Noriko Shimazaki

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.

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.

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.

A sulphoquinovosyl diacylglycerol is a DNA polymerase e inhibitor

Sulphoquinovosyl diacylglycerol (SQDG) was reported as a selective inhibitor of eukaryotic DNA polymerases alpha and beta [Hanashima, Mizushina, Ohta, Yamazaki, Sugawara and Sakaguchi (2000) Jpn. J. Cancer Res. 91, 1073-1083] and an immunosuppressive agent [Matsumoto, Sahara, Fujita, Shimozawa, Takenouchi, Torigoe, Hanashima, Yamazaki, Takahashi, Sugawara et al. (2002) Transplantation 74, 261-267]. The purpose of this paper is to elucidate the biochemical properties of the inhibition more precisely. As expected, SQDG could inhibit the activities of mammalian DNA polymerases such as alpha, delta, eta and kappa in vitro in the range of 2-5 micro M, and beta and lambda in vitro in the range of 20-45 micro M. However, SQDG could inhibit only mammalian DNA polymerases epsilon (pol epsilon) activity at less than 0.04 micro M. SQDG bound more tightly to mammalian pol epsilon than the other mammalian polymerases tested. Moreover, SQDG could inhibit the activities of all the polymerases from animals such as fish and insect, but not of the polymerases from plant and prokaryotes. SQDG should, therefore, be called a mammalian pol epsilon-specific inhibitor or animal polymerase-specific inhibitor. To our knowledge, this represents the first report about an inhibitor specific to mammalian pol epsilon.

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 λ (pol λ) 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 λ (full‐length, i.e. intact pol λ including the BRCA1 C‐ terminal [BRCT] domain) by some derivatives was stronger than that by curcumin, and monoacetylcurcumin (compound 13) was the strongest pol λ inhibitor of all the compounds tested, achieving 50% inhibition at a concentration of 3.9 µm. The compound did not influence the activities of replicative pols such as α, δ, and ɛ. It had no effect on pol β activity either, although the three‐dimensional structure of pol β is thought to be highly similar to that of pol λ. Compound 13 did not inhibit the activity of the C‐terminal catalytic domain of pol λ including the pol β‐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 λ, but did not bind to the C‐terminal region. Based on these results, the pol λ‐inhibitory mechanism of compound 13 is discussed.

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.

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

DNA polymerase lambda (Pol λ) was recently identified as a new member of the family X of DNA polymerases. Here, we show that Pol λ 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 λ showed that the confined C‐terminal region of Pol λ directly binds to PCNA. Furthermore, a synthetic peptide of 20‐mers derived from the C‐terminal region of Pol λ competes with full‐length Pol λ for binding to PCNA. The residues between amino acids 518 and 537 of Pol λ are required for binding to PCNA, and are different from the consensus PCNA interacting motif (PIM). Pol λ associates with PCNA in vivo by immunoprecipitation analysis and EGFP‐tagged Pol λ co‐localizes with PCNA as spots within a nucleus using fluorescent microscopy. Through direct binding, PCNA suppressed the distributive nucleotidyltransferase activity of Pol λ. Pol µ, which also belongs to the family X of DNA polymerases, binds to PCNA by a pivotal amino acid residue.

A histidine in the β-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.

β-Sitosterol-3-O-β-D-glucopyranoside : A eukaryotic DNA polymerase λ 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.

Terminal deoxynucleotidyltransferase forms a ternary complex with a novel chromatin remodeling protein with 82 kDa and core histone

Background: Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances the Ig and TcR gene diversity in the N region at the junctions of variable (V), diversity (D) and joining (J) segments in B‐ and T‐cells. TdT synthesizes the N region in concert with many proteins including DNA‐PKcs, Ku70 and Ku86. To elucidate the molecular mechanism of the N region synthesis, we first attempted to isolate the genes with products that directly interact with TdT.