Journal: Nucleic Acids Research

Article Title: DNA structure and the Werner protein modulate human DNA polymerase delta-dependent replication dynamics within the common fragile site FRA16D

doi: 10.1093/nar/gkp1131

Figure Lengend Snippet: Replication by the pol δ holoenzyme. Effects of the clamp loader RFC on pol δ/PCNA processivity were observed. Reactions proceeded for 30 mins and contained 100 fmol template DNA, 200 fmol pol δ, 250 nM PCNA, 0.003 mg/ml RFC in 20 µl volume as indicated.

Article Snippet: Replication competent HeLa cytoplasmic extracts were purchased from Chimerx (SV40 DNA Replication Assay Kit).

Techniques:

Journal: Nucleic Acids Research

Article Title: DNA structure and the Werner protein modulate human DNA polymerase delta-dependent replication dynamics within the common fragile site FRA16D

doi: 10.1093/nar/gkp1131

Figure Lengend Snippet: WRN stimulates pol δ independently of its helicase or exonuclease activities. ( A ) Reactions contained pol δ/PCNA alone or with, wild-type WRN, or exonuclease-dead WRN (X-WRN) as indicated and ATPγS was included, as shown, to inhibit helicase function. Reactions proceeded for 2, 10 and 30 mins and contained 100 fmol template DNA, 2 pmol pol δ and 100 fmol WRN or X-WRN, as indicated. ( B ) Replication extension by WRN fragments. 1, GST control, 2. WRN 500–946 , or 3. WRN 949–1432 were added (100 fmol) to pol δ/PCNA reactions. Gels in ‘B’ are cropped prior to the start of the fragile site sequence. Dashed lines: boundary of CFS insert.

Article Snippet: Replication competent HeLa cytoplasmic extracts were purchased from Chimerx (SV40 DNA Replication Assay Kit).

Techniques: Control, Sequencing

Journal: Nucleic Acids Research

Article Title: DNA structure and the Werner protein modulate human DNA polymerase delta-dependent replication dynamics within the common fragile site FRA16D

doi: 10.1093/nar/gkp1131

Figure Lengend Snippet: CFS replication in HeLa cell extracts. ( A ) Replication-competent cell-free extracts were used for synthesis of regions 1–5 in both ‘A’ and ‘B’ orientations. Extract (140 µg protein) was supplemented with 4 mM ATP, 40 mM phosphocreatine and 0.625 units creatine phosphokinase. DNA synthesis reactions were stopped 1 min after the addition of 100 fmol primer-template DNA and 100 µM dNTPs. The fragile regions are enclosed in black bars. Arrows indicate sites of replication stalling at specific motifs: (a), near group of small inverted repeats; (b), base of [AT/TA] 24i stem; (c), within poly(dA); (d), end of poly(dT); (e), base of long inverted repeat (see Supplmentary Figure S1 for sequences). Histogram shows percent of product that has completely replicated through the indicated FRA16D region, quantitated as described above. ( B ) Western analyses of HeLa replication-competent (RC) extract (20 µg), compared to U2OS cytoplamic RC extract and nuclear fractions (for full western blots, Supplementary Figure S7 ). Blots were probed with the indicated antibodies.

Article Snippet: Replication competent HeLa cytoplasmic extracts were purchased from Chimerx (SV40 DNA Replication Assay Kit).

Techniques: DNA Synthesis, Western Blot

Journal: Nature methods

Article Title: PROBER Identifies Proteins Associated with Programmable Sequence-Specific DNA in Living Cells

doi: 10.1038/s41592-022-01552-w

Figure Lengend Snippet: a, PROBER-WB of canonical YY1, NF-κB, STAT1 DNA motifs and their nucleotide composition-matched scrambled controls + /− TNF-α or IFN-γ probed together with YY1, RelA, or STAT1 antibodies. b, Heatmap of a representing fold enrichment of signals, n = 1 biological replicate. c, PROBER-WB showing effect of single-copy, duplicate, triplicate, quadruplicate and quintuplicate YY1 motifs on fold enrichment, d, quantification of c representing mean enrichment, n = 2 biologically independent replicates. e, Trimeric NF-κB motif and matched scrambled sequences were cloned in pBait with different lengths of flanks at both ends to obtain inserts of varying sizes. The NF-κB motifs are shown as “N”, position of UAS elements are also shown. f, PROBER-WB showing the effect of insert size on RelA enrichment, and g, quantification of f representing mean enrichment, n = 2 biologically independent replicates. Note that increasing distance between NF-κB motifs and UAS beyond 25-nt stuffer flanks results in loss of enrichment because it puts the TFs outside BASU labeling radius. h, Nuclear plasmid copies after transfection of HEK293T cells with varying amounts of bait plasmid and 3 μg pSprayer; an SV40 Ori-less derivative of pBait with trimeric YY1 motif was used eliminate copy number increase by endogenous large T antigen, and i, resulting YY1 PROBER fold enrichment in response to bait copy number in h. n = 1 biological replicate.

Article Snippet: The pDriver was created by subcloning the SV40 Large T-antigen from pD2160-v1–03 (ATUM) under a CMV promoter.

Techniques: Clone Assay, Labeling, Plasmid Preparation, Transfection