Allosteric communication in DNA polymerase clamp loaders relies on a critical hydrogen-bonded junction
- Department of Molecular and Cell Biology, University of California, Berkeley, United States
- California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, United States
- Howard Hughes Medical Institute, University of California, Berkeley, United States
- Biophysics Graduate Group, University of California, Berkeley, United States
- Howard Hughes Medical Institute, Rockefeller University, United States
- Department of Chemistry, University of California, Berkeley, United States
- Physical Biosciences Division, Lawrence Berkeley National Laboratory, United States
Figures
Figure 1
Clamp-loader complex of T4 bacteriophage.
(A) Crystal structure (left) and schematic diagram (right) of the clamp loader. (B) The clamp loading cycle, from left to right, showing the key stages of loading the sliding clamp around …
Figure 2
Clamp-loader activity assay through phage propagation.
(A) Schematic depicts the generation of T4del. The clamp-loader locus in wild-type T4 bacteriophage, containing genes for the sliding clamp and the ATPase and clasp subunits of the clamp loader, is …
Figure 3 with 1 supplement
Validation of the high-throughput phage-propagation assay with a library targeting the 10-residue region including the Walker B motif of the ATPase subunit.
(A) The location of the 10-residue region in the structure of the clamp loader. (B) Relative fitness values of all 32 codons from the NNS substitutions at mutationally sensitive positions (107 and …
Figure 3—figure supplement 1
Biochemical measurements for selected mutants of the ATPase subunit.
(A) ATP hydrolysis rates of clamp loader variants with point mutations in the ATPase subunit, measured by a coupled-kinase assay (Goedken et al., 2005) (see Materials and methods for details). (B) …
Figure 4 with 3 supplements
Distribution of relative fitness values from deep mutagenesis of the clamp loader system.
The histograms show the spread of relative fitness values for AAA+ module and collar domain of the ATPase subunit, the clasp subunit and the sliding clamp.
Figure 4—figure supplement 1
Mutational sensitivity of the T4 sliding clamp (gp45).
The fitness scores for all point mutations to the sliding clamp (residue 2–228) are depicted as a heatmap. Gray pixels denote mutations with insufficient counts (fewer than 20 counts) when …
Figure 4—figure supplement 2
Mutational sensitivity of the clasp subunit (gp62) of the T4 clamp loader.
The data are shown as in Figure 4—figure supplement 1.
Figure 4—figure supplement 3
Mutational sensitivity of the collar domain (residue 231–319 of gp44) of the ATPase subunit.
The data are shown as in Figure 4—figure supplement 1.
Figure 5 with 1 supplement
Mutational sensitivity of the AAA+ module of the ATPase subunit.
The fitness scores for all point mutations to the AAA+ module are shown as a heatmap, as in Figure 3C. The secondary structure of the wildtype sequence is indicated above the heatmap, with …
Figure 5—figure supplement 1
Comparison of mutational sensitivity in the AAA+ module of the ATPase subunit to evolutionary sequence conservation.
(A) Receiver-operator characteristic curve illustrating the ability of binarized-sequence-conservation scores (show here for three different threshold values) to predict the mean mutational …
Figure 6 with 2 supplements
Mutationally-sensitive residues form a contiguous network in the structure of the clamp-loader complex, connecting sites interacting with ATP, DNA and the sliding clamp.
(A) Positions with mean mutational effect <= −1.6 are rendered in surface representation and colored blue. (B) Mutationally sensitive residues, viewed from the top (collar domain not shown) without …
Figure 6—figure supplement 1
Mutational sensitivity of residue 118 in the ATPase subunit of the T4 clamp loader, tested with a small library of 20 amino acid substitutions.
Figure 6—figure supplement 2
Expression levels of mCherry tagged wildtype and Q118N variant of the clamp loader, observed by flow cytometry.
Figure 7
The central coupler in the bacteriophage T4 clamp loader.
(A) The subunit at C position of the T4 clamp loader is shown, with the surface of the central coupler displayed. (B) The structure of the complete clamp-loader complex is shown, with the surfaces …
Figure 8 with 1 supplement
Molecular dynamics simulations of the of the wildtype T4 clamp loader and Q118A mutant.
(A) The central-coupler unit of the clamp loader is shown in green. Pairs of residues in subunit C that show correlated motions in simulations of the wildtype structure (B) and Q118A structure (C) …
Figure 8—figure supplement 1
Subunit C heavy-atom root-mean-square fluctuations from AAA+ module aligned trajectories, for wildtype and Q118A trajectories.
Figure 9 with 3 supplements
Screen for second-site suppressor mutations, in the Q118N background.
(A) The distribution of fitness scores (relative to that for the Q118N mutant) from two independent trials. G143N is the best performing mutant in both trials. (B) The top four best performing …
Figure 9—figure supplement 1
Elution profile of mCherry-tagged clamp loader variants in size-exclusion chromatography.
Figure 9—figure supplement 2
ATPase activity of mCherry-tagged clamp loader variants, in the presence of clamp and DNA.
Figure 9—figure supplement 3
Ability of mCherry-tagged clamp loader variants to carry out DNA replication with M13 phage ssDNA as template.
Figure 10
The central-coupler unit in different AAA+ proteins.
The central-coupler unit of subunit C is shown in green for (A) the human RFC structure (PDB ID: 6vvo [Gaubitz et al., 2020]), (B) the ClpX structure (PDB ID: 6vfs [Ripstein et al., 2020]), (C) the …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Bacteriophage T4) | T4del | This paper | https://benchling.com/s/seq-Vg4DZh83BOrbx3RgpaaC | |
| Recombinant DNA reagent | CRISPR plasmid | This paper | https://benchling.com/s/seq-abaKV7JgTgAghRyR9ZIY | |
| Recombinant DNA reagent | Donor plasmid | This paper | https://benchling.com/s/seq-Z2Bo2vnShDbLtji83VHm | |
| Recombinant DNA reagent | Helper plasmid | This paper | https://benchling.com/s/seq-KdFkydUFMBrlRkdpORA3 | |
| Recombinant DNA reagent | Recombination plasmid | This paper | https://benchling.com/s/seq-bRQ2OUu39lSrs6I0gfmu | |
| Commercial assay or kit | MiSeq 500 cycles kit | Illumina | Cat. #: MS-102–2003 | |
| Software, algorithm | FLASH | doi:10.1093/bioinformatics/btr507 | ||
| Software, algorithm | Analysis scripts | This paper | https://github.com/kuriyan-lab/cl1 |
Additional files
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Source data 1
Relative enrichment values of single mutants of the ATPase subunit of the clamp loader.
- https://cdn.elifesciences.org/articles/66181/elife-66181-data1-v2.xlsx
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Source data 2
Relative enrichment values of single mutants of the sliding clamp.
- https://cdn.elifesciences.org/articles/66181/elife-66181-data2-v2.xlsx
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Source data 3
Relative enrichment values of single mutants of the clasp subunit of the clamp loader.
- https://cdn.elifesciences.org/articles/66181/elife-66181-data3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/66181/elife-66181-transrepform-v2.pdf
