Exonuclease 3

CO-FISH assay was performed as described [59 (link)], with slight modification. mESCs or mEpiSCs were incubated with BrdU (10 μM) for 12 h. Nocodazole at 0.3 μg/ml was added for 3 h prior to cell harvest, and metaphase spreads were prepared by a routine method. Chromosome slides were treated with RNase A, fixed with 4% formaldehyde, then stained with Hoechst 33258 (0.5 mg/ml), incubated in 2 × SSC (Invitrogen) for 15 min, and exposed to 365 nm UV light (Stratalinker 1800UV irradiator) for 40 min. BrdU-substituted DNA was digested with Exonuclease III (Takara). Slides were then dehydrated through ethanol series and air-dried. PNA-FISH was performed with FITC-OO-(CCCTAA)₃ (Panagene, F1009). Slides were hybridized, washed, dehydrated, mounted, and counterstained with 1.25 μg/ml DAPI in VectaShield antifade medium. Digital images were captured using CCD camera on Zeiss Imager Z2 microscope. To analyze telomere sister chromatid exchange (T-SCE), one signal at each end of the chromosome was counted as no T-SCE, while two signals at both chromatids on one chromosome end were identified as one T-SCE. At least 15 metaphase spreads were counted for the frequency of T-SCE. Pairwise comparisons for statistical significance were made by t-tests.

To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions43 (link). After heat inactivation, the exosomal DNA was purified by Proteinase K (Wako) treatment. The amount of dsDNA was determined using an Agilent High Sensitivity DNA kit (Agilent Technologies) or a QuantiFluor dsDNA System (Promega).

To determine the genome type of halovirus VOLN27B, the genome of VOLN27B was isolated from the virus stock according to the method described by Summer [23 (link)] using Wizard DNA Clean-Up System (Promega, USA). Genomic DNA (1 μg) was digested with DNase I (5 U) (TaKaRa, Japan), Exonuclease III (20 U) (TaKaRa, Japan), and Mung Bean Nuclease (4 U) (TaKaRa, Japan) at 37°C for 1 h, and the resulting product was checked by DNA electrophoresis. An equal amount of undigested plasmid pBR322 (NEB, USA) was used as a circular dsDNA standard, and the EcoRI (TaKaRa, Japan) cleaved pBR322 was used as a linear dsDNA standard. These untreated and EcoRI leaved pBR322 plasmids were also used as controls in treatments nucleases DNase I, Exonuclease III, and Mung Bean Nuclease.

CO-FISH assay was performed based on the method described⁶⁶ , with minor modification. Subconfluent ES cells were incubated with BrdU (10 μM) for 10-12 h. Nocodazole was added to enrich metaphase 2 h prior to cell harvest, and metaphase spreads were prepared as described above for telomere QFISH. Chromosome slides were treated with RNaseA, fixed with 4% formaldehyde, then stained with Hoechst 33258 (0.5 mg/ml) for 15 min and exposed to 365 nm UV light for 40 min. The BrdU-substituted DNA was digested with Exonuclease III (Takara). The slides were then dehydrated through ethanol series and air-dried. PNA-FISH was performed with FITC-OO-(CCCTAA)₃ (Panagene, F1009). Slides were hybridized, washed, dehydrated, and counterstained with VectaShield antifade medium (Vector), containing 1.25 μg/ml DAPI. Digital images were captured using a CCD camera on a Zeiss Axio-Imager Z1 microscope.

DNA adenine methyltransferase (MTase), S-Adenosyl-L-methionine (SAM), Uracil-DNA glycosylase (UDG), Dpn I, Nb.BtsI and Exonuclease III were all bought from from Takara Biotech-nology Co., Ltd. (Dalian, China). poly (9,9-bis(6′-N,N,N-trimethylammonium)hexyl)-fluorenylenephenylenedibromide (PFP) and 5-fluorouracil were bought from Yuanye Co., Ltd. (Shanghai, China). Two oligonucleotides were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). Carboxyfluorescein-labeled DNA (F-DNA): 5′-AGGAAGACGTACGTATCTTCC TCTAATGA-FAM-3′, HP-DNA: 5′-CCCAACGGATCATTAGAGGAAGATACGTACAA TGATCCGTTGGTATAT-3′.