The DDE motif in RAG-1 is contributed in trans to a single active site that catalyzes the nicking and transesterification steps of V(D)J recombination

The process of assembling immunoglobulin and T-cell receptor genes from variable (V), diversity (D), and joining (J) gene segments, called V(D)J recombination, involves the introduction of DNA breaks at recombination signals. DNA cleavage is catalyzed by RAG-1 and RAG-2 in two chemical steps: first-strand nicking, followed by hairpin formation via direct transesterification. In vitro, these reactions minimally proceed in discrete protein-DNA complexes containing dimeric RAG-1 and one or two RAG-2 monomers bound to a single recombination signal sequence. Recently, a DDE triad of carboxylate residues essential for catalysis was identified in RAG-1. This catalytic triad resembles the DDE motif often associated with transposase and retroviral integrase active sites. To investigate which RAG-1 subunit contributes the residues of the DDE triad to the recombinase active site, cleavage of intact or prenicked DNA substrates was analyzed in situ in complexes containing RAG-2 and a RAG-1 heterodimer that carried an active-site mutation targeted to the same or opposite RAG-1 subunit mutated to be incompetent for DNA binding. The results show that the DDE triad is contributed to a single recombinase active site, which catalyzes the nicking and transesterification steps of V(D)J recombination by a single RAG-1 subunit opposite the one bound to the nonamer of the recombination signal undergoing cleavage (cleavage in trans). The implications of a trans cleavage mode observed in these complexes on the organization of the V(D)J synaptic complex are discussed.     

Modulation of RAG/DNA complex by HSP70 in V(D)J recombination

V(D)J recombination, a site-specific gene rearrangement process, requires two RAG1 and RAG2 proteins specifically recognizing recombination signal sequences and forming DNA double-strand breaks. The broken DNA ends tightly bound to RAG proteins are joined by repair proteins. Here, we found that heat shock protein 70 was associated with RAG2 following two-step affinity chromatography purification. It was also co-immunoprecipitated with RAG2 in pro-B cells. Purified HSP70 protein disrupted RAG/DNA complexes assembled in vitro and also inhibited the V(D)J cleavage (both nick and hairpin formation) in a dose-dependent manner. This HSP70 action required ATP energy. These data suggest that HSP70 might play a crucial role in disassembling RAG/DNA complexes stably formed during V(D)J recombination.     

The amino terminus of bacteriophage lambda integrase is involved in protein-protein interactions during recombination

Bacteriophage lambda integrase (Int) catalyzes at least four site-specific recombination pathways between pairs of attachment (att) sites. Protein-protein contacts between monomers of Int are presumed to be important for these site-specific recombination events for several reasons: Int binds to the att sites cooperatively, catalytic Int mutants can complement each other for strand cleavage, and crystal structures for two other recombinases in the Int family (Cre from phage P1 and Int from Haemophilus influenzae phage HP1) show extensive protein-protein contacts between monomers. We have begun to investigate interactions between Int monomers by three approaches. First, using a genetic assay, we show that regions of protein-protein interactions occur throughout Int, including in the amino-terminal domain. This domain was previously thought to be important only for high-affinity protein-DNA interactions. Second, we have found that an amino-terminal His tag reduces cooperative binding to DNA. This disruption in cooperativity decreases the stable interaction of Int with core sites, where catalysis occurs. Third, using protein-protein cross-linking to investigate the multimerization of Int during recombination, we show that Int predominantly forms dimers, trimers, and tetramers. Moreover, we show that the cysteine at position 25 is present at or near the interface between monomers that is involved in the formation of dimers and tetramers. Our evidence indicates that the amino-terminal domain of Int is involved in protein-protein interactions that are likely to be important for recombination.     

Evidence for ATP binding and double-stranded DNA binding by Escherichia coli RecF protein.

RecF protein is one of the important proteins involved in DNA recombination and repair. RecF protein has been shown to bind single-stranded DNA (ssDNA) in the absence of ATP (T. J. Griffin IV and R. D. Kolodner, J. Bacteriol. 172:6291-6299, 1990; M. V. V. S. Madiraju and A. J. Clark, Nucleic Acids Res. 19:6295-6300, 1991). In the present study, using 8-azido-ATP, a photo-affinity analog of ATP, we show that RecF protein binds ATP and that the binding is specific in the presence of DNA. 8-Azido-ATP photo-cross-linking is stimulated in the presence of DNA (both ssDNA and double-stranded DNA [dsDNA]), suggesting that DNA enhances the affinity of RecF protein for ATP. These data suggest that RecF protein possesses independent ATP- and DNA-binding sites. Further, we find that stable RecF protein-dsDNA complexes are obtained in the presence of ATP or ATP-gamma-S [adenosine-5'-O-(3-thio-triphosphate)]. No other nucleoside triphosphates served as necessary cofactors for dsDNA binding, indicating that RecF is an ATP-dependent dsDNA-binding protein. Since a mutation in a putative phosphate-binding motif of RecF protein results in a recF mutant phenotype (S. J. Sandler, B. Chackerian, J. T. Li, and A. J. Clark, Nucleic Acids Res. 20:839-845, 1992), we suggest on the basis of our data that the interactions of RecF protein with ATP, with dsDNA, or with both are physiologically important for understanding RecF protein function in vivo.     

Fine structure and activity of discrete RAG-HMG complexes on V(D)J recombination signals

Two lymphoid cell-specific proteins, RAG-1 and RAG-2, initiate V(D)J recombination by introducing DNA breaks at recombination signal sequences (RSSs). Although the RAG proteins themselves bind and cleave DNA substrates containing either a 12-RSS or a 23-RSS, DNA-bending proteins HMG-1 and HMG-2 are known to promote these processes, particularly with 23-RSS substrates. Using in-gel cleavage assays and DNA footprinting techniques, I analyzed the catalytic activity and protein-DNA contacts in discrete 12-RSS and 23-RSS complexes containing the RAG proteins and either HMG-1 or HMG-2. I found that both the cleavage activity and the pattern of protein-DNA contacts in RAG-HMG complexes assembled on 12-RSS substrates closely resembled those obtained from analogous 12-RSS complexes lacking HMG protein. In contrast, 23-RSS complexes containing both RAG proteins and either HMG-1 or HMG-2 exhibited enhanced cleavage activity and displayed an altered distribution of cleavage products compared to 23-RSS complexes containing only RAG-1 and RAG-2. Moreover, HMG-dependent heptamer contacts in 23-RSS complexes were observed. The protein-DNA contacts in RAG-RSS-HMG complexes assembled on 12-RSS or 23-RSS substrates were strikingly similar at comparable positions, suggesting that the RAG proteins mediate HMG-dependent heptamer contacts in 23-RSS complexes. Results of ethylation interference experiments suggest that the HMG protein is positioned 5' of the nonamer in 23-RSS complexes, interacting largely with the side of the duplex opposite the one contacting the RAG proteins. Thus, HMG protein plays the dual role of bringing critical elements of the 23-RSS heptamer into the same phase as the 12-RSS to promote RAG binding and assisting in the catalysis of 23-RSS cleavage.     

Cooperative recruitment of HMGB1 during V(D)J recombination through interactions with RAG1 and DNA

During V(D)J recombination, recombination activating gene (RAG)1 and RAG2 bind and cleave recombination signal sequences (RSSs), aided by the ubiquitous DNA-binding/-bending proteins high-mobility group box protein (HMGB)1 or HMGB2. HMGB1/2 play a critical, although poorly understood, role in vitro in the assembly of functional RAG–RSS complexes, into which HMGB1/2 stably incorporate. The mechanism of HMGB1/2 recruitment is unknown, although an interaction with RAG1 has been suggested. Here, we report data demonstrating only a weak HMGB1–RAG1 interaction in the absence of DNA in several assays, including fluorescence anisotropy experiments using a novel Alexa488-labeled HMGB1 protein. Addition of DNA to RAG1 and HMGB1 in fluorescence anisotropy experiments, however, results in a substantial increase in complex formation, indicating a synergistic binding effect. Pulldown experiments confirmed these results, as HMGB1 was recruited to a RAG1–DNA complex in a RAG1 concentration-dependent manner and, interestingly, without strict RSS sequence specificity. Our finding that HMGB1 binds more tightly to a RAG1–DNA complex over RAG1 or DNA alone provides an explanation for the stable integration of this typically transient architectural protein in the V(D)J recombinase complex throughout recombination. These findings also have implications for the order of events during RAG–DNA complex assembly and for the stabilization of sequence-specific and non-specific RAG1–DNA interactions.     

A Dual Interaction between the DNA Damage Response Protein MDC1 and the RAG1 Subunit of the V(D)J Recombinase

The first step in V(D)J recombination is the formation of specific DNA double-strand breaks (DSBs) by the RAG1 and RAG2 proteins, which form the RAG recombinase. DSBs activate a complex network of proteins termed the DNA damage response (DDR). A key early event in the DDR is the phosphorylation of histone H2AX around DSBs, which forms a binding site for the tandem BRCA1 C-terminal (tBRCT) domain of MDC1. This event is required for subsequent signal amplification and recruitment of additional DDR proteins to the break site. RAG1 bears a histone H2AX-like motif at its C terminus (R1Ct), making it a putative MDC1-binding protein. In this work we show that the tBRCT domain of MDC1 binds the R1Ct motif of RAG1. Surprisingly, we also observed a second binding interface between the two proteins that involves the Proline-Serine-Threonine rich (PST) repeats of MDC1 and the N-terminal non-core region of RAG1 (R1Nt). The repeats-R1Nt interaction is constitutive, whereas the tBRCT-R1Ct interaction likely requires phosphorylation of the R1Ct motif of RAG1. As the C terminus of RAG1 has been implicated in inhibition of RAG activity, we propose a model in which phosphorylation of the R1Ct motif of RAG1 functions as a self-initiated regulatory signal.     

Protein-DNA complexes containing DNA-dependent protein kinase in crude extracts from human and rodent cells

The DNA-dependent protein kinase (DNA-PK) is composed of a large catalytic subunit (DNA-PKcs) and a DNA-binding protein, Ku. Cells lacking DNA-PK activity are radiosensitive and are defective in DNA double-strand break repair and V(D)J recombination. Although much information regarding the interactions of Ku with DNA ends is available, relatively little is known about the interaction of DNA-PKcs with DNA-bound Ku. Here we show, using electrophoretic mobility shift assays, that chemical crosslinkers enhance the formation of protein-DNA complexes containing DNA-PKcs, Ku and other proteins in extracts from cells of normal human cell lines. Extracts from cells of the radiosensitive human cell line M059J, which lacks DNA-PKcs, are not competent to form these protein-DNA complexes, while addition of purified DNA-PKcs protein restores complex formation. This assay may be useful for screening for DNA-PK function in cells of human cell lines and for identifying proteins that interact with the DNA-PK-DNA complex. We also show that Ku protein in rodent cells can interact with human DNA-PKcs; however, this assay may be less useful for studying Ku/DNA-PKcs interactions in cells of rodent cell lines due to the low abundance of DNA-PKcs in these cells.     

Recognition sequence of a highly conserved DNA binding protein RBP-J kappa.

DNA binding specificity of the RBP-J kappa protein was extensively examined. The mouse RBP-J kappa protein was originally isolated as a nuclear protein binding to the J kappa type V(D)J recombination signal sequence which consisted of the conserved heptamer (CACTGTG) and nonamer (GGTTTTTGT) sequences separated by a 23-base pair spacer. Electrophoretic mobility shift assay using DNA probes with mutations in various parts of the J kappa recombination signal sequence showed that the RBP-J kappa protein recognized the sequence outside the recombination signal in addition to the heptamer but did not recognize the nonamer sequence and the spacer length at all. Database search identified the best naturally occurring binding motif (CACTGTGGGAACGG) for the RBP-J kappa protein in the promoter region of the m8 gene in the Enhancer of split gene cluster of Drosophila. The binding assay with a series of m8 motif mutants indicated that the protein recognized mostly the GTGGGAA sequence and also interacted weakly with ACT and CG sequences flanking this hepta-nucleotide. Oligonucleotides binding to the RBP-J kappa protein were enriched from a pool of synthetic oligonucleotides containing 20-base random sequences by the repeated electrophoretic mobility shift assay. The enriched oligomer shared a common sequence of CGTGGGAA. All these data indicate that the RBP-J kappa protein recognizes a unique core sequence of CGTGGGAA and does not bind to the V(D)J recombination signal without the flanking sequence.     

A RAG-1/RAG-2 tetramer supports 12/23-regulated synapsis,cleavage, and transposition of V(D)J recombination signals

Initiation of V(D)J recombination involves the synapsis and cleavage of a 12/23 pair of recombination signal sequences by RAG-1 and RAG-2. Ubiquitous nonspecific DNA-bending factors of the HMG box family, such as HMG-1, are known to assist in these processes. After cleavage, the RAG proteins remain bound to the cut signal ends and, at least in vitro, support the integration of these ends into unrelated target DNA via a transposition-like mechanism. To investigate whether the protein complex supporting synapsis, cleavage, and transposition of V(D)J recombination signals utilized the same complement of RAG and HMG proteins, I compared the RAG protein stoichiometries and activities of discrete protein-DNA complexes assembled on intact, prenicked, or precleaved recombination signal sequence (RSS) substrates in the absence and presence of HMG-1. In the absence of HMG-1, I found that two discrete RAG-1/RAG-2 complexes are detected by mobility shift assay on all RSS substrates tested. Both contain dimeric RAG-1 and either one or two RAG-2 subunits. The addition of HMG-1 supershifts both complexes without altering the RAG protein stoichiometry. I find that 12/23-regulated recombination signal synapsis and cleavage are only supported in a protein-DNA complex containing HMG-1 and a RAG-1/RAG-2 tetramer. Interestingly, the RAG-1/RAG-2 tetramer also supports transposition, but HMG-1 is dispensable for its activity.     

Function and disruption of DNA methyltransferase 3a cooperative DNA binding and nucleoprotein filament formation

The catalytic domain of Dnmt3a cooperatively multimerizes on DNA forming nucleoprotein filaments. Based on modeling, we identified the interface of Dnmt3a complexes binding next to each other on the DNA and disrupted it by charge reversal of critical residues. This prevented cooperative DNA binding and multimerization of Dnmt3a on the DNA, as shown by the loss of cooperative complex formation in electrophoretic mobility shift assay, the loss of cooperativity in DNA binding in solution, the loss of a characteristic 8- to 10-bp periodicity in DNA methylation and direct imaging of protein-DNA complexes by scanning force microscopy. Non-cooperative Dnmt3a-C variants bound DNA well and retained methylation activity, indicating that cooperative DNA binding and multimerization of Dnmt3a on the DNA are not required for activity. However, one non-cooperative variant showed reduced heterochromatic localization in mammalian cells. We propose two roles of Dnmt3a cooperative DNA binding in the cell: (i) either nucleofilament formation could be required for periodic DNA methylation or (ii) favorable interactions between Dnmt3a complexes may be needed for the tight packing of Dnmt3a at heterochromatic regions. The complex interface optimized for tight packing would then promote the cooperative binding of Dnmt3a to naked DNA in vitro.     

The RAG1 Homeodomain Recruits HMG1 and HMG2 To Facilitate Recombination Signal Sequence Binding and To Enhance the Intrinsic DNA-Bending Activity of RAG1-RAG2

V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.     

DNA binding of Xrcc4 protein is associated with V(D)J recombination but not with stimulation of DNA ligase IV activity

Mammalian cells are protected from the effects of DNA double-strand breaks by end-joining repair. Cells lacking the Xrcc4 protein are hypersensitive to agents that induce DNA double-strand breaks, and are unable to complete V(D)J recombination. The residual repair of broken DNA ends in XRCC4-deficient cells requires short sequence homologies, thus possibly implicating Xrcc4 in end alignment. We show that Xrcc4 binds DNA, and prefers DNA with nicks or broken ends. Xrcc4 also binds to DNA ligase IV and enhances its joining activity. This stimulatory effect is shown to occur at the adenylation of the enzyme. DNA binding of Xrcc4 is correlated with its complementation of the V(D)J recombination defects in XRCC4-deficient cells, but is not required for stimulation of DNA ligase IV. Thus, the ability of Xrcc4 to bind to DNA suggests functions independent of DNA ligase IV.     

DNA-dependent protein kinase complex: a multifunctional protein in DNA repair and damage checkpoint

DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase that is activated upon DNA damage generated by ionizing radiation or UV-irradiation. It is a three-protein complex consisting of a 470-kDa catalytic subunit (DNA-PKcs) and the regulatory DNA binding subunits, Ku heterodimer (Ku70 and Ku80). Mouse and human cells deficient in DNA-PKcs are hypersensitive to ionizing radiation and defective in V(D)J recombination, suggesting a role for the kinase in double-strand break repair and recombination. The Ku heterodimer binds to double-strand DNA breaks produced by either DNA damage or recombination, protects DNA ends from degradation, orients DNA ends for re-ligation, and recruits its catalytic subunit and additional factors necessary for successful end-joining. DNA-PK is also involved in an early stage of damage-induced cell cycle arrest, however, it remains unclear how the enzyme senses DNA damage and transmits signals to downstream gene(s) and proteins.     

Influence of DNA stiffness in protein-DNA recognition

Protein-DNA recognition plays an essential role in the regulation of gene expression. The protein-DNA binding specificity is based on direct atomic contacts between protein and DNA and/or the conformational properties of DNA. In this work, we have analyzed the influence of DNA stiffness (E) to the specificity of protein-DNA complexes. The average DNA stiffness parameters for several protein-DNA complexes have been computed using the structure based sequence dependent stiffness scale. The relationship between DNA stiffness and experimental protein-DNA binding specificity has been brought out. We have investigated the importance of DNA stiffness with the aid of experimental free energy changes (DeltaDeltaG) due to binding in several protein-DNA complexes, such as, ETS proteins, 434, lambda, Mnt and trp repressors, 434 cro protein, EcoRV endonuclease V and zinc fingers. We found a correlation in the range 0.65-0.97 between DeltaDeltaG and E in these examples. Further, we have qualitatively analyzed the effect of mutations in the target sequence of lambda repressor and we observed that the DNA stiffness could correctly identify 70% of the correct bases among the considered nine positions.     

DNA-binding properties of a lac repressor mutant incapable of forming tetramers

The interaction of proteins bound to sites widely separated on the genome is a recurrent motif in both prokaryotic and eukaryotic regulatory systems. Lac repressor mediates the formation of "DNA loops" by the simultaneous interaction of a single protein tetramer with two DNA-binding sites. The DNA-binding properties of a Lac repressor mutant (LacIadi) deficient in the association of protein dimers to tetramers was investigated. The results of quantitative footprint and gel mobility-shift titrations suggest that the wild-type Lac repressor (LacI+) binds cooperatively to two operator sites separated by 11 helical turns on a linear DNA restriction fragment by the formation of a "looped complex." LacIadi binds to this two-site operator non-cooperatively and without formation of a looped complex. These results demonstrate that the dimer-tetramer association of LacI+ is directly responsible for its cooperative binding and its ability to mediate formation of a looped complex. The Iadi mutation disrupts the monomer-dimer as well as eliminating the dimer-tetramer association equilibria while the DNA binding affinity of LacIadi to a single site is unchanged relative to the wild-type protein. These results suggest that DNA binding and dimer-tetramer association are functionally unlinked. The similarity of the DNA-binding properties of LacIadi and Gal repressor, a protein believed to function by mediating the formation of a looped complex, are discussed.