Bromination of Azoxybenzene

departmental bulletin pape

Precise Measurement of B Meson Lifetimes with Hadronic Decay Final States

journal articl

Activation-induced deaminase (AID) localizes to the nucleus in brief pulses

Activation-induced deaminase (AID) converts C to U and 5-methyl-C to T. These mutagenic activities are critical to immunoglobulin (Ig) gene diversification and epigenetic reprogramming, but they must be tightly controlled to prevent compromising cell fitness. AID acts in the nucleus but localizes predominately to the cytoplasm. To address this apparent paradox, we have carried out time-lapse imaging of AID in single living B cells and fibroblasts. We demonstrate that AID enters the nucleus in brief (30 min) pulses, evident in about 10% of cells in the course of a single cell cycle (24 hr imaging). Pulses do not depend on AID catalytic activity, but they are coordinated with nuclear accumulation of P53. Pulsing may protect cells from pathologic consequences of excess exposure to AID, or enable AID to synchronize its activity with transcription of genes that are AID targets or with nuclear entry of factors that act at sites of AID-catalyzed DNA deamination to promote Ig gene diversification or epigenetic reprogramming.</div

Timing and frequency of attenuation of the AID^F193A-mCherry nuclear signal.

(A) Timelines of attenuation events that reduce the nuclear signal in HT1080 AIDF193A-mCherry transfectants. Details and notations as in Fig 2A. (B) Above, summary of data on AIDF193A-mCherry pulses (see also S1 Table). Below, pie diagram showing the number of cells with indicated number of pulses per cell (total number of cells = 49), based on the timeline in panel (A). (C) Frames captured by live cell imaging showing attenuation of AIDF193A-mCherry nuclear signal. Arrows point to cells in which signal attenuation was observed. Attenuation events captured in images occur in: S8 Movie, cell at upper left of frames 91–94 (left images); S9 Movie, cell at top center of frames 97–100 (right image; this cell undergoes division at frame 88).</p

Nuclear P53 increases in response to nuclear pulses of AID.

(A) Tracings of nuclear fluorescent signals of four GM639 AID-mCherry P53-GFP double transfectants over the duration of 24 hr time lapse imaging. Cell 0 did not pulse; cells 1–3 exhibited one, three and five pulses, respectively, indicated by arrowheads. (B) Quantification of fold change in nuclear P53-GFP signal in Cells 0, 1, 2 and 3 over the course of 24 hr of imaging. (C) Frames captured by live cell imaging, showing P53-GFP (top), AID-mCherry (middle), and merged (bottom) images of Cells 2 and 3 over the course of two pulses, the second and third pulse in Cell 2 and the first and fifth pulse in Cell 3. Arrows point to the cell that pulses. Pulses occur in: Cell 1, S12 Movie, cell at bottom center of frames 18–22; Cell 2, S13 Movie, cell at top right of frames 33–35 for the second pulse and frames 89–91 for the third pulse; and Cell 3, S13 Movie, cell at top right of frames 15–17 for the first pulse and 108–110 for the fifth pulse.</p

Attenuation of the AID^F193A-mCherry nuclear signal occurs in synchrony with AID-GFP pulses.

(A) Frames captured by live cell imaging, showing AID-GFP (top), AIDF193A-mCherry (middle), and merged (bottom) images over the course of a pulse. Arrows point to cell that pulses. Pulses captured in images occur in: Cell1, S10 Movie, cell at top left of frames 26–29; Cell 2, S10 Movie, cell at center of frames 17–20; Cell 3, S11 Movie, cell at top center of frames 136–139, which undergoes division in frame 100. (B) Tracings of nuclear signals during two separate pulses spanning indicated frames for each of the three cells shown in panel (A).</p

Distinct outcomes of an AID-initiated DNA nick in G1 and S/G2M phases.

<p>(A) Deamination of a C/G base pair by AID creates a U/G mispair at which UNG2 or MSH2/MSH6 will act to create a DNA nick. In either case, the repair processes that remove U generates a DNA nick as an intermediate. (B) In G1 phase, a nick can undergo mutagenic repair resulting in SHM. In S phase, if replication occurs before repair the nick will be converted to a replicative DSB. This may activate ATM and DNA-PK, cause γ-H2AX accumulation, a DNA damage response, checkpoint activation and potential loss of viability.</p

Timing and frequency of AID-mCherry nuclear pulses.

(A) Timelines of nuclear pulses in HT1080 AID-mCherry transfectants relative to cell division. Diagrams are aligned to start of imaging (t = 0); and ranked by order of cell division (indicated by bifurcation of line). For each cell, AID-mCherry positive nuclear signals were scored and indicated as rectangles along the line denoting the 24 hr time course of live cell imaging. Lines are truncated prior to 24 hr for cells that exited the visual field. The number on the left indicates the number of pulses per cell. Commas (,) separate the tallies for mother and daughter cells and slashes (/) separate the tallies for daughter cells. (B) Above, summary of data on AID-mCherry pulses (see also S1 Table). Below, pie diagram indicating the number of cells with indicated number of pulses per cell (total number of cells = 36), based on the timeline in panel (A).</p

Nuclear pulses are independent of AID catalytic activity.

(A) Timelines of nuclear pulses in HT1080 AID-mCherry transfectants relative to cell division. Details and notations as in Fig 2A. (B) Above, summary of data on AIDH56A-mCherry pulses (see also S1 Table). Below, pie diagram indicating the number of cells with indicated number of pulses per cell (total number of cells = 46), based on the timeline in panel A. (C) Representative frames captured by live cell imaging of nuclear pulses in HT1080 AIDH56A-mCherry transfectants at 10 min intervals. Arrows point to cells that pulse. Movies including these frames are provided in Supporting Information. Pulses captured in images above occur in: S6 Movie, cell at top center, frames 2–5 (upper images); S7 Movie, cell at center left of frames 37–40 (lower images).</p

Phosphorylation and catalytic activity regulate AID abundance and nuclear stability.

<p>(A) Nuclear and cytoplasmic mCherry signals from indicated mutants, as determined by HCS analysis, expressed in arbitrary units. Population average signals are shown; black bars represent SEM of the population, which are too small to discern. (B) Representative HCS analysis of kinetics of response of nuclear (solid lines) and cytoplasmic (dashed lines) signals of indicated AID-mCherry mutants to treatment with LMB, relative to untreated cells. Each point represents a population average, and black bars represent SEM of the population, which are too small to discern. Dotted line represents no change (fold change of 1). (C) Representative HCS analysis of kinetics of response of nuclear (solid lines) and cytoplasmic (dashed lines) signals of indicated AID-mCherry mutants to treatment with LMB in G1, S and G2/M phase cells. Each point represents a population average, and black bars represent SEM of the population, which are too small to discern. Dotted line represents no change (fold change of 1). Analysis of WT AID was carried out in the same experiment and is shown at right for comparison.</p