Inducible DNA breaks in Ig S regions are dependent on AID and UNG

Class switch recombination (CSR) occurs by an intrachromosomal deletion whereby the IgM constant region gene (Cμ) is replaced by a downstream constant region gene. This unique recombination event involves formation of double-strand breaks (DSBs) in immunoglobulin switch (S) regions, and requires activation-induced cytidine deaminase (AID), which converts cytosines to uracils. Repair of the uracils is proposed to lead to DNA breaks required for recombination. Uracil DNA glycosylase (UNG) is required for most CSR activity although its role is disputed. Here we use ligation-mediated PCR to detect DSBs in S regions in splenic B cells undergoing CSR. We find that the kinetics of DSB induction corresponds with AID expression, and that DSBs are AID- and UNG-dependent and occur preferentially at G:C basepairs in WRC/GYW AID hotspots. Our results indicate that AID attacks cytosines on both DNA strands, and staggered breaks are processed to blunt DSBs at the initiating ss break sites. We propose a model to explain the types of end-processing events observed

Listeriolysin O Is Necessary and Sufficient to Induce Autophagy during Listeria monocytogenes Infection

Recent studies have suggested that autophagy is utilized by cells as a protective mechanism against Listeria monocytogenes infection.However we find autophagy has no measurable role in vacuolar escape and intracellular growth in primary cultured bone marrow derived macrophages (BMDMs) deficient for autophagy (atg5-/-). Nevertheless, we provide evidence that the pore forming activity of the cholesterol-dependent cytolysin listeriolysin O (LLO) can induce autophagy subsequent to infection by L. monocytogenes. Infection of BMDMs with L. monocytogenes induced microtubule-associated protein light chain 3 (LC3) lipidation, consistent with autophagy activation, whereas a mutant lacking LLO did not. Infection of BMDMs that express LC3-GFP demonstrated that wild-type L. monocytogenes was encapsulated by LC3-GFP, consistent with autophagy activation, whereas a mutant lacking LLO was not. Bacillus subtilis expressing either LLO or a related cytolysin, perfringolysin O (PFO), induced LC3 colocalization and LC3 lipidation. Further, LLO-containing liposomes also recruited LC3-GFP, indicating that LLO was sufficient to induce targeted autophagy in the absence of infection. The role of autophagy had variable effects depending on the cell type assayed. In atg5-/- mouse embryonic fibroblasts, L. monocytogenes had a primary vacuole escape defect. However, the bacteria escaped and grew normally in atg5-/- BMDMs.We propose that membrane damage, such as that caused by LLO, triggers bacterial-targeted autophagy, although autophagy does not affect the fate of wild-type intracellular L. monocytogenes in primary BMDMs

Mutations occur in the Ig Smu region but rarely in Sgamma regions prior to class switch recombination

Nucleotide substitutions are found in recombined Ig switch (S) regions and also in unrecombined (germline, GL) Smicro segments in activated splenic B cells. Herein we examine whether mutations are also introduced into the downstream acceptor S regions prior to switch recombination, but find very few mutations in GL Sgamma3 and Sgamma1 regions in activated B cells. These data suggest that switch recombination initiates in the Smicro segment and secondarily involves the downstream acceptor S region. Furthermore, the pattern and specificity of mutations in GL and recombined Smicro segments differ, suggesting different repair mechanisms. Mutations in recombined Smicro regions show a strong bias toward G/C base pairs and WRCY/RGYW hotspots, whereas mutations introduced into the GL Smicro do not. Additionally, induction conditions affect mutation specificity within the GL Smicro segment. Mutations are most frequent near the S-S junctions and decrease rapidly with distance from the junction. Finally, we find that mice expressing a transgene for terminal deoxynucleotidyl transferase (TdT) have nucleotide insertions at S-S junctions, indicating that the recombining DNA ends are accessible to end-processing enzyme activities

Autophagy is induced by wild-type, Δ<i>actA</i>, Δ<i>plcAB</i> but not by Δ<i>hly</i> or heat-killed <i>L. monocytogenes</i>.

<p>(<b>A</b>) Western blot of LC3I and LC3II in wild-type and <i>atg5^−/−</i> BMDMs infected with wild-type <i>L. monocytogenes</i>. All images are from a single gel. (<b>B</b>) Western blot of LC3I and LC3II in wild-type BMDMs at 60 minutes post-infection. BMDMs were infected with wild-type, Δ<i>hly</i>, Δ<i>actA</i>, Δ<i>plcAB</i> mutant <i>L. monocytogenes</i> as well as heat-killed <i>L. monocytogenes</i>. Images taken from a single gel, cropped and combined. Data shown are representative of results obtained in three independent experiments.</p

LC3-GFP co-localizes with wild-type <i>L. monocytogenes</i>, but does not co-localize with Δ<i>hly</i>.

<p>(<b>A</b>) Percent of BMDMs that contain LC3-GFP, colocalizing with wild-type and mutant (Δ<i>hly</i>) <i>L. monocytogenes</i> (red) up to three hours post-infection. (<b>B</b>) Photomicrographs of LC3-GFP BMDMs 40 minutes post-infection showing colocalization of wild-type <i>L. monocytogenes</i> (red) with LC3-GFP. Graphs are a representative of three independent experiments, standard deviations have been included. Images are representative of results obtained in three independent experiments. Bar = 2 µm.</p

<i>B. subtilis</i> expressing cytolysins induced LC3I lipidation and LC3-GPF colocalization in BMDMs.

<p>(<b>A</b>) Western blot for LC3I lipidation in BMDMs infected with either wild-type <i>B. subtilis</i> or <i>B. subtilis</i> expressing LLO. (<b>B</b>) LC3 colocalization in LC3-GFP BMDMs infected with either wild-type <i>B. subtilis</i> or <i>B. subtilis</i> engineered to express LLO or PFO. Solid line, closed circles: LC3-GFP BMDMs infected with <i>B. subtilis</i> expressing PFO; dashed line, closed squares: LC3-GFP BMDMs infected with <i>B. subtilis</i> expressing LLO; solid line, open circles: LC3-GFP BMDMs infected with <i>B. subtili</i>. Graphs are a representative of three independent experiments, standard deviations have been included. (<b>C</b>) Photomicrographs: LC3-GFP BMDMs 40 minutes post-infection with either wild-type or cytolysin expressing <i>B. subtilis</i> (red). Images are representative of results obtained in 3 independent experiments. Bar = 2 µm. (<b>D</b>) Stills from time-lapse video microscopy of <i>B. subtilis</i> (red) expressing LLO in LC3-GFP BMDMs. Bar = 3.4 µm.</p

LLO activity is sufficient for autophagy activation, as measured by LC3-GFP colocalization.

<p>(<b>A</b>) Photomicrographs: liposomes containing, wild-type LLO or heat-killed LLO. Texas Red (TR) = red dye in both wild-type and heat-killed LLO containing liposomes. Images are representative of results obtained in three independent experiments. Bar = 1 µm. (<b>B</b>) Percentage of colocalization of LC3-GFP with LLO-containing liposomes over a 30 minute time course. Closed line, closed circles: liposomes containing wild-type LLO; dashed line, closed squares: liposomes containing heat-killed LLO. Data shown are representative of results obtained in 3 independent experiments.</p