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
- Program in Structural Biology and Biochemistry, University of Colorado Anschutz Medical Campus, United States
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, United States
- Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program, United States
- Department of Biology, New York University, United States
- Department of Pharmacology, University of Colorado School of Medicine, United States
Figures
Figure 1 with 3 supplements
The yKER region favors binding to tetrasome-length DNA and facilitates the function of yCAF-1 in vivo.
(a) Cartoon representing the molecular architecture of the yCAF-1 complex highlighting the protein subunits and functional domains. Domains include the K/E/R-rich (DNA-binding domain), PCNA …
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Figure 1—source data 1
The yKER region favors binding to tetrasome-length DNA and facilitates the function of yCAF-1 in vivo.
Electrophoretic mobility shift assay (EMSA) images (panels b–e) and data analyses (panel j).
- https://cdn.elifesciences.org/articles/83538/elife-83538-fig1-data1-v1.zip
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Figure 1—source data 2
The yKER region favors binding to tetrasome-length DNA and facilitates the function of yCAF-1 in vivo.
Electrophoretic mobility shift assay (EMSA) images (panels f–i and m), data analyses (panels k and l), and flow cytometry data (panel o).
- https://cdn.elifesciences.org/articles/83538/elife-83538-fig1-data2-v1.zip
Figure 1—figure supplement 1
Proteins and mutants used in this study.
Figure 1—figure supplement 2
Quality of proteins used in this study.
4–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) stained with Coomassie Blue of the indicated protein domains purified from bacteria (b, c) or yCAF-1 complexes …
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Figure 1—figure supplement 2—source data 1
Quality of proteins used in this study.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (panels a–c) and western blots (panel d).
- https://cdn.elifesciences.org/articles/83538/elife-83538-fig1-figsupp2-data1-v1.zip
Figure 1—figure supplement 3
Chromatin assembly factor 1 (CAF-1) DNA-binding analysis and in vivo assays.
(a, b) Representative images of electrophoretic mobility shift assays (EMSAs) with 2 nM of the indicated Cy5-labeled DNA and yWHD over a range of protein concentrations of 12–760 nM. (c) …
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Figure 1—figure supplement 3—source data 1
Chromatin assembly factor 1 (CAF-1) DNA-binding analysis and in vivo assays.
Electrophoretic mobility shift assay (EMSA) images (panels a and b), data analyses (panel c), flow cytometry data (panel f), and Phosphorimager image (panel g).
- https://cdn.elifesciences.org/articles/83538/elife-83538-fig1-figsupp3-data1-v1.zip
Figure 2 with 2 supplements
The yKER is a single alpha-helix (SAH) domain that forms a stable complex with DNA.
(a) Ribbon representation of the X-ray crystal structure of yCAF-1 KER region. Cac1 residues 136–222 are shown with side chains of residues Lys, Arg, and His colored in blue and Glu in red. (b) …
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Figure 2—source data 1
The yKER is a single alpha-helix (SAH) domain that forms a stable complex with DNA.
Circular dichroism data (panels c and d).
- https://cdn.elifesciences.org/articles/83538/elife-83538-fig2-data1-v1.zip
Figure 2—figure supplement 1
Analysis of the maltose-binding protein (MBP)-yKER crystal structure.
(a) Diagram showing electron density for a region of the yKER. The 2Fo-Fc composite omit map was contoured at 1 sigma. (b) Crystal packing diagram showing the arrangement of the four molecules in …
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Figure 2—figure supplement 1—source data 1
Maltose-binding protein (MBP)-yKER oligomeric properties.
Electrophoretic mobility shift assay (EMSA) images and data analyses (panels d and e).
- https://cdn.elifesciences.org/articles/83538/elife-83538-fig2-figsupp1-data1-v1.zip
Figure 2—figure supplement 2
DNA-binding properties of the yKER.
(a) Graph of circular dichroism spectra of 40 bp DNA alone and in the presence of yKER. The yKER spectrum was subtracted from the 40 bp + yKER sample to observe only changes in the DNA component. (b)…
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Figure 2—figure supplement 2—source data 1
DNA-binding properties of the yKER.
Circular dichroism data (panel a) and electrophoretic mobility shift assay (EMSA) images (panel b).
- https://cdn.elifesciences.org/articles/83538/elife-83538-fig2-figsupp2-data1-v1.zip
Figure 3 with 1 supplement
The yKER middle region is required for DNA binding and yCAF-1 function in vivo.
(a) Surface representation of two views of the yCAF-1 KER structure with basic residues colored in blue, acidic in red, and polar or hydrophobic in gray. The dashed lines at the top illustrate the …
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Figure 3—source data 1
The yKER middle region is required for DNA binding and yCAF-1 function in vivo.
Circular dichroism data (panel b), electrophoretic mobility shift assay (EMSA) images (panels c–h), flow cytometry data (panel j), and data analyses (panels c–f, j).
- https://cdn.elifesciences.org/articles/83538/elife-83538-fig3-data1-v1.zip
Figure 3—figure supplement 1
The yKER middle region is required for DNA binding.
Representative image of an electrophoretic mobility shift assay (EMSA) showing the binding of a fixed concentration (760 nM) of yCAF-1, yCAF-1 ∆KER, yCAF-1 ∆middle-A, and yCAF-1 KER::Myo7aSAH …
Figure 4
The yKER confers DNA-length selectivity to yCAF-1.
(a–e) Representative images of electrophoretic mobility shift assays (EMSAs) showing DNA binding of yCAF-1, yKER, yWHD, yCAF-1 ∆WHD, or yCAF-1 ED::GSL where each Cy5-labeled DNA fragment is at 1 nM …
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Figure 4—source data 1
The yKER confers DNA-length selectivity to yCAF-1.
Electrophoretic mobility shift assay (EMSA) images and data analyses.
- https://cdn.elifesciences.org/articles/83538/elife-83538-fig4-data1-v1.zip
Figure 5 with 1 supplement
The length and the phase of the yKER single alpha-helix (SAH) modulate yCAF-1 functions in vivo.
(a) Cartoon representing the yCAF-1 2xKER construct along with a representative image of an electrophoretic mobility shift assay (EMSA) showing binding to a set of Cy5-labeled DNA fragments (1 nM …
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Figure 5—source data 1
The length and the phase of the yKER single alpha-helix (SAH) modulate yCAF-1 functions in vivo.
Electrophoretic mobility shift assay (EMSA) images (panel a) and flow cytometry data (panel c).
- https://cdn.elifesciences.org/articles/83538/elife-83538-fig5-data1-v1.zip
Figure 5—figure supplement 1
Analysis of the yKER length.
(a) Representative image of and electrophoretic mobility shift assay (EMSA) experiment of yCAF-1 +N-half, where each Cy5-labeled DNA fragment was at 1 nM concentration and the range of protein …
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Figure 5—figure supplement 1—source data 1
Analysis of the yKER length.
Electrophoretic mobility shift assay (EMSA) images and data analyses (panel a).
- https://cdn.elifesciences.org/articles/83538/elife-83538-fig5-figsupp1-data1-v1.zip
Figure 6 with 1 supplement
Chromatin assembly factor 1 (CAF-1) DNA-length selectivity by the KER is species specific and its function is not conserved in vivo.
(a) Sequence of the KER region from human (CHAF1A, top) and yeast (Cac1, bottom) homologs with positively charged residues Arg and Lys colored in blue, and negatively charged residue Glu and Asp …
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Figure 6—source data 1
DNA-length selectivity by the KER is species specific and its function is not conserved in vivo.
Electrophoretic mobility shift assay (EMSA) images and data analyses (panels b and c) and flow cytometry data and analyses (panel e).
- https://cdn.elifesciences.org/articles/83538/elife-83538-fig6-data1-v1.zip
Figure 6—figure supplement 1
Analysis of the substitution of the yKER with the hKER.
(a) Helical net diagram of the predicted single alpha-helix (SAH) of the hKER (CHAF1A residues 331–441). In this representation, the SAH structure has been split along a helical track and unwound so …
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Figure 6—figure supplement 1—source data 1
Analysis of the substitution of the yKER with the hKER.
Circular dichroism data (panels b and c).
- https://cdn.elifesciences.org/articles/83538/elife-83538-fig6-figsupp1-data1-v1.zip
Figure 7 with 1 supplement
Proposed molecular mechanism model of KER-mediated nascent tetrasome assembly by CAF-1.
(a) The KER safeguards DNA for tetrasome assembly. Because the KER has strong binding affinity toward DNA and is readily competent for binding, recruitment of CAF-1 to DNA through the KER can be an …
Figure 7—figure supplement 1
Cartoon models of KER–DNA association.
(a) The KER is aligned along the length of the DNA. (b) The KER binds in the major groove of DNA in the ‘middle-A’ region similar to basic-leucine zipper proteins. The KER is shown in green and the …
Tables
Table 1
Relative sensitivity to Camptothecin (CPT) and Zeocin of yeast cells harboring chromatin assembly factor 1 (CAF-1) mutations in a rtt106∆ background.
| CPT sensitivity | Zeocin sensitivity | |
|---|---|---|
| Cac1 WT | + | + |
| cac1∆ | +++++ | +++++ |
| ∆KER | ++ | ++ |
| mWHD | +++ | +++ |
| mPIP | +++ | ++ |
| mWHD+mPIP | +++++ | +++++ |
| ∆KER+mWHD | +++++ | +++++ |
| ∆KER+mPIP | +++ | ++ |
| ∆middle-A | ++ | ++ |
| ∆middle-A+mWHD | +++++ | +++++ |
| 2xKER | ++ | +++ |
| 2xKER+mWHD | ++ | +++ |
| ∆145–149 | + | + |
| ∆145–149+mWHD | ++++ | +++++ |
| ∆225–226 | ++ | ++ |
| ∆225–226+mWHD | +++++ | +++++ |
| ∆225–226+mPIP | +++ | +++ |
| KER::hKER | + | ++ |
| KER::hKER+mWHD | ++++ | +++++ |
Additional files
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Supplementary file 1
List of plasmids.
- https://cdn.elifesciences.org/articles/83538/elife-83538-supp1-v1.docx
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Supplementary file 2
List of synthetic DNA oligonucleotides and primers.
- https://cdn.elifesciences.org/articles/83538/elife-83538-supp2-v1.docx
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Supplementary file 3
List of yeast strains.
- https://cdn.elifesciences.org/articles/83538/elife-83538-supp3-v1.docx
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Supplementary file 4
Statistics for crystallographic data collection and refinement.
- https://cdn.elifesciences.org/articles/83538/elife-83538-supp4-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/83538/elife-83538-mdarchecklist1-v1.pdf
