DNA polymerase mu, a candidate hypermutase?

A novel DNA polymerase (Pol mu) has been recently identified in human cells. The amino-acid sequence of Pol mu is 42% identical to that of terminal deoxynucleotidyl transferase (TdT), a DNA-independent DNA polymerase that contributes to antigen-receptor diversity. In this paper we review the evidence supporting the role of Pol mu in somatic hypermutation of immunoglobulin genes, a T-dependent process that selectively occurs at germinal centres: (i) preferential expression in secondary lymphoid organs; (ii) expression associated to developing germinal centres; and (iii) very low base discrimination during DNA-dependent DNA polymerization by Pol mu, a mutator phenotype enormously accentuated by the presence of activating Mn2+ ions. Moreover, its similarity to TdT, together with extrapolation to the crystal structure of DNA polymerase beta complexed (Pol beta) with DNA, allows us to discuss the structural basis for the unprecedented error proneness of Pol mu, and to predict that Pol mu is structurally well suited to participate also in DNA end-filling steps occurring both during V(D)J recombination and repair of DNA double-strand breaks that are processed by non-homologous end-joining.

DNA polymerase mu (Pol mu), homologous to TdT, could act as a DNA mutator in eukaryotic cells.

A novel DNA polymerase has been identified in human cells. Human DNA polymerase mu (Pol mu), consisting of 494 amino acids, has 41% identity to terminal deoxynucleotidyltransferase (TdT). Human Pol mu, overproduced in Escherichia coli in a soluble form and purified to homogeneity, displays intrinsic terminal deoxynucleotidyltransferase activity and a strong preference for activating Mn(2+) ions. Interestingly, unlike TdT, the catalytic efficiency of polymerization carried out by Pol mu was enhanced by the presence of a template strand. Using activating Mg(2+) ions, template-enhanced polymerization was also template-directed, leading to the preferred insertion of complementary nucleotides, although with low discrimination values. In the presence of Mn(2+) ions, template-enhanced polymerization produced a random insertion of nucleotides. Northern-blotting and in situ analysis showed a preferential expression of Pol mu mRNA in peripheral lymphoid tissues. Moreover, a large proportion of the human expressed sequence tags corresponding to Pol mu, present in the databases, derived from germinal center B cells. Therefore, Pol mu is a good candidate to be the mutator polymerase responsible for somatic hyper- mutation of immunoglobulin genes.

Expression of IkappaBalpha in the nucleus of human peripheral blood T lymphocytes.

According to current models the inhibitory capacity of I(kappa)B(alpha) would be mediated through the retention of Rel/NF-kappaB proteins in the cytosol. However, I(kappa)B(alpha) has also been detected in the nucleus of cell lines and when overexpressed by transient transfection. To gain better insight into the potential role of nuclear I(kappa)B(alpha) in a physiological context we have analysed its presence in the nucleus of human peripheral blood T lymphocytes (PBL). We demonstrate the nuclear localization of I(kappa)B(alpha) in PBL by different techniques: Western blot, indirect immunofluorescence and electron microscopy. Low levels of nuclear I(kappa)B(alpha) were detected in resting cells whereas a superinduction was obtained after PMA activation. The nuclear pool of I(kappa)B(alpha) showed a higher stability than cytosolic I(kappa)B(alpha) and was partially independent of the resynthesis of the protein. Unexpectedly, the presence of nuclear I(kappa)B(alpha) did not inhibit NF-kappaB binding to DNA and this phenomenon was not due to the presence of IkappaBbeta at the nuclear level. Immunoprecipitation experiments failed to demonstrate an association between nuclear I(kappa)B(alpha) and NF-kappaB proteins. Our results demonstrate that in resting and PMA-activated human PBL, I(kappa)B(alpha) is present in the nucleus in an apparently inactive form unable to disrupt NF-kappaB binding from DNA.

LTR and tat variability of HIV-1 isolates from patients with divergent rates of disease progression.

The genetic heterogeneity and transcription activity of the human immunodeficiency virus type 1 (HIV-1) LTR region and tat gene have been examined. Comparison involved the relevant genomic regions of viruses isolated from twenty long-term survivors and from ten typical progressors. No significant differences were observed in mutation frequencies among the two groups, although there was a significant higher proportion of synonymous substitutions in the tat gene of viruses from typical progressors. Four LTR sequences showed an insertion of 20-31 residues at the junction between the LTR Nef-coding and the LTR noncoding region. Neither these insertions nor other genetic changes found in these sequences affected the LTR transcription function, as measured in transient expression assays using transfection of both established cell lines and peripheral blood lymphocytes with plasmid DNA. The results did not allow the association of structural or functional alterations in LTR or tat with a degree of disease progression. The results reinforce the concepts of complexity of HIV-1 evolution in infected individuals, and the multifactorial nature of progression to AIDS.

In vitro selective elimination of HIV-infected cells from peripheral blood in AIDS patients by the immunotoxin DAB389CD4.

OBJECTIVES: To study the antiviral efficacy of the recombinant immunotoxin DAB389CD4 against wild-type strains of HIV and to analyse its potential toxicity in non-infected peripheral blood mononuclear cells (PBMC). DESIGN AND METHODS: PBMC from HIV-seropositive patients were cultured in the presence of DAB389CD4. After 30 days in culture, viral load was assessed by quantification of RNA levels in supernatants and HIV-specific polymerase chain reaction (PCR) was performed for measuring proviral DNA as an indicator of remaining virus in cells. To study the toxicity of DAB389CD4, PBMC from healthy donors were isolated and cell viability and lymphocyte proliferation were assessed after immunotoxin treatment. RESULTS: DAB389CD4 presented a strong antiviral activity in five of the six primary isolates decreasing p24 production in cultures to undetectable levels and eliminating selectively HIV-infected cells as measured by HIV DNA-specific PCR. One viral isolate was resistant to DAB389CD4 treatment. The immunotoxin was active against both syncytial and non-syncytial HIV strains. DAB389CD4 was not toxic in non-infected PBMC as measured by different techniques: trypan blue exclusion, methyl thiazol tetrazolium oxidation, lymphocyte proliferation, and CD4 cell count. CONCLUSIONS: DAB389CD4 showed a strong antiviral and specific activity against primary HIV isolates by killing selectively HIV-infected cells without affecting non-infected cells. This antiviral effect produced the eradication of HIV in cultures and indicated the potential use of this drug as a new therapeutic tool in combination with antiretroviral drugs. This immunotoxin would be especially interesting in the context of the marginal populations of HIV-infected cells remaining after successful antiviral treatment.

Absolute dependence on kappa B responsive elements for initiation and Tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes.

The role of NF-kappa B-dependent signals in activating the transcriptional activity of the HIV regulatory region (LTR) was analyzed by systematic comparison of HIV LTR activity in human CD4 T cells purified from peripheral blood and a transformed lymphoblastoid T cell line. In normal CD4 T cells we also analyzed the role played by the viral kappa B responsive elements in HIV replication. Analysis of nuclear extracts of resting, normal T lymphocytes revealed the presence of the p50, but not the p65, NF-kappa B subunit and the induction by phorbol esters of bona fide (p50-p65) NF-kappa B complexes. In parallel, we observed clear enhancer-dependent HIV LTR transactivation comparable in intensity with that observed in lymphoblastoid cells. We show that unstimulated CD4 T lymphocytes offer a cellular environment of very low permissivity to HIV LTR functioning. This was in sharp contrast to the high spontaneous LTR activity observed in lymphoblastoid T cells, where LTR activity was essentially independent of kappa B-responsive elements. Due to the low basal LTR activity in resting T lymphocytes, NF-kappa B-dependent transactivation was a sine qua non event for induction of the HIV LTR. Surprisingly, even the function of HIV Tat in resting CD4 T lymphocytes was found to be absolutely dependent on LTR kappa B responsive elements. The relevance of these observations obtained in transient transfections was confirmed by the incapacity of blood CD4 T lymphocytes infected with an HIV infectious provirus carrying critical point mutations in the kappa B responsive elements to show any detectable transcriptional activity upon cell activation and prolonged culture in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)