Transposition-mediated DNA re-replication in maize

Thank you for sending your work entitled “Transposition-mediated DNA re-replication in maize” for consideration at eLife. Your article has been favorably evaluated by Ian Baldwin (Senior editor) and 3 reviewers.

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The Senior editor and the reviewers discussed their comments before we reached this decision, and we have assembled the following comments to help you prepare a revised submission.

The manuscript describes a detailed analysis of an alternative transposition induced DNA re-replication in the best-studied transposon system to date, namely the Ac/Ds system of maize. A model is described of DNA re-replication mediated by the alternative transposition of Ac/fAc, which in addition to other well-documented chromosomal rearrangements caused by transposition, may have contributed to the dynamics of gene creation and genome expansion. All reviewers agreed that the manuscript was dense but well-written, and that the data supports the main conclusions of work and provides valuable insights into TE behavior. The addition of a new pathway contributing to genome dynamic by re-replication of particular chromosome regions through alternative transposition represents a significant advance in our understanding of transposon proliferation and genome evolution.

The reviewers make a number of points listed below, which if addressed would help to clarify the presentation and broaden the impact of your story. We look forward to your revision and reading how you have responded to these points in a cover letter.

1) The authors argue the generality of TDDCI in the maize genome by a previously characterized bz1-m4-D6856 allele, which was isolated by Barbara McClintock. The actual sequences of bz1-m4-D6856 allele and its progenitor line would provide more solid evidence to support the authors' arguments.

2) The authors argue about the reinsertion frequency of the excised fragment and make efforts to compare with previously reports statistically. About this issue, the reviewer has unpublished data from an enormous population and supports the lower reinsertion value as the authors present in their manuscript. It is a bit wasteful to make big deal out of this.

3) Is there any commonality to the sequence context or genetic/epigenetic context of the RET insertion sites recovered?

1) The authors argue the generality of TDDCI in the maize genome by a previously characterized bz1-m4-D6856 allele, which was isolated by Barbara McClintock. The actual sequences of bz1-m4-D6856 allele and its progenitor line would provide more solid evidence to support the authors' arguments.

We did try to detect a Ds insertion distal to bz1 in stocks containing bz1-m4-D6856 and its presumed progenitor. First, we requested and received seeds of the putative bz1-m4-D6856 progenitor from Dr. Anita Klein, who originally published on this allele (Klein et al., 1988). Unfortunately the seeds were very old and failed to germinate. Next we requested and received seeds of related stocks from Dr. Hugo Dooner, and these we were able to propagate; however, the results of several PCR experiments using bz1- and Ds-homologous primers were negative. This negative result is not too surprising, considering the variation in possible positions of Ds, as well as the uncertainty in identification of the actual progenitor. Moreover, the published account of the origin of bz1-m4-D6856 suggests that the Ds element may have excised during or shortly after the formation of the allele. The detailed description is provided in the following excerpt from Klein, A.S., Clancy, M., Paje-Manalo, L., Furtek, D.B., Hannah, L.C., and Nelson, O.E., Jr (1988) “The mutation bronze-mutable 4 derivative 6856 in maize is caused by the insertion of a novel 6.7-kilobase pair transposon in the untranslated leader region of the bronze-1 gene”, Genetics 120, 779-790:

“The origin of bz-ml D6856 is complex (Figure 1). McClintock (1952) observed that a transposable element at one locus would “spread” to adjacent loci. In the maize line she was studying, Ds, in the presence of Ac (Activator), caused chromosome breaks immediately distal to the shrunken (sh) locus. From this stock, McClintock isolated new mutable alleles of the flanking genes, C-I (dominant colorless) or Bz. The original bz-m4 allele was isolated in that study (B. McClintock, personal communication). This bz-m4 line was stably recessive for the shrunken (sh) trait. Later McClintock demonstrated that recombination between sh and bz in this stock was substantially reduced, indicating that the unstable bz-ml allele arose concommitantly with a deletion of chromosomal material in the interval between these loci (McClintock 1965; Dooner 1981). In the presence of Ac, the original bz-m4 allele formed dicentric chromosomes at a high frequency. In a subsequent generation this bz-m4 reverted to a Bz’ allele (B. McClintock, personal communication). This was unstable, indicating that a Ds element was near or at the Bz’ allele. Subsequently, again with Ac present, a gamete from a Bz’-m plant, culture #6771, mutated to an unusual dark bronze, recessive allele which also had a reduced frequency of dicentric formation. This allele was bz-ml D6856.”

Revised eLife text: “This model presupposes the existence of a Ds element (the leftmost element in Figure 8B) distal to the tandem repeats in bz1-m4-D6856 and its progenitor allele. No such element was reported on the original bz1-m4-D6856 genomic clones (Klein et al., 1988; Dowe et al., 1990). Efforts in our lab to identify a Ds element in this position in bz1-m4-D6856 and related stocks have been unsuccessful. However, McClintock's description of the origin of bz1-m4-D6856 (as reported in Klein et al., 1988) indicates that the bz1-m4 progenitor produced a high frequency of dicentric chromosomes, while the bz1-m4-D6856 derivative exhibited low dicentric frequency. Dicentric chromosome formation is a characteristic feature of alternative transposition reactions, such as RET, involving two nearby Ac/Ds elements (Huang and Dooner, 2008; Yu et al., 2010). The switch from high to low dicentric frequency observed by McClintock would be consistent with excision of the “missing” Ds shortly after the formation of the bz1-m4-D6856 allele.”

2) The authors argue about the reinsertion frequency of the excised fragment and make efforts to compare with previously reports statistically. About this issue, the reviewer has unpublished data from an enormous population and supports the lower reinsertion value as the authors present in their manuscript. It is a bit wasteful to make big deal out of this.

We thank the reviewer for relating their unpublished data regarding reinsertion values. Per the reviewer’s suggestion we have shortened this section, while keeping the main point that the repair of broken chromatid ends is surprisingly efficient.

Revised eLife text: “Our model proposes that DNA re-replication aborts to produce chromatids terminated by broken ends, which are joined together to restore chromosome linearity (Figures 1, 6, 7 and 9). If the chromatid DSBs were not repaired, the cell would die and that event would not be recovered in our screen. From a population of ∼2000 plants, we isolated 16 alleles that carry a duplication and/or insertion structure. Nine of these 16 alleles (56%) have only a duplication (Zhang et al., 2013), which indicates that the target site was replicated at the time of RET (Figure 1–figure supplement 1); whereas seven alleles have an insertion, which indicates the target site was unreplicated (Figures 1, 6 and 7). The frequency of insertion into an unreplicated target site is 7/16 (44%), which is similar to a previous estimate of Ac insertion into unreplicated sites (Greenblatt and Brink, 1962). Thus the products of insertion into unreplicated target sites are not significantly under-represented in our sample, suggesting that repair of re-replication-generated DSBs is quite efficient in mitotic S phase cells.”

3) Is there any commonality to the sequence context or genetic/epigenetic context of the RET insertion sites recovered?

In all respects, the RET insertion sites appear to be similar to those observed for standard Ac/Ds transposition. We have included a sentence to this effect in the Discussion:

“Moreover, the RET reinsertion sites have the same characteristic features as for standard Ac/Ds transposition, including preferential insertion into nearby, hypomethylated, gene-rich regions (Greenblatt and Brink, 1962; Chen et al., 1992; Vollbrecht et al., 2010), and formation of 8-bp Target Site Duplications lacking sequence specificity (Vollbrecht et al., 2010).”