(A) Sperm chromatin was added to interphase egg extracts supplemented with either buffer (+Buffer), 20 µM UbVS (+UbVS), or 20 µM UbVS and 58 µM ubiquitin (+UbVS + Ub). Samples were analyzed by …
Figure 1 Original blots.
Figure 1—figure supplement 1 Original blots.
(A) Schematic illustration of PAF15-USP7 binding experiment. USP7 recognizes P/A/ExxS or KxxxK motifs in its substrates via TRAF or ubiquitin-like (UBL) domains, respectively. PAF15 has three …
Figure 2 Original blots.
(A) Superimposition of plots of enthalpy changes in the interaction between hUSP7561-1102 and full-length hPAF15 wild-type (WT) or K93A/K96A mutant by isothermal titration calorimetry (ITC) …
(A) Sperm chromatin was added to Mock- or PAF15-depleted interphase extracts supplemented with either buffer (+Buffer), wild-type rPAF15-3xFLAG (+WT) or rPAF15 SAKA-3xFLAG (+SAKA). Chromatin …
Figure 3 Original blots.
(A) rPAF15 WT-3xFLAG or SAKA-3xFLAG were added to PAF15-depleted extracts. The extracts used in Figure 3A were analyzed by immunoblotting using the antibodies indicated. (B) rUSP7 WT-3xFLAG or …
Figure 3—figure supplement 1 Original blots.
(A) Sperm chromatin was added to interphase extracts. Replicating chromatin was digested by micrococcal nuclease (MNase). Immunoprecipitation was performed by PAF15, USP7, and DNMT1 antibodies from …
Figure 4 Original blots.
(A) rDNMT1WT-3xFLAG or 4KA-3xFLAG were added to DNMT1-depleted extracts. The extracts used in Figure 4C were analyzed by immunoblotting using the antibodies indicated. (B) Mock-, PAF15-, DNMT1-, or …
Figure 4—figure supplement 1 Original blots.
(A) Sperm chromatin was added to Mock- or DNMT1-depleted interphase extracts supplemented with either buffer (+Buffer), wild-type rDNMT1-3xFLAG (+WT), or catalytic mutant rDNMT1 C1101S-3xFLAG …
Figure 5 Orignal blots.
(A) rDNMT1 WT-3xFLAG or C1101S-3xFLAG were added to DNMT1-depleted extracts. The extracts used in Figure 5A were analyzed by immunoblotting using the antibodies indicated. (B) rDNMT1 WT-3xFLAG or …
Figure 5—figure supplement 1 Original blots.
(A) Sperm chromatin was added to Mock-, USP7-, ATAD5-, and USP7/ATAD5-depleted interphase extracts and isolated at indicated time points. Chromatin bound proteins were analyzed by immunoblotting. (B)…
Figure 6 Original blots.
(A) Mock-, USP7-, ATAD5-, or USP7/ATAD5 co-depleted extracts were used in Figure 6A and analyzed by immunoblotting using the antibodies indicated. (B) rhATAD5-RFCs was added to ATAD5-depleted …
Figure 6—figure supplement 1 Original blots.
Sperm chromatin and radiolabeled S-(methyl-3H)-adenosyl-L-methionine were added to either Mock- and USP7-, ATAD5-, or USP7/ATAD5 co-depleted extracts. Purified DNA samples were analyzed to determine …
Figure 7 DNA methylation.
(A) Mock-, USP7-, ATAD5-, or USP7/ATAD5 co-depleted extracts used in Figure 7 were analyzed by immunoblotting using the antibodies indicated. (B) Sperm chromatin and radiolabeled (α-32P) dCTP were …
Figure 7—figure supplement 1 Original blots, DNA replication, and methylation data.
(A) Immunoprecipitation (IP) of endogenous mPAF15 from whole cell lysates of wt and mPAF15 SAKA mouse embryonic stem cells (mESCs) using an anti-PAF15 antibody. Bound fractions were subjected to …
Figure 8 Original blots and DNA methylation data.
(A) Schematic representation of the CRISPR/Cas9 gene editing strategy. The restriction enzyme recognition site for restriction fragment length polymorphism (RFLP) screening is shown. (B) Genotyping …
Figure 8—figure supplement 1 Original gel and sequencing data.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (insect cells) | Sf9 | This paper | Cell line maintained in M. Nakanishi Lab | |
Cell line (mouse cells) | mESC J1 line | This paper | Cell line maintained in H. LeonhardtLab | |
Antibody | Anti-Xenopus PAF15 (Rabbit polyclonal) | PMID:32145273 | PMID:32145273 | WB (1:500) Nakanishi Lab |
Antibody | Anti-Xenopus DNMT1 (Rabbit polyclonal) | PMID:24013172 | PMID:24013172 | WB (1:500) Nakanishi Lab |
Antibody | Anti-USP7 (Rabbit polyclonal) | Thermo Fisher Scientific | Cat# A300-033A, RRID:AB_203276 | WB (1:1000) |
Antibody | Anti-Xenopus UHRF1 (Rabbit polyclonal) | PMID:24013172 | WB (1:500) Nakanishi Lab | |
Antibody | Anti-histone H3 (Rabbit polyclonal) | abcam | Cat# ab1791, RRID:AB_302613 | WB (1:3000) |
Antibody | Anti-PCNA (mouse monoclonall) | Santa Cruz Biotechnology | Cat# sc-56, RRID:AB_628110 | WB (1:1000) |
Antibody | Anti-Xenopus ATAD5 (rabbit polyclonal) | This study | WB (1:1000) | |
Antibody | Anti-tubulin (mouse monoclonal) | Sigma-Aldrich | Cat# T9026, RRID:AB_477593 | WB (1:2000) |
Antibody | Anti-PAF15 (mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-390515 | WB (1:500) |
Antibody | HRP-anti-mouse IgG (rabbit polyclonal) | Sigma-Aldrich | Cat# A9044, RRID:AB_258431 | WB (1:5000) |
Recombinant DNA reagent | pGEX4T-3-xPAF15 | PMID:32145273 | PMID:32145273 | Nakanishi Lab |
Recombinant DNA reagent | pVL1392-xUSP7-3xFLAG | This study | Expression and purification of xUSP7 in insect cells | |
Recombinant DNA reagent | pVL1392-xDNMT1-3xFLAG | PMID:29053958 | Nakanishi Lab | |
Recombinant DNA reagent | pGEX-4T-3-mDPPA3 | PMID:33235224 | Nakanishi Lab | |
Recombinant DNA reagent | pGEX-6P-1-hUSP7 (561–1102) | This study | Expression and purification of hUSP7 fragment in bacteria cells | |
Recombinant DNA reagent | pGEX-4T-1-SUMO-hPAF15-FLAG | This study | Expression and purification of full-length hPAF15 in bacteria cells | |
Recombinant DNA reagent | pCSII-EF-mini-AzamiGreen ATAD5 | This study | Expression and purification of full-length hATAD5 in human 293T cells | |
Recombinant DNA reagent | pCSII-EF-RFC2, 3, 4 and 5 | This study | Expression and purification of full-length hRFC complex in human 293T cells | |
Peptide, recombinant protein | Ubiquitin | R&D systems (Boston biochem) | U-100H | 58 μM |
Peptide, recombinant protein | Recombinant Human Ubiquitin Vinyl Sulfone Protein | R&D systems (Boston biochem) | U-202 | 20 μM |
Commercial assay or kit | EZ DNA Methylation-Gold Kit | Zymo | D5005 | |
Chemical compound and drug | FT-671 | MedChem Express | HY-107985 | 100 μM |
MS-based quantification of GST interacting protein.
MS-based quantification of xPAF15 interacting proteins.
Oligonucleotides used in this study.