DNA replication errors are a major source of adaptive gene amplification

Reviewer #1 (Public Review):

Summary:

The work by Chuong et al. provides important new insights into the contribution of different molecular mechanisms in the dynamics of CNV formation. It will be of interest to anyone curious about genome architecture and evolution from yeast biologists to cancer researchers studying genome rearrangements.

Strengths:

Their results are especially striking in that the "simplest" mechanism of GAP1 amplification-non-allelic homologous recombination between the flanking Ty-LTR elements is not the most common route taken by the cells, emphasizing the importance of experimentally testing what might seem on the surface to be obvious answers. One of the important developments of their work is the use of their neural network simulation-based inference (nnSBI) model to derive rates of amplicon formation and their fitness effects.

Weaknesses:

The manuscript reads as though two different people wrote two different sections of the manuscript - an experimental evolutionist and a computational scientist. If the goal is to reach both groups of readers, there needs to be more explanation of both types of work. I found the computational sections to be particularly dense but even the experimental sections need clearer explanations and more specific examples of the rearrangements found. I will point out these areas in the detailed remarks to the authors. While I have no reason to question their conclusions, I couldn't independently verify the results that ODIRA was the majority mechanism since the sequence of amplified clones was not made available during the review. I've encouraged the authors to include specific, detailed sequence information for both ODIRA events as well as the specific clones where GAP1 was amplified but the flanking gene GFP was not.

Reviewer #2 (Public Review):

Summary:

This study examines how local DNA features around the amino acid permease gene GAP1 influence adaptation to glutamine-limited conditions through changes in GAP1 Copy Number Variation (CNV). The study is well motivated by the observation of numerous CNVs documented in many organisms, but difficulty in distinguishing the mechanisms by which they are formed, and whether or how local genomic elements influence their formation. The main finding is convincing and is that a nearby Autonomous Replicating Sequence (ARS) influences the formation of GAP1 CNVs and this is consistent with a predominate mechanism of Origin Dependent Inverted Repeat Amplification (ODIRA). These results along with finding and characterizing other mechanisms of GAP1 CNV formation will be of general interest to those studying CNVs in natural systems, experimental evolution, and in tumor evolution. While the results are limited to a single CNV of interest (GAP1), the carefully controlled experimental design and quantification of CNV formation will provide a useful guide to studying other CNVs and CNVs in other organisms.

Strengths:

The study was designed to examine the effects of two flanking genomic features next to GAP1 on CNV formation and adaptation during experimental evolution. This was accomplished by removing two Long Terminal Repeats (LTRs), removing a downstream ARS, and removing both LTRs and the ARS. Although there was some heterogeneity among replicates, later shown to include the size and breakpoints of the CNV and the presence of an unmarked CNV, both marker-assisted tracking of CNV formation and modeling of CNV rate and fitness effects showed that deletion of the ARS caused a clear difference compared to the control and the LTR deletion.

The consequence of deletion of local features (LTR and ARS) was quantified by genome sequencing of adaptive clones to identify the CNV size, copy number and infer the mechanism of CNV formation. This greatly added value to the study as it showed that i) ODIRA was the most common mechanism but ODIRA is enhanced by a local ARS, ii) non-allelic homologous recombination (NAHR) is also used but depends on LTRs, and iii) de novo insertion of transposable elements mediate NAHR in strains with both ARS and LTR deletions. Together, these results show how local features influence the mechanism of CNV formation, but also how alternative mechanisms can substitute when primary ones are unavailable.

Weaknesses:

The CNV mutation rate and its effect on fitness are hard to disentangle. The frequency of the amplified GFP provides information about mutation rate differences as well as fitness differences. The data and analysis show that each evolved population has multiple GAP1 CNV lineages within it, with some being unmarked by GFP. Thus, estimates of CNV fitness are more of a composite view of all CNV amplifications increasing in frequency during adaptation. Another unknown but potential complication is whether the local (ARS, LTR) deletions influence GAP1 expression and thus the fitness gain of GAP1 CNVs. The neural network simulation-based inference does a good job at estimating both mutation rates and fitness effects, while also accounting for unmarked CNVs. However, the model does not account for the population heterogeneity of CNVs and their fitness effects. Despite these limitations of distinguishing mutation rate and fitness differences, the authors' conclusions are well supported in that the LTR and ARS deletions have a clear impact on the CNV-mediated evolutionary outcome and the mechanism of CNV formation.

Reviewer #3 (Public Review):

Summary:

The authors represent an elegant and detailed investigation into the role of cis-elements, and therefore the underlying mechanisms, in gene dosage increase. Their most significant finding is that in their system copy number increase frequently occurs by what they call replication errors that result from the origin of replication firing.

The authors somewhat quantitatively determine the effect of the presence of a proximal origin of replication or LTR on the different CNV scenarios.

Strengths:

(1) A clever and elegant experimental design.

(2) A quantitative determination of the effect of a proximal origin of replication or LTR on the different CNV scenarios. Measuring directly the contribution of two competing elements.

(3) ODIRA can occur by firing of a distal ARS element.

(4) Re-insertion of Ty elements is interesting.

Weaknesses:

(1) Overall, the research does not considerably advance the current knowledge. The research does not investigate what the maximum distance between ARS for ODIRA is to occur. This is an important point since ODIRA was previously described. A considerable contribution to the field would be to understand under what conditions ODIRA wins NAHR.

(2) The title and some sentences in the abstract give a wrong impression of the generality and the novelty of the observations presented. Below are some examples of much earlier work that dealt with mechanisms of CNV and got different conclusions. The Lobachev lab (Cell 2006) published a different scenario years ago, with a very different mechanism (hair-pin capped breaks). The Argueso lab found something different (NAHR) (Genetics 2013).

In fact, the CUP1 system presents a good example of this point. The Houseley group showed a complex replication transcription-based mechanism (NAR 2022, cited), the Argueso group showed Ty-based amplification and the Resnick group showed aneuploidy-based amplification. While aneuploidy is a minor factor here the numerous works in Candida albicans, Cryptococcus neoformans, and Yeast suggest otherwise (Selmecki et al Science 2006, Yona et al PNAS 2013, Yang et al Microbiology Spectrum 2021).

(3) The authors added a mathematical model to their experimental data. For me, it was very difficult to understand the contribution of the model to the research. I anticipated, for example, that the model would make predictions that would be tested experimentally. For example, " ARSΔ and ALLΔ are predicted to be almost eliminated by generation 116, as the average predicted WT proportion is 0.998 and 0.999" But to my understanding without testing the model.