Journal: Cell Cycle

Article Title: Genomic and chromatin features shaping meiotic double-strand break formation and repair in mice

doi: 10.1080/15384101.2017.1361065

Figure Lengend Snippet: Crossover asymmetry in hotspots with polymorphisms in putative PRDM9 binding sites. (A) Model for crossover asymmetry. A sequence polymorphism in a PRDM9 binding site may affect relative DSB frequencies on the 2 hotspot alleles, manifested as asymmetry in the locations of crossover breakpoints. If DSBs are preferentially formed on the B6 chromosome of a B6 × DBA hybrid mouse, crossover breakpoints will tend to lie to the left of the hotspot center when recombinant products are assayed after PCR amplification in the B6-to-DBA orientation, and will tend to lie to the right when amplified in the DBA-to-B6 orientation. (B and C) Examples of crossover hotspots with (B) or without (C) crossover asymmetry. (i) B6 (top) and DBA (bottom) sequences of putative 36-bp binding sites for PRDM9 B6 at hotspot centers. The nucleotides shaded in yellow in HS59.5 highlight a polymorphism between the B6 and DBA haplotypes. In HS61.1 , the PRDM9 motif shown is on the Crick strand. (ii) SPO11-oligo maps. Red lines indicate SPO11-oligo hotspots. (iii) Crossover breakpoints (densities expressed as centiMorgans (cM) per Mb) mapped by allele-specific PCR on sperm DNA in the B6-to-DBA (top) and DBA-to-B6 (bottom) orientation. , Ticks represent tested polymorphisms. (iv) Cumulative distributions of crossover breakpoints with fitted Gaussian curves. The number indicates the distance between the 2 curves at the midpoint for each cumulative plot. Vertical dashed lines indicate hotspot centers. For hotspot HS61.1 , zero values in both orientations at outlier position -1130 bp are not shown. (D) Crossover asymmetry is associated with presence of polymorphisms in putative PRDM9 binding sites at hotspots (Table S3). Crossover asymmetry was defined for each locus as the absolute difference between the midpoints of cumulative crossover breakpoint maps in the 2 orientations.

Article Snippet: For crossover breakpoint analyses, cumulative breakpoint distributions with fitted Gaussian curves were determined using GraphPad Prism version 7.

Techniques: Binding Assay, Sequencing, Recombinant, Amplification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Molecular strategy for blocking isopeptide bond formation in nascent pilin proteins

doi: 10.1073/pnas.1807689115

Figure Lengend Snippet: Using AFM to detect the presence, absence, or the blocking of isopeptide bonds in the Spy0128 construct. (A) The native intramolecular isopeptide bond in Spy0128 prevents the protein from unfolding and extending under force. Unfolding sawtooth pattern obtained by stretching the unmodified Spy0128 construct. These traces only show the three I91 fingerprints with short initial extensions (Li, defined as the extension to the first I91 unfolding) and contour length increments of 29 nm, measured using fits of the worm-like chain model of polymer elasticity (solid lines). Data are from ref. 24. (B) Mutant Spy0128 (E258A) where the isopeptide bond is not formed. In this case, the sawtooth patterns show two additional high-force unfolding events that always follow the I91 fingerprints and have large contour length increments of ∼50 nm. Data from ref. 24. (C) Spy0128 constructs modified by the isopeptide blocker show a very different type of sawtooth pattern, with long initial extensions marked by a series of random low-force peaks observed always before the I91 fingerprint. (D) Histogram of initial extensions (Li) measured from sawtooth patterns obtained from Spy0128 constructs that were coexpressed with the isopeptide blocker (n = 698). A Gaussian fit (solid line) marks three distinct peaks at 41, 80, and 130 nm. The peak at 41 nm corresponds to unmodified Spy0128 constructs. The peaks at 80 and 130 nm closely correspond to the extensions predicted if one or both isopeptide bonds were blocked and the modified protein was unable to fold.

Article Snippet: Histograms were fitted using single and multiple Gaussian distributions implemented in Igor 7 (WaveMetrics).

Techniques: Blocking Assay, Construct, Polymer, Mutagenesis, Modification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Molecular strategy for blocking isopeptide bond formation in nascent pilin proteins

doi: 10.1073/pnas.1807689115

Figure Lengend Snippet: Mechanical properties of HaloTag-purified Spy0128 constructs. Using Promega paramagnetic beads, we enrich the fraction of proteins that have been modified by the blocking peptide. The proteins are subsequently detached from the beads using the TEV protease. From these purified proteins we obtained three types of sawtooth patterns: (A) Unmodified Spy0128 constructs with short initial extensions (Li < 50 nm) and (B) showing a single modification (Li ∼ 50–100 nm) or (C) two modifications (Li > 100 nm). (D) Histogram of initial extensions (n = 453). A Gaussian fit identifies three peaks at 34, 86, and 135 nm. (E) Two-dimensional histogram showing the density of unfolding forces (FU) versus the contour length increments (∆Lc) for the isopeptide-blocked Spy0128. Modified pilin proteins unfold at forces below 100 pN and do not show any well-defined unfolding pathway. Remarkably, 14% of the traces (n = 40) show long initial extensions without any unfolding event, suggesting that the modified Spy0128 extends as an unstructured polymer. (F) Mutant Spy0128 E258A unfolds through well-defined unfolding pathways with high mechanical stability, either through a single event with FU ∼ 300 pN or through an intermediate with FU1 ∼ 180 pN and FU2 ∼ 110 pN. Data from ref. 24.

Article Snippet: Histograms were fitted using single and multiple Gaussian distributions implemented in Igor 7 (WaveMetrics).

Techniques: Purification, Construct, Modification, Blocking Assay, Polymer, Mutagenesis