Journal: Nature Communications

Article Title: RTF2 controls replication repriming and ribonucleotide excision at the replisome

doi: 10.1038/s41467-024-45947-z

Figure Lengend Snippet: a Schematic of the iPOND set-up in SV40-immortlized Rtf2 -/- MEFs expressing HA-FLAG empty vector (EV) or RTF2 cDNA. EdU pulses were normalized to replication tract lengths. Nascent DNA was purified using streptavidin beads against biotin-conjugated EdU. b Peptide Spectral Match (#PSM) values for indicated proteins from experiment in ( a ). c Representative iPOND immunoblot from p53 -/- MEFs at 72 hr after Cre. d Left: Representative images of RNASEH2A-EdU nascent proximity ligation assay (nPLA) co-stained with PCNA in SV40-immortalized Rtf2 -/- MEFs expressing HA-FLAG EV or RTF2 cDNA. Right: Quantification of RNASEH2A-EdU foci in PCNA-positive cells. e Representative iPOND immunoblot in DDI-depleted U2OS cells. Proteins purified using streptavidin beads recognizing nascent DNA were detected by immunoblot. f Representative iPOND immunoblot in CRISPR-edited RNASEH2A KO or WT HeLa cells. Proteins purified using streptavidin beads recognizing nascent DNA were detected by immunoblot. Experiments were conducted at least three times in biological replicates with consistent results for ( c )–( f ). Mean is shown with red line for ( d ). Significance was evaluated by Kruskal-Wallis ANOVA with a Dunn’s post-test. P = Parental, RH2A = RNASEH2A, RH2B = RNASEH2B, RH2C = RNASEH2C, RH2 = RNASEH2 complex, Ctrl = Control. Source data are provided as a Source Data file.

Article Snippet: Antibodies used: Mouse monoclonal anti-α-tubulin (clone DM1A), WB:1:5000 (MilliporeSigma, Cat# T9026, RRID:AB_477593); Mouse monoclonal anti-Poly (ADP-Ribose) Polymer antibody [10H], WB:1:100 (Abcam, Cat # ab14459, RRID:AB_301239); Mouse monoclonal anti-PCNA (PC10), WB: 1:1000 (Santa Cruz, Cat# sc-56, RRID:AB_628110); Mouse monoclonal anti-RNASEH2A, WB:1:500 (Santa Cruz, Cat# sc-515475); Mouse monoclonal anti-RTF2 (clone OTI1E8), WB: 1:1000 (LS Bio, Cat# LS-C340588); Mouse Monoclonal anti-vinculin, Unconjugated, Clone hVIN-1, WB: 1:5000 (MilliporeSigma, Cat# V9131, RRID:AB_477629); Rabbit monoclonal anti-MCM7 (D10A11) XP, WB: 1:1000 (Cell Signaling, Cat# 3735 S, RRID:AB_2142705); Rabbit polyclonal anti-c20orf43 (RTF2), WB: 1:500 (Novus, Cat# NBP2-30645); Rabbit polyclonal anti-GFP, 1:1000 (Abcam, Cat# ab290 RRIDL AB_303395); Rabbit polyclonal anti-RNASEH2A, 1:1000 (Abcam, Cat# ab83943, RRID:AB_1861175); Rabbit polyclonal anti-RNASEH2C (AbClonal, Cat# A13884, RRID:AB_2760737); Rabbit polyclonal anti-phospho-RPA32 (S4/8), WB:1:1000 (Bethyl, Cat# A300-245A, RRID:AB_210547); Rabbit polyclonal anti-RPA32, WB 1:2000 (Bethyl, Cat# A300-244A, RRID:AB_185548); Rabbit polyclonal anti-PRIM1 (Proteintech, Cat # 10773-1-AP, RRID:AB_2237549).

Techniques: Expressing, Plasmid Preparation, Purification, Western Blot, Proximity Ligation Assay, Staining, CRISPR, Control

Journal: Nature Communications

Article Title: RTF2 controls replication repriming and ribonucleotide excision at the replisome

doi: 10.1038/s41467-024-45947-z

Figure Lengend Snippet: a Representative images from GFP-EdU nPLA co-stained with PCNA in p53 -/- MEFs expressing GFP-tagged wildtype (WT) or PIP box mutant (PIPm, RNASEH2B F300A;F301A ) RNASEH2B at 72 hr after Cre. b Quantification of GFP (RNASEH2B)-EdU foci in PCNA-positive cells. Note that endogenous RNase H2 is present in these cells. c Representative immunoblots following GFP immunoprecipitation of exogenously-expressed GFP-RNASEH2B-WT or GFP-RNASEH2B-PIP mutant from HEK293T cells. d Representative immunoblots following immunoprecipitation of RTF2 from HEK293T cells. e Representative immunoblot from immunoprecipitation of recombinant RNase H2 complex and RTF2 expressed in E. coli . Protein amount (pmol) indicated above each lane; range of RTF2 is 0.5, 1, 2, 4 pmol. Images for input and pulldown of RTF2 are separate exposures of the same blot. f Representative immunoblot from immunoprecipitation of recombinant GFP-tagged RTF2 and RNase H2 complex expressed in E. coli . Protein amount (pmol) indicated above each lane; range of RTF2 is 0.5, 1, 2, 4 pmol. Experiments were conducted at least three times in biological replicates with consistent results for ( a )–( f ). Mean is shown with red line for ( b ). Significance was evaluated by Kruskal-Wallis ANOVA with a Dunn’s post-test. WT = wildtype, PIPm = PIP box mutant, RH2A = RNASEH2A, RH2B = RNASEH2B, RH2C = RNASEH2C. Source data are provided as a Source Data file.

Article Snippet: Antibodies used: Mouse monoclonal anti-α-tubulin (clone DM1A), WB:1:5000 (MilliporeSigma, Cat# T9026, RRID:AB_477593); Mouse monoclonal anti-Poly (ADP-Ribose) Polymer antibody [10H], WB:1:100 (Abcam, Cat # ab14459, RRID:AB_301239); Mouse monoclonal anti-PCNA (PC10), WB: 1:1000 (Santa Cruz, Cat# sc-56, RRID:AB_628110); Mouse monoclonal anti-RNASEH2A, WB:1:500 (Santa Cruz, Cat# sc-515475); Mouse monoclonal anti-RTF2 (clone OTI1E8), WB: 1:1000 (LS Bio, Cat# LS-C340588); Mouse Monoclonal anti-vinculin, Unconjugated, Clone hVIN-1, WB: 1:5000 (MilliporeSigma, Cat# V9131, RRID:AB_477629); Rabbit monoclonal anti-MCM7 (D10A11) XP, WB: 1:1000 (Cell Signaling, Cat# 3735 S, RRID:AB_2142705); Rabbit polyclonal anti-c20orf43 (RTF2), WB: 1:500 (Novus, Cat# NBP2-30645); Rabbit polyclonal anti-GFP, 1:1000 (Abcam, Cat# ab290 RRIDL AB_303395); Rabbit polyclonal anti-RNASEH2A, 1:1000 (Abcam, Cat# ab83943, RRID:AB_1861175); Rabbit polyclonal anti-RNASEH2C (AbClonal, Cat# A13884, RRID:AB_2760737); Rabbit polyclonal anti-phospho-RPA32 (S4/8), WB:1:1000 (Bethyl, Cat# A300-245A, RRID:AB_210547); Rabbit polyclonal anti-RPA32, WB 1:2000 (Bethyl, Cat# A300-244A, RRID:AB_185548); Rabbit polyclonal anti-PRIM1 (Proteintech, Cat # 10773-1-AP, RRID:AB_2237549).

Techniques: Staining, Expressing, Mutagenesis, Western Blot, Immunoprecipitation, Recombinant