Real-time analysis of RAG complex activity in V(D)J recombination.

Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS-RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination.

Histone methylation and V(D)J recombination.

V(D)J recombination is the process by which the diversity of antigen receptor genes is generated and is also indispensable for lymphocyte development. This recombination event occurs in a cell lineage- and stage-specific manner, and is carefully controlled by chromatin structure and ordered histone modifications. The recombinationally active V(D)J loci are associated with hypermethylation at lysine4 of histone H3 and hyperacetylation of histones H3/H4. The recombination activating gene 1 (RAG1) and RAG2 complex initiates recombination by introducing double-strand DNA breaks at recombination signal sequences (RSS) adjacent to each coding sequence. To be recognized by the RAG complex, RSS sites must be within an open chromatin context. In addition, the RAG complex specifically recognizes hypermethylated H3K4 through its plant homeodomain (PHD) finger in the RAG2 C terminus, which stimulates RAG catalytic activity via that interaction. In this review, we describe how histone methylation controls V(D)J recombination and discuss its potential role in lymphoid malignancy by mistargeting the RAG complex.

Modeling of the RAG reaction mechanism.

In vertebrate V(D)J recombination, it remains unclear how the RAG complex coordinates its catalytic steps with binding to two distant recombination sites. Here, we test the ability of the plausible reaction schemes to fit observed time courses for RAG nicking and DNA hairpin formation. The reaction schemes with the best fitting capability (1) strongly favor a RAG tetrameric complex over a RAG octameric complex; (2) indicate that once a RAG complex brings two recombination signal sequence (RSS) sites into synapsis, the synaptic complex rarely disassembles; (3) predict that the binding of both RSS sites (synapsis) occurs before catalysis (nicking); and (4) show that the RAG binding properties permit strong distinction between RSS sites within active chromatin versus nonspecific DNA or RSS sites within inactive chromatin. The results provide general insights for synapsis by nuclear proteins as well as more specific testable predictions for the RAG proteins.

Mechanistic basis for RAG discrimination between recombination sites and the off-target sites of human lymphomas.

During V(D)J recombination, RAG targeting to correct sites versus off-target sites relies on both DNA sequence features and on chromatin marks. Kinetic analysis using the first highly active full-length purified RAG1/RAG2 complexes has now allowed us to define the important catalytic features of this complex. We found that the overall rate of nicking, but not hairpinning, is critical for the discrimination between correct (optimal) versus off-target (suboptimal) sites used in human T-cell lymphomas, and we show that the C-terminal portion of RAG2 is required for this. This type of kinetic analysis permits us to analyze only the catalytically active RAG complex, in contrast to all other methods, which are unavoidably confounded by mixture with inactive RAG complexes. Moreover, we can distinguish the two major features of any enzymatic catalysis: the binding constant (K(D)) and the catalytic turnover rate, k(cat). Beyond a minimal essential threshold of heptamer quality, further suboptimal heptamer deviations primarily reduce the catalytic rate constant k(cat) for nicking. Suboptimal nonamers reduce not only the binding of the RAG complex to the recombination site (K(D)) but also the catalytic rate constant, consistent with a tight interaction between the RAG complex and substrate during catalysis. These features explain many aspects of RAG physiology and pathophysiology.

[Survey of difference in appearance between multiple-specifications press-through-package drug products and an attempt to evaluate their effectiveness based on dispensing incidents].

To survey the difference in appearance between multiple-specification press-through-package (PTP) drug products and to attempt to evaluate their effectiveness as discriminating factors based on dispensing incidents. Front and back sides of, respectively, 153 and 134 PTP drug products of multiple specifications stockpiled in the author's pharmacy were surveyed for differences in wording and appearance between specifications of the same type of drug. Fifty six dispensing incidents with 40 sets occurred over a year and they were analyzed for the appearance similarity of the front side. The difference factors detected in the 40 sets of "mix-ups" were also reviewed after similarity-omitted counting. We identified six factors with difference in appearance: color-related (letter front or patterns, sheet, medicine) and shape- or pattern-related details (sheet and medicine sizes, patterns). Multiple differences on the front packaging of the same type of drug were identified in 93% of the sets, while only one difference was found in about half of the sets on the back, indicating that pharmaceutical companies placed more emphasis on the front side to discriminate their features. When reviewed by similarity-omitted counting, the ratio of sets with only one difference in the 40 mix-ups was higher than those to 128 sets of non-mix-ups, the total sets except the mix-ups, while the ratio of sets with two differences was lower. In addition, the ratio of sets in which only color-related factors differed in the 40 mix-ups was higher than that in the corresponding category to the 128 sets of non-mix-ups. Various discriminating factors were used in combination on the front side of multiple-specification PTP drug products. A combined use of shape- or pattern-related and color-related factors probably reduces dispensing incidents among products with multiple specifications. However, further accumulation of incident data and multifactor analysis of those data seem necessary to clarify the function of difference in appearance in dispensing incidents.

Nonhomologous DNA end joining (NHEJ) and chromosomal translocations in humans.

Double-strand breaks (DSBs) arise in dividing cells about ten times per cell per day. Causes include replication across a nick, free radicals of oxidative metabolism, ionizing radiation, and inadvertent action by enzymes of DNA metabolism (such as failures of type II topoisomerases or cleavage by recombinases at off-target sites). There are two major double-strand break repair pathways. Homologous recombination (HR) can repair double-strand breaks, but only during S phase and typically only if there are hundreds of base pairs of homology. The more commonly used pathway is nonhomologous DNA end joining, abbreviated NHEJ. NHEJ can repair a DSB at any time during the cell cycle and does not require any homology, although a few nucleotides of terminal microhomology are often utilized by the NHEJ enzymes, if present. The proteins and enzymes of NHEJ include Ku, DNA-PKcs, Artemis, DNA polymerase mu (Pol micro), DNA polymerase lambda (Pol lambda), XLF (also called Cernunnos), XRCC4, and DNA ligase IV. These enzymes constitute what some call the classical NHEJ pathway, and in wild type cells, the vast majority of joining events appear to proceed using these components. NHEJ is present in many prokaryotes, as well as all eukaryotes, and very similar mechanistic flexibility evolved both convergently and divergently. When two double-strand breaks occur on different chromosomes, then the rejoining is almost always done by NHEJ. The causes of DSBs in lymphomas most often involve the RAG or AID enzymes that function in the specialized processes of antigen receptor gene rearrangement.

H3K4me3 stimulates the V(D)J RAG complex for both nicking and hairpinning in trans in addition to tethering in cis: implications for translocations.

The PHD finger of the RAG2 polypeptide of the RAG1/RAG2 complex binds to the histone H3 modification, trimethylated lysine 4 (H3K4me3), and in some manner increases V(D)J recombination. In the absence of biochemical studies of H3K4me3 on purified RAG enzyme activity, the precise role of H3K4me3 remains unclear. Here, we find that H3K4me3 stimulates purified RAG enzymatic activity at both the nicking (2- to 5-fold) and hairpinning (3- to 11-fold) steps of V(D)J recombination. Remarkably, this stimulation can be achieved with free H3K4me3 peptide (in trans), indicating that H3K4me3 functions via two distinct mechanisms. It not only tethers the RAG enzyme complex to a region of DNA, but it also induces a substantial increase in the catalytic turnover number (k(cat)) of the RAG complex. The H3K4me3 catalytic stimulation applies to suboptimal cryptic RSS sites located at H3K4me3 peaks that are critical in the inception of human T cell acute lymphoblastic lymphomas.

A histidine in the beta-CASP domain of Artemis is critical for its full in vitro and in vivo functions.

Artemis is a key factor of the nonhomologous end-joining (NHEJ) pathway, which is critical for DNA double-strand break (DSB) repair in eukaryotic cells. It belongs to the beta-CASP family of nucleases, forming a distinct group within the metallo-beta-lactamase superfamily. Proteins of this group are specific for nucleic acids and contain an original domain, the beta-CASP domain, which serves as a cap covering the active site displayed by the metallo-beta-lactamase domain.Here, we have identified in the highly divergent sequences of the beta-CASP domains from DNA-specific nucleases two conserved residues (Artemis E213 and H254), which are not present in RNA-specific enzymes, and shown that H254 plays a key role in the Artemis function, as it is critical for its full activity in vitro. Moreover, inherited mutation of H254 results in radiosensitive severe combined immune deficiency (RS-SCID) in humans. This residue might play a key role in specificity towards DNA, if not directly in zinc binding.

A biochemically defined system for coding joint formation in V(D)J recombination.

V(D)J recombination is one of the most complex DNA transactions in biology. The RAG complex makes double-stranded breaks adjacent to signal sequences and creates hairpin coding ends. Here, we find that the kinase activity of the Artemis:DNA-PKcs complex can be activated by hairpin DNA ends in cis, thereby allowing the hairpins to be nicked and then to undergo processing and joining by nonhomologous DNA end joining. Based on these insights, we have reconstituted many aspects of the antigen receptor diversification of V(D)J recombination by using 13 highly purified polypeptides, thereby permitting variable domain exon assembly by using this fully defined system in accord with the 12/23 rule for this process. The features of the recombination sites created by this system include all of the features observed in vivo (nucleolytic resection, P nucleotides, and N nucleotide addition), indicating that most, if not all, of the end modification enzymes have been identified.

XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps.

XRCC4 and DNA ligase IV form a complex that is essential for the repair of all double-strand DNA breaks by the nonhomologous DNA end joining pathway in eukaryotes. We find here that human XRCC4:DNA ligase IV can ligate two double-strand DNA ends that have fully incompatible short 3' overhang configurations with no potential for base pairing. Moreover, at DNA ends that share 1-4 annealed base pairs, XRCC4:DNA ligase IV can ligate across gaps of 1 nt. Ku can stimulate the joining, but is not essential when there is some terminal annealing. Polymerase mu can add nucleotides in a template-independent manner under physiological conditions; and the subset of ends that thereby gain some terminal microhomology can then be ligated. Hence, annealing at sites of microhomology is very important, but the flexibility of the ligase complex is paramount in nonhomologous DNA end joining. These observations provide an explanation for several in vivo observations that were difficult to understand previously.

Cholesterol hemisuccinate: a selective inhibitor of family X DNA polymerases.

Cholesterol hemisuccinate (compound 5), which consists of succinic acid esterified to the beta-hydroxyl group of cholesterol, selectively and strongly inhibited the activities of mammalian DNA polymerases (pols) such as pol beta, pol lambda, and terminal deoxynucleotidyltransferase (TdT), which are family X pols, in vitro, and the IC50 values were 2.9, 6.3, and 6.5 microM, respectively. The compound moderately suppressed the activities of other mammalian pols such as pol A (i.e., pol gamma), pol B (i.e., pols alpha, delta, and epsilon), and pol Y (i.e., pols iota, eta, and kappa) with 50% inhibition observed at concentrations of 131, 89.2-98.0, and 120-125 microM, respectively. The compound had no influence on the activities of plant pols alpha and beta, prokaryotic pols and other DNA metabolic enzymes tested. Since other cholesterol-related compounds such as cholesterol, cholesteryl chloride, cholesteryl bromide, cholesteryl acetate, and cholesteryl-5alpha, 6alpha-epoxide (compounds 1-4 and 6, respectively) did not influence the activities of any enzymes tested, the hemisuccinate group of compound 5 could be important for inhibition of the pol X family. Surface plasmon resonance analysis demonstrated that compound 5 bound selectively to the C-terminal 31 kDa domain of pol beta and pol lambda containing a pol beta-like region. On the basis of these results, the inhibitory mechanism of compound 5 on the pol X family was discussed.

Beta-sitosterol-3-O-beta-D-glucopyranoside: a eukaryotic DNA polymerase lambda inhibitor.

Beta-sitosterol-3-O-beta-D-glucopyranoside (compound 1), a steroidal glycoside isolated from onion (Allium cepa L.) selectively inhibited the activity of mammalian DNA polymerase lambda (pol lambda) in vitro. The compound did not influence the activities of replicative DNA polymerases such as alpha, delta and epsilon, but also showed no effect even on the activity of pol beta which is thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. Since parts of compound 1 such as beta-sitosterol (compound 2) and D-glucose (compound 3) did not influence the activities of any enzymes tested, the converted structure of compounds 2 and 3 might be important for pol lambda inhibition. The inhibitory effect of compound 1 on both intact pol lambda (i.e. residues 1-575) and a truncated pol lambda lacking the N-terminal BRCA1 C-terminus (BRCT) domain (133-575, del-1 pol lambda) was dose-dependent, and 50% inhibition was observed at a concentration of 9.1 and 5.4 microM, respectively. The compound 1-induced inhibition of del-1 pol lambda activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol lambda inhibitory mechanism of compound 1 is discussed.

Structural relationship of curcumin derivatives binding to the BRCT domain of human DNA polymerase lambda.

We previously reported that phenolic compounds, petasiphenol and curcumin (diferuloylmethane), were a selective inhibitor of DNA polymerase lambda (pol lambda) in vitro. The purpose of this study was to investigate the molecular structural relationship of curcumin and 13 chemically synthesized derivatives of curcumin. The inhibitory effect on pol lambda (full-length, i.e. intact pol lambda including the BRCA1 C- terminal [BRCT] domain) by some derivatives was stronger than that by curcumin, and monoacetylcurcumin (compound 13) was the strongest pol lambda inhibitor of all the compounds tested, achieving 50% inhibition at a concentration of 3.9 microm. The compound did not influence the activities of replicative pols such as alpha, delta, and epsilon. It had no effect on pol beta activity either, although the three-dimensional structure of pol beta is thought to be highly similar to that of pol lambda. Compound 13 did not inhibit the activity of the C-terminal catalytic domain of pol lambda including the pol beta-like core, in which the BRCT motif was deleted from its N-terminal region. MALDI-TOF MS analysis demonstrated that compound 13 bound selectively to the N-terminal domain of pol lambda, but did not bind to the C-terminal region. Based on these results, the pol lambda-inhibitory mechanism of compound 13 is discussed.

5-(Hydroxymethyl)-2-furfural: a selective inhibitor of DNA polymerase lambda and terminal deoxynucleotidyltransferase.

5-(Hydroxymethyl)-2-furfural (HMF), a pyrolysate of carbohydrate isolated from instant coffee (Coffea arabica L.), selectively inhibits the activities of mammalian DNA polymerase lambda (pol lambda) and terminal deoxynucleotidyltransferase (TdT) which are family X pols, in vitro. The compound influenced neither the activities of replicative DNA polymerases such as alpha, delta, and epsilon, nor even the activity of pol beta which is from the same family and thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. Since parts of HMF such as furan, furfuryl alcohol, and 2-furaldehyde did not influence the activities of any enzymes tested, the substituted form of furan with a hyroxymethyl group and a formyl group might be important for the inhibition of pol lambda and TdT. The inhibitory effect of HMF on intact pol lambda (i.e., residues 1-575), a truncated pol lambda lacking the N-terminal BRCA1 C-terminus domain (133-575, del-1 pol lambda) and another truncated pol lambda lacking the N-terminal proline-rich region (245-575, del-2 pol lambda) was dose-dependent, and 50% inhibition was observed at a concentration of 26.1, 10.3, and 4.6 microM, respectively. The IC(50) value of HMF for TdT was the same as that for del-2 pol lambda (5.5 microM). The HMF-induced inhibition of both pol lambda and TdT activities was competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, HMF was suggested to bind to the pol beta-like region of pol lambda and TdT.

Inhibitory effect of tocotrienol on eukaryotic DNA polymerase lambda and angiogenesis.

Tocotrienols, vitamin E compounds that have an unsaturated side chain with three double bonds, selectively inhibited the activity of mammalian DNA polymerase lambda (pol lambda) in vitro. These compounds did not influence the activities of replicative pols such as alpha, delta, and epsilon, or even the activity of pol beta which is thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. Since delta-tocotrienol had the strongest inhibitory effect among the four (alpha- to delta-) tocotrienols, the isomer's structure might be an important factor in the inhibition of pol lambda. The inhibitory effect of delta-tocotrienol on both intact pol lambda (residues 1-575) and a truncated pol lambda lacking the N-terminal BRCA1 C-terminus (BRCT) domain (residues 133-575, del-1 pol lambda) was dose-dependent, with 50% inhibition observed at a concentration of 18.4 and 90.1microM, respectively. However, del-2 pol lambda (residues 245-575) containing the C-terminal pol beta-like region was unaffected. Tocotrienols also inhibited the proliferation of and formation of tubes by bovine aortic endothelial cells, with delta-tocotrienol having the greatest effect. These results indicated that tocotrienols targeted both pol lambda and angiogenesis as anti-cancer agents. The relationship between the inhibition of pol lambda and anti-angiogenesis by delta-tocotrienol was discussed.

Monoacetylcurcumin: a new inhibitor of eukaryotic DNA polymerase lambda and a new ligand for inhibitor-affinity chromatography.

We previously reported that a phenolic compound, curcumin (diferuloylmethane), was a selective inhibitor of DNA polymerase lambda (pol lambda) in vitro [Y. Mizushina, M. Hirota, C. Murakami, T. Ishidoh, S. Kamisuki, N. Shimazaki, M. Takemura, M. Perpelescu, M. Suzuki, H. Yoshida, F. Sugawara, O. Koiwai, K. Sakaguchi, Some anti-chronic inflammatory compounds are DNA polymerase lambda-specific inhibitors, Biochem. Pharmacol. 66 (2003) 1935-1944.]. We also found that monoacetylcurcumin ([1E,4Z,6E]-7-(4''-acetoxy-3''-methoxyphenyl)-5-hydroxy-1-(4'-hydroxy-3'-methoxyphenyl)hepta-1,4,6-trien-3-on), a chemically synthesized derivative of curcumin, was a stronger pol lambda inhibitor than curcumin, achieving 50% inhibition at a concentration of 3.9microM. Monoacetylcurcumin did not influence the activities of replicative pols such as alpha, delta, and epsilon, and showed no effect even on the activity of pol beta, the three-dimensional structure of which is thought to be highly similar to that of pol lambda. The compound-induced inhibition of pol lambda activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. Monoacetylcurcumin did not inhibit the activity of the C-terminal catalytic domain of pol lambda including the pol beta-like core, in which the BRCT motif was deleted. The compound did not influence the activities of prokaryotic pols or other mammalian DNA metabolizing enzymes such as calf primase of pol alpha, calf terminal deoxynucleotidyl transferase, human telomerase, human immunodeficiency virus type-1 reverse transcriptase, T7 RNA polymerase, T4 polynucleotide kinase, and bovine deoxyribonuclease I. Therefore, we concluded that monoacetylcurcumin is a selective inhibitor of pol lambda and could be used as a chromatographic ligand to purify pol lambda. We then made a monoacetylcurcumin-conjugated column with epoxy-activated Sepharose 6B. In the column, pol lambda of full length was selectively adsorbed and eluted.

DNA polymerase lambda directly binds to proliferating cell nuclear antigen through its confined C-terminal region.

DNA polymerase lambda (Pol lambda) was recently identified as a new member of the family X of DNA polymerases. Here, we show that Pol lambda directly binds to proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA replication and repair enzymes, both in vitro and in vivo. A pull-down assay using deletion mutants of Pol lambda showed that the confined C-terminal region of Pol lambda directly binds to PCNA. Furthermore, a synthetic peptide of 20-mers derived from the C-terminal region of Pol lambda competes with full-length Pol lambda for binding to PCNA. The residues between amino acids 518 and 537 of Pol lambda are required for binding to PCNA, and are different from the consensus PCNA interacting motif (PIM). Pol lambda associates with PCNA in vivo by immunoprecipitation analysis and EGFP-tagged Pol lambda co-localizes with PCNA as spots within a nucleus using fluorescent microscopy. Through direct binding, PCNA suppressed the distributive nucleotidyltransferase activity of Pol lambda. Pol micro, which also belongs to the family X of DNA polymerases, binds to PCNA by a pivotal amino acid residue.

Structural analysis of epolactaene derivatives as DNA polymerase inhibitors and anti-inflammatory compounds.

Epolactaene (compound 1), a neuritogenic compound found in human neuroblastoma cells, was found to show anti-inflammatory activity in vivo in this study. DNA polymerases and DNA topoisomerase II (topo II) were some of the major molecular targets of compound 1. Since the agent seems to be a potential pharmaceutical medicine, we synthesized derivatives chemically and obtained seven compounds, 1 to 7 to screen clinically more efficient epolactaene derivatives. A comparison of its structural derivatives revealed that the long alkyl side chain seemed to have an important role in the inhibitory effect. Notably, C18-alkyl chain conjugated epolactaene (compound 5) was the strongest inhibitor of DNA polymerase alpha, beta, lambda (pol alpha, beta, lambda) and topo II, with IC50 values of 13, 135, 4.4 and 5 microM, respectively, and 500 microg of compound 5 caused a marked reduction in TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation (inhibitory effect, 65.0%). Compound 5 did not influence the activities of plant or prokaryotic DNA polymerases, or of other DNA metabolic enzymes such as telomerase, RNA polymerase and deoxyribonuclease I. Based on these results, the relationship among the three-dimensional structure of epolactaene derivatives and the inhibition of polymerases and topo II, and anti-inflammation is discussed.

A biochemically defined system for mammalian nonhomologous DNA end joining.

Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs). Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions. Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice. The pol lambda and pol mu contributions require their BRCT domains and are both physically and functionally dependent on Ku. This indicates a specific biochemical function for Ku in NHEJ at incompatible DNA ends. The XRCC4:DNA ligase IV complex is able to ligate one strand that has only minimal base pairing with the antiparallel strand. This important aspect of the ligation leads to an iterative strand-processing model for the steps of NHEJ.