposition. We characterized in detail the structural changes at the p cluster that resulted from genomic instability. Because structure determines function, we linked genomic rearrangements at the P1-wr cluster to functional consequences. At the P1wr locus, structural changes caused regulatory/transcriptional modifications that in turn give rise to phenotypic alterations. of various sizes, but also insertions of sequences (filler DNA) that are often copied from sites close to the DSB. NHEJ does not require sequence similarities for the incorporation of filler DNA into the break. Taken together, NHEJ does not preserve genetic information and genomic integrity at the break site. Only few cases of filler DNA suggesting a DSB break repair have been reported. HR seems to play a minor role in DSB repair in somatic tissues. Homologous sequences used as template for t (...truncated)