So Maezawa

BPOZ-2 directly binds to eEF1A1 to promote eEF1A1 ubiquitylation and degradation and prevent translation.

Bood POZ containing gene type 2 (BPOZ‐2), which contains ankyrin repeats, NLS, BTB/POZ domains and LXXLL motifs, is an adaptor protein for the E3 ubiquitin ligase scaffold protein CUL3. We isolated a cDNA encoding eukaryotic elongation factor 1A1 (eEF1A1) as a BPOZ‐2 binding protein by screening a human thymus cDNA library using a yeast two‐hybrid system. eEF1A1 is essential for translation and is also involved in the 26S proteasome‐dependent degradation of misfolded or unfolded proteins. The binding between BPOZ‐2 and eEF1A1 was confirmed by pull‐down and immunoprecipitation assays in vitro and in vivo, respectively. BPOZ‐2 binds to eEF1A1 through the ankyrin repeats and both BTB/POZ domains in BPOZ‐2 and Domains I and III in eEF1A1. BPOZ‐2 and eEF1A1 over‐expressed in HEK 293T cells co‐localized as speckles within the cytoplasm. BPOZ‐2 promoted eEF1A1 ubiquitylation and degradation, suggesting that eEF1A1 is a substrate of BPOZ‐2. BPOZ‐2 inhibited GTP binding to eEF1A1 and prevented translation in in vitro translation assay using rabbit reticulocytes.

Identification of functional domains in TdIF1 and its inhibitory mechanism for TdT activity

TdT interacting factor 1 (TdIF1) was identified as a protein that binds to terminal deoxynucleotidyltransferase (TdT) to negatively regulate TdT activity. TdT is a template‐independent DNA polymerase that catalyzes the incorporation of deoxynucleotides to the 3′‐hydroxyl end of DNA templates to increase the junctional diversity of immunoglobulin or T‐cell receptor (TcR) genes. Here, using bioinformatics analysis, we identified the TdT binding, DNA binding and dimerization regions, and nuclear localization signal (NLS) in TdIF1. TdIF1 bound to double‐stranded DNA (dsDNA) through three DNA binding regions: residues 1–75, the AT‐hook‐like motif (ALM) and the predicted helix‐turn‐helix (HTH) motif. ALM in TdIF1 preferentially bound to AT‐rich DNA regions. NLS was of the bipartite type and overlapped ALM. TdIF1 bound to the Pol β‐like region in TdT and blocked TdT access to DNA ends. In the presence of dsDNA, however, TdIF1 bound to dsDNA to release TdT from the TdIF1/TdT complex and to exhibit TdT activity, implying that active TdT released microenvironmentally concentrates around AT‐rich DNA to synthesize DNA.

Bood POZ containing gene type 2 is a human counterpart of yeast Btb3p and promotes the degradation of terminal deoxynucleotidyltransferase

Bood POZ containing gene type 2 (BPOZ‐2) is involved in the growth suppressive effect of the phosphatase and tensin homologue (PTEN). We showed that BPOZ‐2 is a human counterpart of yeast Btb3p, which is a putative adaptor for Pcu3p‐based ubiquitin ligase. BPOZ‐2 bound to E3 ligase CUL3 in vitro and in vivo. BPOZ‐2 itself was ubiquitinated through the CUL3‐based E3 ligase mainly within the nucleus and degraded by the 26S proteasome. Although BPOZ‐2 was mainly expressed within the cytoplasm, it accumulated within the nucleus in the presence of the specific 26S proteasome inhibitor MG132. BPOZ‐2 may be recruited to the nucleus from the cytoplasm. Terminal deoxynucleotidyltransferase (TdT) was detected as a BPOZ‐2‐binding protein using a yeast two‐hybrid system by screening a human thymus cDNA library. TdT, BPOZ‐2, and CUL3 formed a ternary complex in vivo. TdT was ubiquitinated only within the nucleus and degraded by the 26S proteasome. The ubiqutination or degradation of TdT was markedly promoted by co‐expression of BPOZ‐2 and CUL3 or BPOZ‐2 in 293T cells, respectively.

BRCT domain of DNA polymerase μ has DNA-binding activity and promotes the DNA polymerization activity.

DNA polymerase μ (pol μ) catalyzes nonhomologous end‐joining in DNA double‐stranded break repair. Pol μ consists of an amino‐terminal BRCA1 carboxyl‐terminal homology (BRCT) domain and a pol β‐like region, which contains the catalytic site. By DNA cellulose column chromatography, using full‐length pol μ and five different deletion mutants, we found that the amino‐terminal region has double‐stranded DNA (dsDNA)‐binding activity. Pol μ without BRCT domain reduces the DNA polymerization activity when compared to full‐length pol μ. Observation by atomic force microscopy showed that full‐length pol μ binds to the ends and middle part of dsDNA. Pol μ lacking the amino‐terminal region or with a mutation within the BRCT domain bound only to DNA ends, whereas the amino‐terminal region with the BRCT domain bound to both the ends and the middle part of dsDNA (mpdDNA). Terminal deoxynucleotidyltransferase, which, like pol μ, belongs to the X family DNA polymerases, also bound to mpdDNA through its amino‐terminal region.

TdIF2 is a nucleolar protein that promotes rRNA gene promoter activity

Terminal deoxynucleotidyltransferase (TdT) interacting factor 2 (TdIF2) is an acidic protein that binds to TdT. TdIF2 binds to DNA and core histones and contains an acidic‐amino acid‐rich region in its C‐terminus. It has therefore been suggested to function as a histone chaperone within the nucleus. TdIF2 localized within the nucleolus in HEK 293T cells, and its N‐terminal (residues 1–234) and C‐terminal (residues 606–756) regions were crucial for the nucleolar localization. A chromatin immunoprecipitation (ChIP) assay showed that TdIF2 associated with the promoter of human ribosomal RNA genes (hrDNAP), and an in vitro luciferase assay system showed that it promoted hrDNAP activity. Using the yeast two‐hybrid system with TdIF2 as the bait, we isolated the cDNA encoding HIV Tat interactive protein 60 (Tip60), which has histone acetyltransferase (HAT) activity, as a TdIF2‐binding protein. TdIF2 bound to Tip60 in vitro and in vivo, inhibited the Tip60 HAT activity in vitro and co‐localized with Tip60 within the nucleolus. In addition, TdIF2 promotes upstream binding factor (UBF) acetylation in vivo. Thus, TdIF2 might promote hrDNAP activity by suppressing Tip60’s HAT activity and promoting UBF acetylation.

TdT interacting factor 1 enhances TdT ubiquitylation through recruitment of BPOZ-2 into nucleus from cytoplasm

We isolated human cDNA clone encoding Bood POZ containing gene type 2 (BPOZ‐2) as a gene with a product that binds to TdT interacting factor 1 (TdIF1) using a yeast two‐hybrid system. BPOZ‐2 is an adaptor for E3 ligase CUL3 and participates in developmental processes. The binding between BPOZ‐2 and TdIF1 was confirmed by GST pull‐down and immunoprecipitation assays using specific antibodies against BPOZ‐2 and TdIF1 in vitro and in vivo. Although when BPOZ‐2 solely was expressed in COS7 cells, BPOZ‐2 was observed mainly within the cytoplasm, co‐transfection of pEGFP‐BPOZ‐2 and pDsRed‐TdIF1 into COS7 cells resulted in co‐localization of EGFP‐BPOZ‐2 and DsRed‐TdIF1 within the nucleus. TdIF1 may recruit BPOZ‐2 into the nucleus from the cytoplasm by directly binding to BPOZ‐2. BPOZ‐2 enhanced TdT ubiquitylation when TdIF1 was expressed together with BPOZ‐2 in 293T cells, strongly suggesting that the recruitment of BPOZ‐2 into the nucleus from the cytoplasm is significant for the TdT ubiquitylation within the nucleus.

Truncated UDP-glucuronosyltransferase (UGT) from a Crigler–Najjar syndrome type II patient colocalizes with intact UGT in the endoplasmic reticulum

Mutations in the gene encoding bilirubin UDP-glucuronosyltransferase (UGT1A1) are known to cause Crigler–Najjar syndrome type II (CN-II). We previously encountered a patient with a nonsense mutation (Q331X) on one allele and with no other mutations in the promoter region or other exons, and proposed that CN-II is inherited as a dominant trait due to the formation of a heterologous subunit structure comprised of the altered UGT1A1 gene product (UGT1A1-p.Q331X) and the intact UGT1A1. Here, we investigated the molecular basis of CN-II in this case by expressing UGT1A1-p.Q331X in cells. UGT1A1-p.Q331X overexpressed in Escherichia coli or mammalian cells directly bound or associated with intact UGT1A1 in vitro or in vivo, respectively. Intact UGT1A1 was observed as a dimer using atomic force microscopy. Fluorescent-tagged UGT1A1-p.Q331X and intact UGT1A1 were colocalized in 293T cells, and fluorescence recovery after photobleaching analysis showed that UGT1A1-p.Q331X was retained in the endoplasmic reticulum (ER) without rapid degradation. These findings support the idea that UGT1A1-p.Q331X and UGT1A1 form a dimer and provide an increased mechanistic understanding of CN-II.

Ubiquitylation of Terminal Deoxynucleotidyltransferase Inhibits Its Activity

Terminal deoxynucleotidyltransferase (TdT), which template-independently synthesizes DNA during V(D)J recombination in lymphoid cells, is ubiquitylated by a BPOZ-2/Cul3 complex, as the ubiquitin ligase, and then degraded by the 26 S proteasome. We show here that TdT is ubiquitylated by the Cul3-based ubiquitylation system in vitro. Because TdT could also be ubiquitylated in the absence of Cul/BPOZ-2, we determined that it could also be directly ubiquitylated by the E2 proteins UbcH5a/b/c and UbcH6, E3-independently. Furthermore, the ubiquitylated TdT inhibited its nucleotidyltransferase activity.

Double-strand break repair based on short-homology regions is suppressed under terminal deoxynucleotidyltransferase expression, as revealed by a novel vector system for analysing DNA repair by nonhomologous end joining.

We have constructed a novel, nonhomologous end‐joining (NHEJ) assay vector (NAV), containing mKate2, Venus and ccdB genes. Cotransfection of NAV with a construct expressing the restriction enzyme I‐SceI generated a double‐strand break (DSB) in NAV that excised mKate2 and ccdB. Repair of this DSB produced an intact vector that expressed Venus, a green fluorescent protein. Because cells bearing the repaired NAV lacked the ccdB gene which slows cell proliferation, the cultures were enriched in cells containing repaired DSBs. DNA sequence analysis of the DSB junctions indicated that the repair was carried out mainly by using the closest homology sequence. Use of the NAV yielded rapid results within 3 days after transfection. We then used the NAV to analyse NHEJ in cells overexpressing terminal deoxynucleotidyltransferase (TdT). The results indicated that TdT suppresses DNA repair that is based on short (one‐ or two‐base) homology regions, to efficiently add deoxynucleotides during VDJ recombination in lymphoid cells.