Suppression of Signal Transducer and Activator of Transcription 3–Dependent B Lymphocyte Terminal Differentiation by Bcl-6

Lymphocytes usually differentiate into effector cells within days after antigen exposure, except in germinal centers where terminal differentiation is delayed while somatic hypermutation creates high-affinity antibody mutants. Here we investigate whether arrest of terminal differentiation can be mediated by BCL-6, a transcriptional repressor that is expressed by germinal center B cells and is required for this phase of B cell development. We find that BCL-6 suppresses the differentiation of transformed and primary B cells to plasma cells by inhibiting the signal transducer and activator of transcription 3–dependent expression of the major regulator of plasma cell development, the B lymphocyte–induced maturation protein (Blimp-1). This function of BCL-6 as a repressor of B lymphocyte differentiation may also underlie the association between chromosomal translocations of its gene and B cell lymphomas

DNA communications by Type III restriction endonucleases—confirmation of 1D translocation over 3D looping

DNA cleavage by Type III restriction enzymes is governed strictly by the relative arrangement of recognition sites on a DNA substrate—endonuclease activity is usually only triggered by sequences in head-to-head orientation. Tens to thousands of base pairs can separate these sites. Long distance communication over such distances could occur by either one-dimensional (1D) DNA translocation or 3D DNA looping. To distinguish between these alternatives, we analysed the activity of EcoPI and EcoP15I on DNA catenanes in which the recognition sites were either on the same or separate rings. While substrates with a pair of sites located on the same ring were cleaved efficiently, catenanes with sites on separate rings were not cleaved. These results exclude a simple 3D DNA-looping activity. To characterize the interactions further, EcoPI was incubated with plasmids carrying two recognition sites interspersed with two 21res sites for site-specific recombination by Tn21 resolvase; inhibition of recombination would indicate the formation of stable DNA loops. No inhibition was observed, even under conditions where EcoPI translocation could also occur

Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

A new Type III restriction endonuclease designated PstII has been purified from Providencia stuartii. PstII recognizes the hexanucleotide sequence 5 0-CTGATG(N)25-26/27-28-3 0. Endonuclease activity requires a substrate with two copies of the recognition site in head-to-head repeat and is dependent on a low level of ATP hydrolysis ( 40 ATP/site/min). Cleavage occurs at just one of the two sites and results in a staggered cut 25–26 nt downstream of the top strand sequence to generate a two base 5 0-protruding end. Methylation of the site occurs on one strand only at the first adenine of 5 0-CATCAG-3 0. Therefore, PstII has characteristic Type III restriction enzyme activity as exemplified by EcoPI or EcoP15I. Moreover, sequence asymmetry of the PstII recognition site in the T7 genome acts as an historical imprint of Type III restriction activity in vivo. In contrast to other Type I and III enzymes, PstII has a more relaxed nucleotide specificity and can cut DNA with GTP and CTP (but not UTP). We also demonstrate that PstII and EcoP15I cannot interact and cleave a DNA substrate suggesting that Type III enzymes must make specific protein–protein contacts to activate endonuclease activity

Type III restriction enzymes communicate in 1D without looping between their target sites

To cleave DNA, Type III restriction enzymes must communicate the relative orientation of two asymmetric recognition sites over hundreds of base pairs. The basis of this long-distance communication, for which ATP hydrolysis by their helicase domains is required, is poorly understood. Several conflicting DNA-looping mechanisms have been proposed, driven either by active DNA translocation or passive 3D diffusion. Using single-molecule DNA stretching in combination with bulk-solution assays, we provide evidence that looping is both highly unlikely and unnecessary, and that communication is strictly confined to a 1D route. Integrating our results with previous data, a simple communication scheme is concluded based on 1D diffusion along DNA