The amino-terminal domain of HIV-2 integrase has a remarkable hybrid structure combining features of a three-helix bundle fold with a zinc-binding HHCC motif. This structure shows no similarity with any of the known zinc-finger structures. The strictly conserved residues of the HHCC motif of retroviral integrases are involved in metal coordination, whereas many other well conserved hydrophobic residues are part of the protein core.

Transcriptional regulation of vertebrate histone genes during the cell cycle is mediated by several factors interacting with a series of cis-acting elements located in the 5' regions of these genes. The arrangement of these promoter elements is different for each gene. However, most histone H4 gene promoters contain a highly conserved sequence immediately upstream of the TATA box (H4 subtype consensus sequence), and this region in the human H4 gene F0108 is involved in cell cycle control. The sequence-specific interaction of nuclear factor HiNF-D with this key proximal promoter element of the H4-FO108 gene is cell cycle regulated in normal diploid cells (J. Holthuis, T. A. Owen, A. J. van [5825][5826][5827][5828][5829][5830][5831] 1991). The H4.A gene fails to interact with factors HiNF-M and HiNF-D owing to two independent sets of specific nucleotide variants, indicating differences in protein-DNA interactions between these H4 genes. Cytosine methylation of a highly conserved CpG dinucleotide interferes with binding of HiNF-P/H4TF-2 to both the H4-FO108 and H4.A promoters, but no effect is observed for either HiNF-M or HiNF-D binding to the H4-FO108 gene. Thus, strong evolutionary conservation of the H4 consensus sequence may be related to combinatorial interactions involving overlapping and interdigitated recognition nucleotides for several proteins, whose activities are regulated independently. Our results also suggest molecular complexity in the transcriptional regulation of distinct human H4 genes.Stringent cell cycle regulation (41) of S-phase-related genes, including thymidine kinase, thymidylate synthase, dihydrofolate reductase, and histone genes, occurs at many transcriptional and posttranscriptional levels (reviewed in reference 22). Functional redundancy of these multilevel gene regulatory processes ensures a tight coupling between availability of enzymes required for DNA synthesis, histone proteins, and DNA replication in proliferating cells. The stringency with which control of gene expression is mediated is abrogated during neoplastic transformation, which may ultimately affect diverse transcriptional components regulating S-phase-related genes. For example, the interactions of nuclear factor Yi (14) with the thymidine kinase promoter and of nuclear factor HiNF-D (64) with the histone H4 promoter are regulated differently in several normal diploid and tumor cells (4, 23). HiNF-D is a proliferation-specific DNA-binding activity (64), and its nuclear abundance is cell cycle regulated in normal diploid cells (23 which is located in a histone gene cluster at chromosomal region 1q21 (1, 54). This gene is expressed in a cell cycledependent manner both as an endogenous gene in human cells (44) and when introduced as an episomal gene into murine cells (16). The 5' region of this gene mediates cell cycle-regulated transcription in cell culture (44), contributes to proliferation-specific expression in transgenic mice (58), and contains two in vivo domains of protein-DNA interactions (designated H4 site I...

The integrase protein (IN) of human immunodeficiency virus type 1 removes two nucleotides from both 3' ends of the viral DNA (donor cleavage) and subsequently couples the newly generated 3' OH groups to phosphates in the target DNA (integration). The sequence requirements of IN for cleavage as well as for integration of viral DNA substrates have previously been studied by mutational analyses and by adduct interference assays. We extended these studies by analysis of heteroduplex oligonucleotide substrates and by missing-base analysis. We found for some base pairs that mutation of only one of the two bases and not the other affected IN activity. These base pairs center around the cleavage site. Besides donor cleavage and integration, IN can also perform "intermolecular disintegration," which has been described as the reversal of the integration reaction. We found that this reaction is independent of viral DNA sequences. In addition, the optimum spacing between the integration sites in intermolecular disintegration does not reflect the spacing found in vivo. These results indicate that this reaction is not the exact reversal of integration but rather is a sequence-independent phosphoryl transfer reaction between gapped DNA duplex molecules.

X-ray absorption spectroscopy (XAS), including extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) analysis, has been carried out at the ZnKedge of the N-terminal part of the integrase protein of the human immunodeficiency virus, type 2 (HIV-2), and of some zinc coordination compounds. In the presence of excess β-mercaptoethanol, which was present in the NMR structure elucidation of the protein [Eijkelenboomet al.(1997),Curr. Biol.7, 739–746; (2000),J. Biomol. NMR,18, 119–28], the protein spectrum was nearly identical to that recorded in its absence. Comparison of the XANES of the protein with that of model compounds and literature data permits the conclusion that the Zn ion is four-coordinated. The major shell of the EXAFS provides evidence for a mixed (N or O as well as S) coordination sphere, while the minor shells indicate imidazole coordination. Our approach to the analysis of the EXAFS, including quantification of the imidazole by multiple scattering simulations withEXCURV92, was validated on the model compounds. An important result is that with multiple scattering simulations using restraints on the parameters of the imidazole rings the number of imidazoles and their orientation could be determined. The integrase spectra can be fitted with two sulfur ligands at 2.26 Å (Debye–Waller-type factor 0.009 Å2) and two imidazole ligands with the N atoms at 1.99 Å (Debye–Waller-type factor 0.005 Å2). The XAS-derived geometry is fully consistent with that found in the NMR structure determination and, allowing for the volume contraction due to the temperature difference between the experiments, justifies the restraints applied in the structure calculation (Zn—S and Zn—N distances of 2.3 Å and 2.0 Å, respectively).