Periodic accumulation and degradation of RAG2 (recombination-activating gene 2) protein controls the cellcycle-dependent V(D)J recombination of lymphocyte antigen receptor genes. Here we show the molecular mechanism of RAG2 degradation. The RAG2 protein is translocated from the nucleus to the cytoplasm and degraded through the ubiquitin/proteasome system. RAG2 translocation is mediated by the Thr-490 phosphorylation of RAG2. Inhibition of this phosphorylation by p27Kip1 stabilizes the RAG2 protein in the nucleus. These results suggest that RAG2 sequestration in the cytoplasm and its subsequent degradation by the ubiquitin/proteasome system upon entering the S phase is an integral part of G0/G1-specific V(D)J recombination.During B and T cell development, the genes encoding the variable region of immunoglobulin (Ig) and the T cell receptor (TCR) are assembled from germ line-variable (V), diversity (D), and joining (J) gene segments by V(D)J recombination (1-3). V(D)J recombination is initiated by the introduction of sitespecific double-strand breaks (DSBs) between two recombining gene segments and their flanking recombination signal sequences (RSSs). The essential components of this reaction are RAG1 and RAG2 (recombination-activating genes 1 and 2) proteins, which are expressed specifically in the developing B and T lineage cells under V(D)J recombination (4, 5). Subsequent reaction steps employ more generally expressed proteins involved in non-homologous end joining (6, 7): the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the Ku70/80 dimer, XRCC4, and DNA Ligase IV. More proteins may be involved in V(D)J recombination to ensure the strict regulation of this system. Several lines of evidence suggest that V(D)J recombination is restricted to the G0/G1 stage of the cell cycle and that periodic accumulation and destruction of the RAG2 protein regulate cell-cycle-dependent V(D)J recombination (8). According to Lee and Desiderio, Thr-490 of RAG2 is phosphorylated by cyclin A/CDK2 when the cells are at the G1-S transition of the cell cycle, thereby triggering the rapid degradation of RAG2 (9). However, the precise molecular mechanism of the degradation process remains elusive.The ubiquitin/proteasome system plays a major role in target-specific protein degradation and in the regulation of protein expression levels (10, 11). The formation of ubiquitin-protein conjugates proceeds via a three-step cascade. First, a ubiquitinactivating enzyme (E1) activates ubiquitin, which is then transferred by a ubiquitin-conjugating enzyme (E2) to a ubiquitin ligase (E3) with which the substrate protein is associated. Finally, E3 catalyzes the conjugation of ubiquitin to the substrate protein. Proteins polyubiquitinated by these enzymes are subjected to degradation by the 26S proteasome. Recent reports have suggested that many proteins, such as p53, IB, ␤-catenin, and p27Kip1, are degraded by the ubiquitin/proteasome pathway (10 -13).p27Kip1, a cyclin-dependent kinase inhibitor, increases RAG2 stability by inhibiting ...

Immunoglobulin heavy-chain (IgH) class switch recombination (CSR) is initiated by DNA breakage in the switch (S) region featuring tandem repetitive nucleotide sequences. Various studies have demonstrated that S-region transcription and splicing proceed to genomic recombination and are indispensable for CSR in vivo, although the precise molecular mechanism is largely unknown. Here, we show the novel physical property of the in vitro transcribed S-region RNA by direct visualization using an atomic force microscope (AFM). The S-region sense RNA, but not the antisense RNA, forms a persistent hybrid with the template plasmid DNA and changes the plasmid conformation from supercoil to open circle in the presence of spermidine. In addition, the S-region transcripts generate globular forms and are assembled on the template DNA into a large aggregate that may stall replication and increase the recombinogenicity of the S-region DNA.Two subsequent DNA recombination steps are involved in immunoglobulin (Ig) production (1-3). First, V(D)J recombination assembles the V, D, and J segments into a variable region exon and contributes to the diversification of antigen-binding sites. Second, class switch recombination (CSR) 1 converts the constant (C H ) region of the immunoglobulin heavy chain (IgH) from C to other classes and alters physiological functions that differ in their properties of complement fixation, binding to class-specific Fc receptors, and polymerization. Thus, class switching is a mechanism for diversifying Ig effector functions with the same antigen specificity.The mouse IgH locus of the IgM-expressing B lymphocyte contains a rearranged V(D)J exon and 8 C H exons of the following organization: 5Ј-V(D)J-C-C␦-C␥3-C␥1-C␥2b-C␥2a-C⑀-C␣-3Ј. CSR is initiated by DNA breakage in the S-region featuring tandem repetitive nucleotide sequences that are located upstream of each C H exon except for the C␦ exon. In the case of CSR from IgM to IgE, DNA breakage initially occurs in both S and S⑀ and results in deletion of the intervening region containing the C to C␥2a exons, and religation of S and S⑀ causes juxtaposition of the V(D)J exon and the C⑀ exon. Despite the recent discovery of the activation-induced deaminase (AID) as an indispensable factor for CSR (2, 4), the molecular mechanism of CSR still remains elusive. In particular, the initiation step of DNA cleavage is completely unknown.Previous studies have revealed that the generation of germline transcripts from the sites upstream of each S-region is a prerequisite for CSR, and the higher order DNA structure involving S-region RNAs that are spliced out from the primary transcripts has been proposed as a critical factor for CSR (1, 5-7). Moreover, biochemical studies, including an agarose gel analysis, suggested that a persistent RNA/DNA hybrid is formed between S-region transcripts and its template DNA (8 -11). Thus, the S-region RNA may be catalytically involved in DNA breakage and CSR, and the characterization of the RNA/DNA hybrid may be critical for the understandi...

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Rolling circle amplification (RCA) of plasmid DNA using random hexamers and bacteriophage phi29 DNA polymerase is an increasingly applied technique for amplifying template DNA for DNA sequencing. We analyzed this RCA reaction at a single-molecular level by atomic force microscopy (AFM) and found that multibranched amplified products containing tandem repeats of a circle unit are formed within 1 h. We also used the RCA product of a GFP expression vector for the protein expression in cells, and found that the crude RCA product from one bacterial colony is sufficient for the GFP expression. Thus, the RCA reaction is useful in amplifying DNA for both DNA sequencing and protein expression.