Author response:
<blockquote>
Reviewer #1:
Summary:
The authors address the role of the centromere histone core in force transduction by the kinetochore.
Strengths:
They use a hybrid DNA sequence that combines CDEII and CDEIII as well as Widom 601 so they can make stable histones for biophysical studies (provided by the Widom sequence) and maintain features of the centromere (CDE II and III).
Weaknesses:
The main results are shown in one figure (Figure 2). Indeed the Centromere core of Widom and CDE II and III contribute to strengthening the binding force for the OA-beads. The data are very nicely done and convincingly demonstrate the point. The weakness is that this is the entire paper. It is certainly of interest to investigators in kinetochore biology, but beyond that, the impact is fairly limited in scope.
</blockquote>
This reviewer might have missed that this is a Research Advance, not an article. Research Advances are limited in scope by definition and provide a new development that builds on research reported in a prior paper. They can be of any length. Our Research Advance builds on our prior work, Hamilton et al., 2020 and provides the new result that native centromere sequences strengthen the attachment of the kinetochore to the nucleosome.
<blockquote>
Reviewer #2:
Summary:
This paper provides a valuable addendum to the findings described in Hamilton et al. 2020 (<a href="https://doi.org/">https://doi.org/</a> 10.7554/eLife.56582). In the earlier paper, the authors reconstituted the budding yeast centromeric nucleosome together with parts of the budding yeast kinetochore and tested which elements are required and sufficient for force transmission from microtubules to the nucleosome. Although budding yeast centromeres are defined by specific DNA sequences, this earlier paper did not use centromeric DNA but instead the generic Widom 601 DNA. The reason is that it has so far been impossible to stably reconstitute a budding yeast centromeric nucleosome using centromeric DNA.
In this new study, the authors now report that they were able to replace part of the Widom 601 DNA with centromeric DNA from chromosome 3. This makes the assay more closely resemble the in vivo situation. Interestingly, the presence of the centromeric DNA fragment makes one type of minimal kinetochore assembly, but not the other, withstand stronger forces.
</blockquote>
We thank the reviewer for their careful and positive assessment of our work.
<blockquote>
Which kinetochore assembly turned out to be affected was somewhat unexpected, and can currently not be reconciled with structural knowledge of the budding yeast centromere/kinetochore. This highlights that, despite recent advances (e.g. Guan et al., 2021; Dendooven et al., 2023), aspects of budding yeast kinetochore architecture and function remain to be understood and that it will be important to dissect the contributions of the centromeric DNA sequence.
</blockquote>
We couldn’t agree more.
<blockquote>
Given the unexpected result, the study would become yet more informative if the authors were able to pinpoint which interactions contribute to the enhanced force resistance in the presence of centromeric DNA.
Strength:
The paper demonstrates that centromeric DNA can increase the attachment strength between budding yeast microtubules and centromeric nucleosomes.
Weakness:
How centromeric DNA exerts this effect remains unclear.
</blockquote>

Yamini Dalal

eLife Assessment
Centromeres are specific sites on chromosomes that are essential for mitosis and genome fidelity. This valuable work extends previous studies to convincingly show that the centromere-histone core contributes to force transduction through the kinetochore. The centromere mainly strengthens one of the two paths of force transduction, influenced by the centromeric DNA sequence, the mechanism for which remains to be determined. This work will be of interest to those studying cell division and chromosome segregation.

anonymous

Reviewer #1 (Public review):
Summary:
The authors address the role of the centromere histone core in force transduction by the kinetochore.
Strengths:
They use a hybrid DNA sequence that combines CDEII and CDEIII as well as Widom 601 so they can make stable histones for biophysical studies (provided by the Widom sequence) and maintain features of the centromere (CDE II and III).
Weaknesses:
The main results are shown in one figure (Figure 2). Indeed the Centromere core of Widom and CDE II and III contribute to strengthening the binding force for the OA-beads. The data are very nicely done and convincingly demonstrate the point. The weakness is that this is the entire paper. It is certainly of interest to investigators in kinetochore biology, but beyond that, the impact is fairly limited in scope.

Reviewer #2 (Public review):
Summary:
This paper provides a valuable addendum to the findings described in Hamilton et al. 2020 (<a href="https://doi.org/">https://doi.org/</a> 10.7554/eLife.56582). In the earlier paper, the authors reconstituted the budding yeast centromeric nucleosome together with parts of the budding yeast kinetochore and tested which elements are required and sufficient for force transmission from microtubules to the nucleosome. Although budding yeast centromeres are defined by specific DNA sequences, this earlier paper did not use centromeric DNA but instead the generic Widom 601 DNA. The reason is that it has so far been impossible to stably reconstitute a budding yeast centromeric nucleosome using centromeric DNA.
In this new study, the authors now report that they were able to replace part of the Widom 601 DNA with centromeric DNA from chromosome 3. This makes the assay more closely resemble the in vivo situation. Interestingly, the presence of the centromeric DNA fragment makes one type of minimal kinetochore assembly, but not the other, withstand stronger forces.
Which kinetochore assembly turned out to be affected was somewhat unexpected, and can currently not be reconciled with structural knowledge of the budding yeast centromere/kinetochore. This highlights that, despite recent advances (e.g. Guan et al., 2021; Dendooven et al., 2023), aspects of budding yeast kinetochore architecture and function remain to be understood and that it will be important to dissect the contributions of the centromeric DNA sequence.
Given the unexpected result, the study would become yet more informative if the authors were able to pinpoint which interactions contribute to the enhanced force resistance in the presence of centromeric DNA.
Strength:
The paper demonstrates that centromeric DNA can increase the attachment strength between budding yeast microtubules and centromeric nucleosomes.
Weakness:
How centromeric DNA exerts this effect remains unclear.

Force transmission through the inner kinetochore is enhanced by centromeric DNA sequences

Figures and data

Recombinantly wrapped NCPs with W601 or CCEN DNA.

Statistical analyses for microtubule binding assay¹

Kinetochore assemblies built on CCEN-NCPs and OA form stronger attachments to microtubules.

Plasmids used for expression of kinetochore proteins¹

Kinetochore protein purification buffers.

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