Association of terminal deoxynucleotidyl transferase with Ku.

Terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of nucleotides at the junctions of rearranging Ig and T cell receptor gene segments, thereby generating antigen receptor diversity. Ku is a heterodimeric protein composed of 70- and 86-kDa subunits that binds DNA ends and is required for V(D)J recombination and DNA double-strand break (DSB) repair. We provide evidence for a direct interaction between TdT and Ku proteins. Studies with a baculovirus expression system show that TdT can interact specifically with each of the Ku subunits and with the heterodimer. The interaction between Ku and TdT is also observed in pre-T cells with endogenously expressed proteins. The protein-protein interaction is DNA independent and occurs at physiological salt concentrations. Deletion mutagenesis experiments reveal that the N-terminal region of TdT (131 amino acids) is essential for interaction with the Ku heterodimer. This region, although not important for TdT polymerization activity, contains a BRCA1 C-terminal domain that has been shown to mediate interactions of proteins involved in DNA repair. The induction of DSBs in Cos-7 cells transfected with a human TdT expression construct resulted in the appearance of discrete nuclear foci in which TdT and Ku colocalize. The physical association of TdT with Ku suggests a possible mechanism by which TdT is recruited to the sites of DSBs such as V(D)J recombination intermediates.

Nucleotide pool imbalance and adenosine deaminase deficiency induce alterations of N-region insertions during V(D)J recombination.

Template-independent nucleotide additions (N regions) generated at sites of V(D)J recombination by terminal deoxynucleotidyl transferase (TdT) increase the diversity of antigen receptors. Two inborn errors of purine metabolism, deficiencies of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), result in defective lymphoid development and aberrant pools of 2'-deoxynucleotides that are substrates for TdT in lymphoid precursors. We have asked whether selective increases in dATP or dGTP pools result in altered N regions in an extrachromosomal substrate transfected into T-cell or pre-B-cell lines. Exposure of the transfected cells to 2'-deoxyadenosine and an ADA inhibitor increased the dATP pool and resulted in a marked increase in A-T insertions at recombination junctions, with an overall decreased frequency of V(D)J recombination. Sequence analysis of VH-DH-JH junctions from the IgM locus in B-cell lines from ADA-deficient patients demonstrated an increase in A-T insertions equivalent to that found in the transfected cells. In contrast, elevation of dGTP pools, as would occur in PNP deficiency, did not alter the already rich G-C content of N regions. We conclude that the frequency of V(D)J recombination and the composition of N-insertions are influenced by increases in dATP levels, potentially leading to alterations in antigen receptors and aberrant lymphoid development. Alterations in N-region insertions may contribute to the B-cell dysfunction associated with ADA deficiency.

Initiation of transcription at the human terminal deoxynucleotidyl transferase gene promoter: a novel role for the TATA binding protein.

Control of initiation of transcription of the human terminal deoxynucleotidyl transferase (TdT) gene was investigated by using an in vitro transcription assay. The precise contribution of discrete basal promoter elements to transcription initiation was determined by testing deletion and substitution mutations. The primary element, contained within the region spanning -34 to -14 bp relative to the transcription start site, accounted for 80% of basal promoter activity. TdT promoter activity required the sequence ACCCT at -24 to -20 bp since a dramatic decrease in transcription initiation was observed after mutation of this sequence, whereas mutation of the adjacent sequence from -32 to -25 bp did not alter promoter activity. The secondary element contained sequences surrounding the transcription start site and had 20% of promoter activity. Deletion of both elements completely abolished transcription initiation. Initiator characteristics of the secondary element were revealed by using the in vitro assay: promoter sequences at the transcription start site were sufficient to direct accurate initiation at a single site. Mutation of the sequence GGGTG spanning the transcription start site resulted in loss of transcription initiation. Both the primary and secondary elements were nonhomologous to corresponding regions from the mouse TdT gene promoter. While the human basal promoter functioned in the absence of TATA consensus sequences or GC-rich SP1 binding sites, it was dependent on active TFIID. In contrast to other TATA-less promoters, purified TATA binding protein substituted for the TFIID complex and restored promoter activity to TFIID-inactivated nuclear extracts.

Identification of a tripartite basal promoter which regulates human terminal deoxynucleotidyl transferase gene expression.

In order to locate the promoter region of the human terminal deoxynucleotidyl transferase gene, serially truncated segments of the 5'-flanking region of the gene were cloned into a chloramphenicol acetyltransferase reporter vector. Transient transfection analyses of the terminal transferase-reporter gene constructs identified the basal promoter region within -34 to +40 base pairs relative to the transcription start site. Three promoter elements were defined in this region. The primary element is within 34 base pairs upstream of the transcription start site. The CAP site is 62 base pairs upstream of the translation start site. The secondary element involves sequences around the transcription start site. The third is located 25 base pairs downstream from the initiation site (+25 to +40). This tripartite basal promoter was not tissue specific; similar patterns of promoter activity were observed in terminal transferase expressing and non-expressing cells. Transfection analyses also indicated the presence of negative regulatory elements upstream of the basal promoter region, and these elements were preferentially active in cells expressing terminal transferase.

Inhibition of reporter gene expression in mammalian cells. Effects of distinct carcinogen lesions in DNA.

The effect of UV photoproducts or benzo[a]pyrene-diol-epoxide-I (BPDE-I) adducts in DNA on the transient expression of a reporter gene was measured in mammalian cells. The plasmid pRSVCAT was UV irradiated or treated with BPDE-I in vitro and co-transfected with undamaged pRSVBGAL into mouse and human fibroblasts. Variations in transfection efficiency among different cell lines were corrected by adjusting the volumes of cell extracts used in the chloramphenicol acetyl transferase (CAT) assays to contain equal beta-galactosidase (BGAL) activity. The expression of the CAT gene was found to decrease exponentially after transfection of pRSVCAT containing increasing numbers of DNA lesions per molecule. The average number of BPDE-I adducts per plasmid molecule was measured by ELISA; the average number of pyrimidine dimers was estimated from the dose kinetics for the disappearance of the supercoiled form of irradiated plasmid DNA treated with Micrococcus luteus UV endonuclease. By expressing the inhibition of CAT activity in terms of the average number of lesions per gene, we were able to compare directly the effects of two different carcinogen lesions on transient transcription. We observed comparable kinetics of inhibition of gene expression by BPDE-I adducts and pyrimidine dimers in DNA. D0 values determined by linear regression analysis of dose-response curves for inhibition of CAT activity were 4.9 BPDE-I adducts or 6.6 pyrimidine dimers per gene in excision-proficient human fibroblasts; the corresponding values in mouse cells were 4.4 BPDE-I adducts or 5.5 pyrimidine dimers. Similar threshold densities of BPDE-I adducts and pyrimidine dimers were observed before inhibition of transcription from pRSVCAT was detected. No threshold was observed in experiments with human fibroblasts deficient in excision repair (xeroderma pigmentosum group A); calculated D0 values were 1.2 pyrimidine dimers of 2.1 BPDE-I adducts. Our results permit direct comparisons of the magnitude of inhibition of gene transcription by distinct DNA lesions, and suggest that BPDE-I adducts and UV-induced cyclobutane pyrimidine dimers in template DNA block transcription with similar efficacy.

Timing of replication of differentially transcribed genes in Syrian hamster embryo fibroblasts.

Syrian hamster embryo cell lines have been used as models of neoplastic progression in vitro. Changes in phenotype and biological properties have been observed in these cell lines in association with modulation of gene transcription. We explored this natural evolution of cells in culture to investigate the reported relationship between transcriptional activity and replication in early S phase. In this study we used two nontumorigenic cell lines that had either retained (supB+) or lost (supB-) the ability to suppress tumorigenicity of malignantly transformed hamster fibroblasts (BP6T). In association with the loss of suppressor gene function, supB- cells have downregulated the expression of the H19 and tropomyosin-I (TM-I) genes, which are actively transcribed in supB+ cells. Synchronous populations of supB+ and supB- cells were pulse-labeled with [3H]thymidine and bromodeoxyuridine at 1-h intervals during S phase; the replicating DNA was isolated by centrifugation in cesium chloride gradients and hybridized to 32P-labeled gene probes. No correlation was found between the timing of gene replication and the status of expression of these two genes. TM-I replicated during the first hour and H19 replicated between the second and third hours of the S phase in the expressing and nonexpressing cell lines. Immunoglobulin gene sequences, known to be late-replicating in fibroblasts, replicated at the end of the S phase. These results suggest that downregulation of transcription is not always accompanied by a concomitant change in time of gene replication from early to late S phase.

Gene replication in the presence of aphidicolin.

DNA replication in the nucleus of eukaryotic cells is restricted to the S phase of the cell cycle, and different genes are duplicated at specific times, according to a well-defined temporal order. We have investigated whether activation of initiation sites, in proximity to genes that are replicated in different portions of the S phase, could be detected when synchronized 10T1/2 cells were maintained in aphidicolin (APC), an inhibitor of DNA polymerases alpha and delta. Cells released from confluence arrest into medium containing 2 micrograms/mL APC progressed into the S phase, and nascent DNA accumulated during incubations of 24 and 32 h. Exposure to APC for 40 or 48 h resulted in growth of the radiolabeled DNA into larger molecules. Replicating DNA was isolated in CsCl gradients and probed with 32P-labeled gene probes for early-replicating genes (e.g., Ha-ras, mos, and myc) and a late-replicating gene (VH Ig). DNA replicated during the 24-h incubation in APC was enriched in Ha-ras gene sequences. The VH Ig gene did not replicate in cells incubated for as long as 56 h with APC. The myc and the mos genes were detected after 32 and 40 h in APC, respectively. The myc gene is replicated in 10T1/2 cells after Ha-ras but before mos. Therefore, the order of activation of these genes was conserved in the presence of APC. The delay in replication of myc and mos correlated well with the slowing of DNA replication by APC.

Pretreatment of primary rat cutaneous epidermal keratinocyte culture with a low concentration of MNNG: effect on DNA cross-linking measured in situ after challenge with bis-2-chloroethyl sulfide.

Bis-2-chloroethyl sulfide- (BCES-) induced DNA cross-links in confluent, primary cultures of newborn rat cutaneous epidermal keratinocytes were detected using an assay that includes in situ unwinding of the DNA followed by separation of single-stranded DNA and double-stranded DNA (DSDNA) with hydroxylapatite. DNA cross-links in BCES-challenged cultures were inferred from increases in the percentage of DNA that remained double-stranded, compared with control cultures, after a 60-min alkaline unwinding incubation. The amount of DNA cross-linking after 5 or 10 microM BCES was increased when keratinocytes were first pretreated with 0.05 microM MNNG for 1 h at 8 a.m., 2 p.m., and 8 p.m. for two consecutive days and challenged with BCES the following morning. This increase was statistically significant (p less than .05, by ANOVA). For example, after 5 microM BCES challenge, cultures not pretreated with MNNG had 114.14% control DSDNA, whereas MNNG pretreated cultures had 122.78% control DSDNA. The level of BCES-induced cross-linking was maximal immediately after 30-min challenge and decreased during postchallenge incubation. At 24 and 48 h post 5, 10, or 20 microM BCES challenge, the level of DSDNA was actually depressed below unchallenged levels. This postchallenge decrease in the level of DSDNA, indicative of SSB in DNA, suggests repair activity by glycosylases and endonucleases. However, completion of repair (i.e., a return to control levels of DSDNA) was not seen in these experiments. The activity that resulted in decreases in the level of DSDNA during postchallenge incubation response was unaffected by MNNG pretreatment.