Title: Transposition-mediated DNA re-replication in maize

1 Every DNA segment in a eukaryotic genome normally replicates once and only once per 2 cell cycle to maintain genome stability. We show here that this restriction can be bypassed 3 through alternative transposition, a transposition reaction that utilizes the termini of two 4 separate, nearby transposable elements (TEs). Our results suggest that alternative 5 transposition during S phase can induce re-replication of the TEs and their flanking 6 sequences. The DNA re-replication can spontaneously abort to generate double-strand 7 breaks, which can be repaired to generate Composite Insertions composed of transposon 8 termini flanking segmental duplications of various lengths. These results show how 9 alternative transposition coupled with DNA replication and repair can significantly alter 10 genome structure and may have contributed to rapid genome evolution in maize and 11 possibly other eukaryotes. 12 13 Introduction 14 Initiation of DNA replication in eukaryotic cells is controlled by the replication licensing system 15 (Blow, 1993; Blow and Dutta, 2005; Truong and Wu, 2011), which ensures that each segment of 16 the genome is replicated only once per cell cycle. The expression and activity of the replication 17 licensing factors are precisely regulated, and misexpression or mutation of these factors can lead 18 to DNA re-replication, genome instability, major chromosomal rearrangements, and 19 tumorigenesis (Green et al., 2010; Green and Li, 2005; Hook et al., 2007; Liontos et al., 2007; 20 Melixetian et al., 2004; Rice et al., 2005; Sugimoto et al., 2009). Misregulation of some histone 21 methyltransferases can also result in DNA re-replication in plants and animals (Fu et al., 2013; 22 Jacob et al., 2010; Tardat et al., 2010). 23 24 Although DNA replication is strictly controlled, some DNA segments can escape this restriction 25 and replicate more than once in a single cell cycle in normal cells. For example, some Class II 26 DNA transposons, including the maize Ac/Ds system, E. coli TN10, and E. coli TN7, are known 27 to transpose during DNA replication (Chen et al., 1987; Peters and Craig, 2001; Roberts et al., 28 1985). If a replicated transposon excises and reinserts into an unreplicated site, the transposon 29 can undergo one additional replication in the same S phase; the re-replication, however, is 30 limited to the TE itself, and does not extend into the TE-flanking regions. 31 32 We and others have previously shown that a pair of Ac termini in reversed orientation can 33 undergo transposition, generating major chromosomal rearrangements such as deletions, 34 inversions, permutations, duplications, and reciprocal translocations (Huang and Dooner, 2008; 35 Zhang and Peterson, 2004; Zhang et al., 2009; Zhang et al., 2006; Zhang et al., 2013), this 36 transposition reaction is termed reversed Ac ends transposition (RET). All the RET-generated 37 genome rearrangements described to date are fully explained by models in which the excised TE 38 termini inserted into target sites that had completed DNA replication. However, it seems 39 reasonable to expect that RET, like standard Ac/Ds transposition, may also occur during DNA 40 replication, and that the excised reversed Ac termini could insert into unreplicated target sites. 41 Here we show that such events do occur, and that they can induce re-replication of the TE and its 42 flanking sequences. This process generates novel structures termed Composite Insertions (CIs) 43 which contain TE sequences and variable lengths of the flanking genomic DNA. 44