The Escherichia coli AlkB protein and human homologs hABH2 and hABH3 are 2-oxoglutarate (2OG)/Fe(II)-dependent DNA/RNA demethylases that repair 1-methyladenine and 3-methylcytosine residues. Surprisingly, hABH1, which displays the strongest homology to AlkB, failed to show repair activity in two independent studies. Here, we show that hABH1 is a mitochondrial protein, as demonstrated using fluorescent fusion protein expression, immunocytochemistry, and Western blot analysis. A fraction is apparently nuclear and this fraction increases strongly if the fluorescent tag is placed at the N-terminal end of the protein, thus interfering with mitochondrial targeting. Molecular modeling of hABH1 based upon the sequence and known structures of AlkB and hABH3 suggested an active site almost identical to these enzymes. hABH1 decarboxylates 2OG in the absence of a prime substrate, and the activity is stimulated by methylated nucleotides. Employing three different methods we demonstrate that hABH1 demethylates 3-methylcytosine in single-stranded DNA and RNA in vitro. Site-specific mutagenesis confirmed that the putative Fe(II) and 2OG binding residues are essential for activity. In conclusion, hABH1 is a functional mitochondrial AlkB homolog that repairs 3-methylcytosine in single-stranded DNA and RNA.

Two EIAs (Medgenix and Quantikine) and a bioassay were used to measure tumor necrosis factor-alpha (TNF alpha) in serum samples from 73 human immunodeficiency virus type 1 (HIV-1)-seropositive patients and in samples from 2 control groups. All clinical groups of HIV-1-infected patients, regardless of concurrent illness, had significantly elevated levels of both types of soluble TNF receptors (sTNFRs) and immunoreactive TNF alpha (Medgenix EIA), with the highest concentrations among the AIDS patients. These TNF parameters were significantly correlated with reduced CD4+ lymphocyte counts. Only a few HIV-1-infected patients had detectable TNF alpha levels measured by the Quantikine EIA. TNF alpha bioactivity was significantly raised only in the AIDS group. Serially measured sTNFRs, expressed as sTNFR slopes, were significantly associated with survival in the patient group. The raised levels of immunoreactive TNF alpha and sTNFRs strongly indicate activation of the TNF alpha system during HIV-1 infection. Levels increase with disease progression and degree of immunodeficiency; thus, serially measured sTNFRs may give useful prognostic information in HIV-1 infection.

Because persistent tumor necrosis factor (TNF)-alpha activation may play a pathogenic role in human immunodeficiency virus infection, TNF component levels were assessed over 78 weeks in plasma and peripheral blood mononuclear cells (PBMC) during highly active antiretroviral therapy (HAART) in 40 HIV-infected patients. HAART induced a significant decline in plasma levels of TNF-alpha and soluble TNF receptors and was associated with a fall in the abnormally increased unstimulated and a rise in the abnormally low Mycobacterium avium complex-purified-protein derivative-stimulated TNF-alpha released from PBMC. However, concentrations of these TNF components were not normalized. Patients with virologic and immunologic treatment failure after 52 weeks had higher levels of several TNF components than other patients early after initiation of therapy, also during periods with adequate virologic response. Although TNF components significantly decreased during HAART, these results support data indicating that full immunologic normalization is not achieved during such therapy. The persistent activation of the TNF system in a subgroup of persons may be involved in treatment failure.

Proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication, DNA repair, cell cycle regulation, chromatin remodeling, and epigenetics. Many proteins interact with PCNA through the PCNA interacting peptide (PIP)-box or the newly identified AlkB homolog 2 PCNA interacting motif (APIM). The xeroderma pigmentosum group A (XPA) protein, with a central but somewhat elusive role in nucleotide excision repair (NER), contains the APIM sequence suggesting an interaction with PCNA. With an in vivo based approach, using modern techniques in live human cells, we show that APIM in XPA is a functional PCNA interacting motif and that efficient NER of UV lesions is dependent on an intact APIM sequence in XPA. We show that XPA−/− cells complemented with XPA containing a mutated APIM sequence have increased UV sensitivity, reduced repair of cyclobutane pyrimidine dimers and (6–4) photoproducts, and are consequently more arrested in S phase as compared to XPA−/− cells complemented with wild type XPA. Notably, XPA colocalizes with PCNA in replication foci and is loaded on newly synthesized DNA in undamaged cells. In addition, the TFIIH subunit XPD, as well as XPF are loaded on DNA together with XPA, and XPC and XPG colocalize with PCNA in replication foci. Altogether, our results suggest a presence of the NER complex in the vicinity of the replisome and a novel role of NER in post-replicative repair.