Journal: Cell Reports

Article Title: Dynamics of the Eukaryotic Replicative Helicase at Lagging-Strand Protein Barriers Support the Steric Exclusion Model

doi: 10.1016/j.celrep.2019.01.086

Figure Lengend Snippet: Single-Molecule Detection of CMG Pausing at a Lagging-Strand Methyltransferase Block (A) Schematic representation of experimental approach used in single-molecule DNA unwinding assays. (B) Images of a sample field of view showing accumulation of EGFP-RPA fluorescence signal at different time points from the addition of EGFP-RPA into the chamber. (C) Example unwinding traces of DNA substrates without a protein barrier. Traces exhibit a signal drop upon completion of unwinding due to dissociation of the leading-strand template (depicted in A). (D) Distribution of average fork rates measured in fully unwound substrates without MH (black) and after bypassing MH Lag (blue). Number of molecules are n(-MH) = 199, n(MH Lag after pause) = 20. (E) Sample unwinding traces of DNA substrates modified with MH Lag . Pausing observed at 800 bp is highlighted with gray rectangle. (F) Distribution of pause durations observed in molecules exhibiting a pausing event (n = 109). The solid line is a fit to a single exponential. See also Figure S6 .

Article Snippet: 5FdC-modified fork templates were mixed with HpaII methyltransferase (M.HpaII) in 1:4 DNA to protein molar ratio in methyltransferase buffer (50 mM Tris-HCl, pH 7.5, 0.5 mM 2-mercaptoethanol (β-ME), 10 mM EDTA, NEB) supplemented with 100 μM S-adenosylmethionine (NEB), and incubated at 37°C for 3 hours.

Techniques: Blocking Assay, Fluorescence, Modification

Journal: Cell Reports

Article Title: Dynamics of the Eukaryotic Replicative Helicase at Lagging-Strand Protein Barriers Support the Steric Exclusion Model

doi: 10.1016/j.celrep.2019.01.086

Figure Lengend Snippet:

Article Snippet: 5FdC-modified fork templates were mixed with HpaII methyltransferase (M.HpaII) in 1:4 DNA to protein molar ratio in methyltransferase buffer (50 mM Tris-HCl, pH 7.5, 0.5 mM 2-mercaptoethanol (β-ME), 10 mM EDTA, NEB) supplemented with 100 μM S-adenosylmethionine (NEB), and incubated at 37°C for 3 hours.

Techniques: Recombinant, Gel Extraction, Purification, Plasmid Preparation, Software

Journal: Molecular Cell

Article Title: Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2

doi: 10.1016/j.molcel.2018.05.018

Figure Lengend Snippet: SETD1A-Mediated H3K4 Methylation Is Required for Fork Protection (A) HeLa cells were transfected with the indicated siRNAs for 72 hr, and WCEs were analyzed by immunoblotting. (B and C) Quantification of PLA signals between EdU and H3K4me1 (B) or H3K4me3 (C) in U-2-OS cells transfected with the indicated siRNAs. Cells were exposed to 4 mM HU for 5 hr where denoted. (D) Stable HeLa cells expressing WT H3.1-GFP or a K4A mutant (clone D1) were treated as described in Figure 3 , and average IdU:CldU ratios were calculated (denoted by an arrow). (E) Cells from (D) were transfected with the indicated siRNAs and treated as above. (F) Cells from (D) were exposed to 50 ng/mL MMC for 24 hr or 4 mM HU for 5 hr and immunostained with antibodies to RAD51, and focus formation was analyzed by fluorescence microscopy. Scale bar, 20 μm. (G) Quantification of PLA signals between EdU and RAD51 in cells from (D). Where denoted, cells were exposed to 4 mM HU for 5 hr. (H) Cells from (D) were exposed to the indicated doses of MMC and left for 14 days, and colonies were stained with methylene blue and enumerated. (I) Cells from (D) were exposed to 50 ng/mL MMC for 20 hr and treated with colcemid, and the incidence of chromosomal damage was analyzed by Giemsa staining and light microscopy. The plots in all cases represent mean data ± SEM from 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also Figure S4 and .

Article Snippet: 24 hr post DNA transfection, pre-existing SNAP-H3.1 was labeled with SNAP-cell 505 star (New England Biolabs) according to the manufacturer’s instructions (‘pulse’).

Techniques: Methylation, Transfection, Western Blot, Expressing, Mutagenesis, Fluorescence, Microscopy, Staining, Light Microscopy

Journal: Molecular Cell

Article Title: Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2

doi: 10.1016/j.molcel.2018.05.018

Figure Lengend Snippet: SETD1A and FANCD2 Promote H3 Mobility to Protect Fork Integrity (A) U-2-OS cells were transfected with the indicated siRNAs and treated as in Figure 3 . Arrows indicate mean ratios. (B) Stable HeLa cells expressing WT H3.1-GFP or a K4A mutant (clone D1) were transfected with the indicated siRNAs and treated as above. (C) U-2-OS cells from (A) were transfected with SNAP-tagged H3.1 and analyzed to reveal levels of pre-existing SNAP-H3.1 (Pulse), background fluorescence (Quench-Pulse), and new H3.1 after a 2-hr release into HU (Quench-HU-Pulse). Cells were analyzed by fluorescence microscopy, and fluorescence intensity per nucleus was quantified using ImageJ. (D) Stable HeLa cells expressing WT H3.1-GFP or a K4A mutant (clone D1) were left untreated or exposed to MMC for 24 hr and analyzed by FRAP. (E) WT DT40 cells or FANCD2 −/− cells expressing the indicated GFP-tagged chFANCD2 variants were treated as described in Figure 3 . Arrows indicate mean ratios. (F) Stable U-2-OS-GFP-RAD51 cells were co-transfected with the indicated siRNA and plasmids encoding either WT or chaperone-dead (R302W) mCherry-FANCD2 and exposed to MMC, and the mobility of GFP-RAD51 was analyzed by FRAP. (G) Model for the role of SET1A-dependent H3K4 methylation in protecting stalled replication forks. Upon fork reversal during replication stress, H3K4me1 by SETD1A acts to restrict CHD4 localization and promote FANCD2-dependent histone mobility. This chaperone activity is vital to stabilize RAD51 nucleofilaments, preventing nucleolytic degradation of stalled forks by DNA2. The plots in (D) and (F) represent mean ± SD relative fluorescence intensities from n = 21–26 and 25–48 cells, respectively. ∗ p < 0.05. See also and and .

Article Snippet: 24 hr post DNA transfection, pre-existing SNAP-H3.1 was labeled with SNAP-cell 505 star (New England Biolabs) according to the manufacturer’s instructions (‘pulse’).

Techniques: Transfection, Expressing, Mutagenesis, Fluorescence, Microscopy, Methylation, Activity Assay

Journal: Molecular Cell

Article Title: Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2

doi: 10.1016/j.molcel.2018.05.018

Figure Lengend Snippet:

Article Snippet: 24 hr post DNA transfection, pre-existing SNAP-H3.1 was labeled with SNAP-cell 505 star (New England Biolabs) according to the manufacturer’s instructions (‘pulse’).

Techniques: Recombinant, In Situ, Mass Spectrometry, Luciferase, Negative Control, Software