TRF2-mediated ORC recruitment underlies telomere stability upon DNA replication stress
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Evaluation Summary:
This paper is of interest to biochemists studying DNA replication and genome maintenance in eukaryotic cells. The work details a structure-function analysis of an interaction between two proteins that are critical for genome stability. A mutation that disrupts this interaction may have no adverse effects under unperturbed conditions but causes telomeric DNA damage when cells experience replication stress. However, the structural nature of the damage and cellular consequences are not sufficiently explored.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Telomeres are intrinsically difficult-to-replicate regions of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2-ORC interaction for telomere maintenance remains largely elusive. Here, we employed a separation-of-function TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that specifically inhibited the TRF2-ORC interaction in human cells without substantially inhibiting TRF2 interactions with its other binding partners. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244–511), which competitively inhibited the TRF2-ORC interaction, increased telomeric DNA damage under replication stress conditions in human cells. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability.
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Evaluation Summary:
This paper is of interest to biochemists studying DNA replication and genome maintenance in eukaryotic cells. The work details a structure-function analysis of an interaction between two proteins that are critical for genome stability. A mutation that disrupts this interaction may have no adverse effects under unperturbed conditions but causes telomeric DNA damage when cells experience replication stress. However, the structural nature of the damage and cellular consequences are not sufficiently explored.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The authors have used a structure based mutational scan of TRF2 to create a separation of function mutant in the protein that would form dimers and gets incorporated into the Shelterin complex, with a defect anticipated to be only in recruiting ORC to telomeres. The mutations used fall within a loop connecting two helices where the side chains of the wt protein are found on the outer surface of the protein. This is well done and data that the mutations are defective in recruiting ORC are convincing especially at the synthetic loci described. This is an advance over what has been done previously that has suggested through Knock down of the entire Trf2 such interactions and roles in DNA replication. The biochemical demonstration that ORC actually touches this region of TRF2 or indeed makes a direct contact to …
Reviewer #1 (Public Review):
The authors have used a structure based mutational scan of TRF2 to create a separation of function mutant in the protein that would form dimers and gets incorporated into the Shelterin complex, with a defect anticipated to be only in recruiting ORC to telomeres. The mutations used fall within a loop connecting two helices where the side chains of the wt protein are found on the outer surface of the protein. This is well done and data that the mutations are defective in recruiting ORC are convincing especially at the synthetic loci described. This is an advance over what has been done previously that has suggested through Knock down of the entire Trf2 such interactions and roles in DNA replication. The biochemical demonstration that ORC actually touches this region of TRF2 or indeed makes a direct contact to TRf2 is missing. Recruitment to chromosomes via co-localization has value but doesn't establish the point.
The major aim of the paper is to test the notion that defects in the recruitment of ORC to telomeres results in DNA replication defects at the telomeres. The manuscript shows that in clones selected there is no DNA damage at telomeres when the mutant TRF2 replaces the wt allele. The control used is a knock -out of the wt (with a CRISPR SS DNA method) and then a replacement wt allele with a marking restriction site. Both the control and the mutants are expressed at lower levels that the parent clone, and more DNA damage is found in the control than parent. This appears confusing and is perhaps a weakness of the paper.
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Reviewer #2 (Public Review):
Higa and coworkers performed a detailed structure-function analysis to identify the interaction sites between ORC1, a member of the origin recognition complex ORC1-6, and TRF2, a telomeric repeat binding factor that interacts with ORC. The authors generated a TRF2-EE mutant in which two aspartic acid residues at positions 111 and 112 are substituted by alanine. This mutant retains the ability to execute all telomere-specific functions as a subunit of the Shelterin complex that protects chromosome ends but is deficient for ORC1 binding. Most of the functional assessments of this mutation are carried out in cell lines that express the mutant form of TRF2 from the endogenous locus. This is a definitive strength of the study. Although the mutation doesn't have any effect on genome integrity under unperturbed …
Reviewer #2 (Public Review):
Higa and coworkers performed a detailed structure-function analysis to identify the interaction sites between ORC1, a member of the origin recognition complex ORC1-6, and TRF2, a telomeric repeat binding factor that interacts with ORC. The authors generated a TRF2-EE mutant in which two aspartic acid residues at positions 111 and 112 are substituted by alanine. This mutant retains the ability to execute all telomere-specific functions as a subunit of the Shelterin complex that protects chromosome ends but is deficient for ORC1 binding. Most of the functional assessments of this mutation are carried out in cell lines that express the mutant form of TRF2 from the endogenous locus. This is a definitive strength of the study. Although the mutation doesn't have any effect on genome integrity under unperturbed conditions, cells exhibit signs of telomeric DNA damage when they are treated with the replication inhibitor hydroxyurea. The authors argue that the damage is caused by the lack of telomeric replication complexes and the inability to rescue stalled replication forks. These claims are based on indirect conclusions, as there aren't any experiments that assess replication through telomeric sequences directly. In a second line of investigation, the group overexpressed a peptide of ORC1 that contains the binding site for TRF2 (ORC244-511) and showed convincingly that this strategy sequesters ORC in a way that it no longer binds to telomeres. This is underscored by a loss of MCM2-7 binding in telomeres. The authors claim that overexpression of the mutant did not affect other genomic regions based on a re-replication assay, not based on direct inspection of origin firing. This is a weakness. Another shortcoming is the limited analysis of the telomeric damage that ensues when cells are treated with hydroxyurea. It remains unclear if the damage is persistent and leads to telomere shortening or telomeric translocations. Some telomeres are processed and become part of micronuclei. This is an interesting observation but could be specific to HeLa cells. All of the presented data is very clean and well controlled.
In conclusion, this is a rigorous study of limited new insight and scope that suggests that the interaction between ORC1 and TRF2 is important to suppress telomeric DNA damage under replication stress conditions.
Concerns:
- It seems as if all of the experiments addressing functional readouts are conducted in HeLa cells although the Materials and Methods describe a variety of different cell lines. It is therefore not clear whether the findings are more generally applicable to other transformed and non-transformed cells. If experiments were performed in multiple cell lines, this needs to be stated more clearly in the figure legends.
- To the best of my knowledge, most telomerase-positive cell lines have telomeres of around 5-7kb (under 10kb). It has been demonstrated by the Schildkraut and de Lange laboratories that short telomeres rarely activate replication origins. Only in longer, upwards of 25-50kb can replication forks be detected that originate within telomeres. It is not clear to me how long the telomeres are in the cell line(s) used in this study.
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Reviewer #3 (Public Review):
In this paper, the authors use gene editing techniques to examine the potential relationship between telomeric binding protein TRF2 and the origin recognition complex protein ORC1. Studies about TRF2 are often difficult, as changes in TRF2 often lead to genome instability. The authors circumvent this through the generation of separation-of-function mutations, to only disrupt TRF2-ORC1 binding. They then demonstrate that overexpression of an ORC1 fragment that binds to TRF2 can lead to replication stress.
Concerns:
• The foremost challenge for this paper is the interpretation of the presented comparisons, due to lack of an effective control. The authors chose to make a separation of function mutation specifically because TRF2 expression level has a direct impact on cell viability. However, their wt control …
Reviewer #3 (Public Review):
In this paper, the authors use gene editing techniques to examine the potential relationship between telomeric binding protein TRF2 and the origin recognition complex protein ORC1. Studies about TRF2 are often difficult, as changes in TRF2 often lead to genome instability. The authors circumvent this through the generation of separation-of-function mutations, to only disrupt TRF2-ORC1 binding. They then demonstrate that overexpression of an ORC1 fragment that binds to TRF2 can lead to replication stress.
Concerns:
• The foremost challenge for this paper is the interpretation of the presented comparisons, due to lack of an effective control. The authors chose to make a separation of function mutation specifically because TRF2 expression level has a direct impact on cell viability. However, their wt control and mutants have decreased TRF2 expression. Moreover, the decreases are not similar between the experimental and the wild type. Concerningly, the differences in the parental line and the wt control clone are quite striking in many of the panels. This becomes quite clear in Figure 2 Supplement 1, Figure 2E, and Figure 3B; it seems that most of the effect is driven by a clonal difference in the wt cell clone rather than a true biological difference. Due to this, it is difficult to tease apart which phenotypes are actually as a result of their mutations, or if it's an issue of TRF2 expression, or preexisting genomic differences in cancer cells that surface when cells are subcloned during the targeting process.
• In addition, a major conclusion of the paper is derived from Figure 6. The authors argue that the separation of function mutants only reveals telomere damage when treated with HU. In Figure 6, it is not clear yet if there is damage at the telomere specifically, or there is damage everywhere including telomeres. This seems to be a central point that would need to be directly shown.
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