A novel single alpha-helix DNA-binding domain in CAF-1 promotes gene silencing and DNA damage survival through tetrasome-length DNA selectivity and spacer function
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eLife assessment
The important paper describes the structure of a single alpha helix in the large subunit of the Chromatin Assembly Factor 1 (CAF-1) that binds DNA. The single alpha-helix DNA interaction is novel and, combined with the CAF-1 Winged Helix Domain, is required for CAF-1 function in vivo for gene silencing and DNA damage response. The data are convincing, but there are additional analyses that may be considered.
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Abstract
The histone chaperone chromatin assembly factor 1 (CAF-1) deposits two nascent histone H3/H4 dimers onto newly replicated DNA forming the central core of the nucleosome known as the tetrasome. How CAF-1 ensures there is sufficient space for the assembly of tetrasomes remains unknown. Structural and biophysical characterization of the lysine/glutamic acid/arginine-rich (KER) region of CAF-1 revealed a 128-Å single alpha-helix (SAH) motif with unprecedented DNA-binding properties. Distinct KER sequence features and length of the SAH drive the selectivity of CAF-1 for tetrasome-length DNA and facilitate function in budding yeast. In vivo, the KER cooperates with the DNA-binding winged helix domain in CAF-1 to overcome DNA damage sensitivity and maintain silencing of gene expression. We propose that the KER SAH links functional domains within CAF-1 with structural precision, acting as a DNA-binding spacer element during chromatin assembly.
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Author Response
Reviewer #3 (Public Review):
The strongest aspects of this study are the structural analysis of the 90 residue KER domain. This is an important advance, discovering a founding member of a novel class of DNA binding motifs, termed a SAH-DBD (single alpha helix-DNA binding domain). Interestingly, they define a subregion of KER (termed "middle-A", residues 155-204 of Cac1) that has nearly the same DNA binding affinity and confers similar in vivo phenotypes as the full KER domain.
This study also shows that the biological role of KER partially overlaps compensatory factors in vivo, both within the same Cac1 protein subunit (e.g. the WHD domain) and also with other proteins acting in parallel (e.g. Rtt106). That is, the presence of either WHD or Rtt106 renders the drug-resistance and silencing assays employed here …
Author Response
Reviewer #3 (Public Review):
The strongest aspects of this study are the structural analysis of the 90 residue KER domain. This is an important advance, discovering a founding member of a novel class of DNA binding motifs, termed a SAH-DBD (single alpha helix-DNA binding domain). Interestingly, they define a subregion of KER (termed "middle-A", residues 155-204 of Cac1) that has nearly the same DNA binding affinity and confers similar in vivo phenotypes as the full KER domain.
This study also shows that the biological role of KER partially overlaps compensatory factors in vivo, both within the same Cac1 protein subunit (e.g. the WHD domain) and also with other proteins acting in parallel (e.g. Rtt106). That is, the presence of either WHD or Rtt106 renders the drug-resistance and silencing assays employed here insensitive to loss of the KER domain.
However, the drug resistance and gene silencing phenotypes are inherently indirect measures of the most important claim of this work, that KER is a molecular ruler for DNA for the purpose of ensuring sufficiently large templates deposition of histone H3/H4 cargoes. Therefore, this study would be of greater impact if the authors more directly tested this measurement idea in assays that directly assess histone deposition. There are multiple options. Since the authors have in hand recombinant wild-type and mutant CAF-1 complexes, one could examine the number and/or spacing of nucleosomes formed during in vitro deposition reactions. Complementary in vivo experiments using the authors' existing mutant strains could be based on the finding that CAF-1 is particularly important for histone deposition onto nascent Okazaki fragments during DNA replication (Smith and Whitehouse, 2012; pmid: 22419157), and that the spacing pattern of nucleosomes on this DNA is greatly perturbed in cac1-delete cells.
Thank you for the suggestion of approaches to obtain data that more directly addresses changes in nucleosome assembly due to CAF-1 KER mutants. We considered using an in vitro nucleosome assembly assay, such as the reconstitution of nucleosomes onto gapped DNA using purified components developed by Kadyrova et al., 2013 (doi: 10.4161/cc.26310). However, they found defects only in the amount of nucleosome assembly and not changes in nucleosome spacing without CAF-1. In addition, we didn’t have the system set up and knew that it would be unlikely to produce data in the time needed for a revision of the manuscript, or even show spacing changes in nucleosomes at all. Therefore, we chose an assay system in yeast that already has been used to assess the impact of CAF-1 DNA binding mutants on nucleosome assembly (Smith and Whitehouse, 2012; pmid: 22419157 and Mattiroli et al., 2017 doi: 10.7554/eLife.22799). This approach, developed by Smith and Whitehouse, uses a degradable Ligase I system in yeast, which reveals Okazaki fragment lengths, and shows a defect when CAF-1 activity is knocked out (Smith and Whitehouse, 2012). This assay also showed that mutations or deletions in the Cac1 WHD DNA binding domain, led to increased lengths of Okazaki fragments (Mattiroli et al., 2017). As the WHD DBD impacts Okazaki fragment lengths, we reasoned that mutations in the KER DBD might also.
We generated numerous new yeast strains that included the degradable Ligase I system and collaborated with Dr. Duncan Smith of (Smith and Whitehouse, 2012; pmid: 22419157) to detect nascent Okazaki fragments in various CAC1 mutants in strains that were RTT106 or rtt106∆. We found that the Okazaki fragment lengths from cac1∆ yeast were larger and less discrete than from CAC1 yeast (as Dr Smith published previously) and that the Okazaki fragments from the cac1∆ rtt106∆ strain were barely detectable, presumably because they were too long to be resolved on the gel. However, the assay did not have sufficient resolution to detect changes between the Okazaki fragment length distribution between wild type CAC1 or the ∆KER, ∆middle-A and 2xKER mutants of CAC1, in either the RTT106 or rtt106∆ background. Therefore, we were unable to detect direct effects of the KER mutants on Okazaki-fragment lengths. We considered using the combination of KER mutants with the WHD mutants, but as this would not directly assess the effects of the KER mutants and CAF-1 proteins lacking the KER and the WHD don’t bind to DNA (Figure 3 in Mattiroli et al., 2017), we didn’t pursue it. As the complete deletion of the KER, shortening of the KER and lengthening of the KER did not give detectable changes in this assay, we also did not pursue the other mutants tested in the manuscript. Although, we are disappointed the experiment did not reveal effects that we had hoped for, this experiment provides support for the redundant functions of CAF-1 and Rtt106 in nucleosome assembly, which has not been shown using this assay. As such, we have added Figure 1-figure supplement 1g and text to the results section, methods section and strain table. We have included Prof. Duncan Smith and his student Anne Seck as authors.
Added text lines 195 to 207: “Finally, to assess the impact of deleting the KER more directly on nucleosome assembly in vivo, we examined histone deposition onto nascent Okazaki fragments during DNA replication as we have shown previously that the length of Okazaki fragment lengths are determined by histone deposition into nucleosomes and is disrupted upon deletion of CAC1 (Smith and Whitehouse, 2012). We compared CAF-1 mutants in the WT yeast background and in yeast lacking Rtt106. We found that the Okazaki fragment length distributions of the ∆KER mutant was indistinguishable from that of WT while that of cac1∆ was disrupted (Figure 1-figure supplement Figure 1-figure supplement 3g). That we did not detect effects on Okazaki-fragment lengths for the yCAF-1 mutants lacking the intact KER is consistent with the results of the viability and silencing assays for KER mutants, which also retained the WHD. Strikingly, the Okazaki fragments from rtt106∆ cac1∆ yeast were highly disrupted (Figure 1-figure supplement Figure 1-figure supplement 3g) further highlighting the redundancy between Rtt106 and Cac1 for assembling histones onto newly replicated DNA. Therefore, t”
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eLife assessment
The important paper describes the structure of a single alpha helix in the large subunit of the Chromatin Assembly Factor 1 (CAF-1) that binds DNA. The single alpha-helix DNA interaction is novel and, combined with the CAF-1 Winged Helix Domain, is required for CAF-1 function in vivo for gene silencing and DNA damage response. The data are convincing, but there are additional analyses that may be considered.
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Reviewer #1 (Public Review):
This study aimed at the identification additional region of Cac1 involved in DNA binding. Previously, it has been shown that Cac1, the large subunit of chromatin assembly factor 1 (CAF-1), contains DNA binding other regions in addition to the known WHD domain. This study shows that the KER region of Cac1 form a single alpha helix based on CD and crystal structure analysis. Furthermore, unlike the SAH motif in other proteins, the Cac1 SAH motif binds DNA. Further, this motif, along with WHD motif, is important for the function of Cac1 in heterochromatin silencing and in response to DNA damage agents in cells, suggesting that these two regions are important for nucleosome assembly. The majority of experiments are well controlled and the results support the confusions. The major concern is that the human KER …
Reviewer #1 (Public Review):
This study aimed at the identification additional region of Cac1 involved in DNA binding. Previously, it has been shown that Cac1, the large subunit of chromatin assembly factor 1 (CAF-1), contains DNA binding other regions in addition to the known WHD domain. This study shows that the KER region of Cac1 form a single alpha helix based on CD and crystal structure analysis. Furthermore, unlike the SAH motif in other proteins, the Cac1 SAH motif binds DNA. Further, this motif, along with WHD motif, is important for the function of Cac1 in heterochromatin silencing and in response to DNA damage agents in cells, suggesting that these two regions are important for nucleosome assembly. The majority of experiments are well controlled and the results support the confusions. The major concern is that the human KER region cannot complement the yeast KER region, likely due to multiple possibilities, which needed to be tested.
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Reviewer #2 (Public Review):
The manuscript illuminates the biological function of the Cac-1 "KER" region within the CAF-1 chromatin assembly factor 1. (This region has a high density of lysine, glutamic acid and arginine residues). The authors present a comprehensive study including quantitative EMSA analyses, analysis of mutants in-vivo, CD, and X-ray crystallography to identify the KER domain as a single alpha-helix element (SAH) that is largely responsible for the ability of the yCAF-1 complex to selectively binding ~40 bp dsDNA fragments over shorter ds oligos, thought to be a 'measuring' function that determines there is sufficient space for assembling H3/H4 tetramers after passage of the DNA replication complex. Moreover, they find that deletions or modifications of the KER domain contribute to yeast phenotypes consistent with a …
Reviewer #2 (Public Review):
The manuscript illuminates the biological function of the Cac-1 "KER" region within the CAF-1 chromatin assembly factor 1. (This region has a high density of lysine, glutamic acid and arginine residues). The authors present a comprehensive study including quantitative EMSA analyses, analysis of mutants in-vivo, CD, and X-ray crystallography to identify the KER domain as a single alpha-helix element (SAH) that is largely responsible for the ability of the yCAF-1 complex to selectively binding ~40 bp dsDNA fragments over shorter ds oligos, thought to be a 'measuring' function that determines there is sufficient space for assembling H3/H4 tetramers after passage of the DNA replication complex. Moreover, they find that deletions or modifications of the KER domain contribute to yeast phenotypes consistent with a deficiency in chromatin assembly. The data in the paper is compelling, supports the conclusions and adds critical new information regarding how CAF-1 functions accomplishes its 'spacing' function in cooperation with DNA replication machinery to deposit H3/H4 dimers onto replicated DNA.
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Reviewer #3 (Public Review):
The strongest aspects of this study are the structural analysis of the 90 residue KER domain. This is an important advance, discovering a founding member of a novel class of DNA binding motifs, termed a SAH-DBD (single alpha helix-DNA binding domain). Interestingly, they define a subregion of KER (termed "middle-A", residues 155-204 of Cac1) that has nearly the same DNA binding affinity and confers similar in vivo phenotypes as the full KER domain.
This study also shows that the biological role of KER partially overlaps compensatory factors in vivo, both within the same Cac1 protein subunit (e.g. the WHD domain) and also with other proteins acting in parallel (e.g. Rtt106). That is, the presence of either WHD or Rtt106 renders the drug-resistance and silencing assays employed here insensitive to loss of the …
Reviewer #3 (Public Review):
The strongest aspects of this study are the structural analysis of the 90 residue KER domain. This is an important advance, discovering a founding member of a novel class of DNA binding motifs, termed a SAH-DBD (single alpha helix-DNA binding domain). Interestingly, they define a subregion of KER (termed "middle-A", residues 155-204 of Cac1) that has nearly the same DNA binding affinity and confers similar in vivo phenotypes as the full KER domain.
This study also shows that the biological role of KER partially overlaps compensatory factors in vivo, both within the same Cac1 protein subunit (e.g. the WHD domain) and also with other proteins acting in parallel (e.g. Rtt106). That is, the presence of either WHD or Rtt106 renders the drug-resistance and silencing assays employed here insensitive to loss of the KER domain.
However, the drug resistance and gene silencing phenotypes are inherently indirect measures of the most important claim of this work, that KER is a molecular ruler for DNA for the purpose of ensuring sufficiently large templates deposition of histone H3/H4 cargoes. Therefore, this study would be of greater impact if the authors more directly tested this measurement idea in assays that directly assess histone deposition. There are multiple options. Since the authors have in hand recombinant wild-type and mutant CAF-1 complexes, one could examine the number and/or spacing of nucleosomes formed during in vitro deposition reactions. Complementary in vivo experiments using the authors' existing mutant strains could be based on the finding that CAF-1 is particularly important for histone deposition onto nascent Okazaki fragments during DNA replication (Smith and Whitehouse, 2012; pmid: 22419157), and that the spacing pattern of nucleosomes on this DNA is greatly perturbed in cac1-delete cells.
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