Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination.

DNA-dependent protein kinase (DNA-PK) is utilized in both DNA double-strand break repair (DSBR) and V(D)J recombination, but the mechanism by which this multiprotein complex participates in these processes is unknown. To evaluate the importance of DNA-PK-mediated protein phosphorylation in DSBR and V(D)J recombination, we assessed the effects of the phosphatidyl inositol 3-kinase inhibitor wortmannin on the repair of ionizing radiation-induced DNA double-strand breaks and V(D)J recombination in the V(D)J recombinase inducible B cell line HDR37. Wortmannin radiosensitized HDR37, but had no affect on V(D)J recombination despite a marked reduction in DNA-PK activity. On the other hand, studies with mammalian expression vectors for wild-type human DNA-PK catalytic subunit (DNA-PKcs) and a kinase domain mutant demonstrated that only the kinase active form of DNA-PKcs can reconstitute DSBR and V(D)J recombination in a DNA-PKcs-deficient cell line (Sf19), implying that DNA-PKcs kinase activity is essential for both DSBR and V(D)J recombination. These apparently contradictory results were reconciled by analyses of cell lines varying in their expression of recombinant wild-type human DNA-PKcs. These studies establish that minimal DNA-PKcs protein levels are sufficient to support V(D)J recombination, but insufficient to confer resistance to ionizing radiation.

Quantification of illegitimate V(D)J recombinase-mediated mutations in lymphocytes of newborns and adults.

We used a direct polymerase chain reaction (PCR) method for quantification of HPRT exons 2 + 3 deletions and t(14;18) translocations as a measure of illegitimate V(D)J recombination. We determined the baseline frequencies of these two mutations in mononuclear leukocyte DNA from the umbilical cord blood of newborns and from the peripheral blood of adults. In an initial group of 21 newborns, no t(14;18) translocations were detected (< 0.049 x 10(-7)). The frequency of HPRT exons 2 + 3 deletions was 0.10 x 10(-7) per mononuclear leukocyte, lower than expected based on the T-cell proportion of this cell fraction (55%-70%) and previous results using the T-cell cloning assay (approximately 2-3 x 10(-7) per clonable T-cell). Phytohemagglutinin (PHA), as used in the T-cell cloning assay, was examined for its effect on the frequencies of these mutation events in mononuclear leukocytes from an additional 11 newborns and from 12 adults. There was no significant effect of PHA on t(14;18) translocations which were rare among the newborns (1 detected among 2.7 x 10(8) leukocytes analyzed), and which occurred at frequencies from < 1 x 10(-7) (undetected) to 1.6 x 10(-4) among the adults. The extremely high frequencies of t(14;18)-bearing cells in three adults were due mainly to in vivo expansion of two to six clones. However, PHA appeared to stimulate a modest (although not significant) increase in the frequency of HPRT exons 2 + 3 deletions in the leukocytes of the newborns, from 0.07 x 10(-7) to 0.23 x 10(-7). We show that both the direct PCR assay and the T-cell cloning assay detect similar frequencies of HPRT exons 2 + 3 deletions when calculations are normalized to blood volume, indicating that the apparent discrepancy is probably due to the different population of cells used in the assays. This direct PCR assay may have utility in characterizing the effects of environmental genotoxic agents on this clinically important recombination mechanism.

Regulation of Cre recombinase activity by the synthetic steroid RU 486.

To create a strategy for inducible gene targeting we developed a Cre-lox recombination system which responds to the synthetic steroid RU 486. Several fusions between Cre recombinase and the hormone binding domain (HBD) of a mutated human progesterone receptor, which binds RU 486 but not progesterone, were constructed. When tested in transient expression assays recombination activities of all fusion proteins were responsive to RU 486, but not to the endogenous steroid progesterone. However, the observed induction of recombination activity by the synthetic steroid varied between the different fusion proteins. The fusion with the highest activity in the presence of RU 486 combined with low background activity in the absence of the steroid was tested after stable expression in fibroblast and embryonal stem (ES) cells. We could demonstrate that its recombination activity was highly dependent on RU 486. Since the RU 486 doses required to activate recombination were considerably lower than doses displaying anti-progesterone effects in mice, this system could be used as a valuable tool for inducible gene targeting.

The Hin dimer interface is critical for Fis-mediated activation of the catalytic steps of site-specific DNA inversion.

BACKGROUND: Hin is a member of an extended family of site-specific recombinases--the DNA invertase/resolvase family--that catalyze inversion or deletion of DNA. DNA inversion by Hin occurs between two recombination sites and requires the regulatory protein Fis, which associates with a cis-acting recombinational enhancer sequence. Hin recombinase dimers bind to the two recombination sites and assemble onto the Fis-bound enhancer to generate an invertasome structure, at which time they become competent to catalyze DNA cleavage and strand exchange. In this report, we investigate the role of the Hin dimer interface in the activation of its catalytic functions. RESULTS: We show that the Hin dimer is formed at an interface that contains putative amphipathic alpha-helices in a manner that is very similar to gamma delta resolvase. Certain detergents weakened cooperative interactions between the subunits of the Hin dimer and dramatically increased the rate of the first chemical step of the reaction--double-strand cleavage events at the center of the recombination sites. Amino-acid substitutions within the dimer interface led to profound changes in the catalytic properties of the recombinase. Nearly all mutations strongly affected the ability of the dimer to cleave DNA and most abolished DNA strand exchange in vitro. Some amino-acid substitutions altered the concerted nature of the DNA cleavage events within both recombination sites, and two mutations resulted in cleavage activity that was independent of Fis activation in vitro. Disulfide-linked Hin dimers were catalytically inactive; however, subsequent to the addition of the Fis-bound enhancer sequence, catalytic activity was no longer affected by the presence of oxidizing agents. CONCLUSIONS: The combined results demonstrate that the Hin dimer interface is of critical importance for the activation of catalysis and imply that interactions with the Fis-bound enhancer may trigger a conformational adjustment within the region that is important for concerted DNA cleavage within both recombination sites, and possibly for the subsequent exchange of DNA strands.

Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro.

We have probed the structural organization of the human immunodeficiency virus type 1 integrase protein by limited proteolysis and the functional organization by site-directed mutagenesis of selected amino acid residues. A central region of the protein was relatively resistant to proteolysis. Proteins with altered amino acids in this region, or in the N-terminal part of the protein that includes a putative zinc-binding motif, were purified and assayed for 3' processing, DNA strand transfer, and disintegration activities in vitro. In general, these mutations had parallel effects on 3' processing and DNA strand transfer, suggesting that integrase may utilize a single active site for both reactions. The only proteins that were completely inactive in all three assays contained mutations at conserved amino acids in the central region, suggesting that this part of the protein may be involved in catalysis. In contrast, none of the mutations in the N-terminal region resulted in a protein that was inactive in all three assays, suggesting that this part of integrase may not be essential for catalysis. The disintegration reaction was particularly insensitive to these amino acid substitutions, indicating that some function that is important for 3' processing and DNA strand transfer may be dispensable for disintegration.

Inhibition of HIV-1 integration protein by aurintricarboxylic acid monomers, monomer analogs, and polymer fractions.

Several aurintricarboxylic acid (ATA) monomers, monomer analogs, and polymer fractions have been tested as inhibitors of HIV-1 integration protein (IN). Both of the ATA monomers and all of the ATA polymer fractions inhibited a selective DNA cleavage reaction catalyzed by IN. The ATA monomer analogs were inactive or had low activity. The activities of the substances as inhibitors of HIV IN correlated in a positive way with their activities as inhibitors of the cytopathic effect of HIV-1 in CEM and HIV-2 in MT4 cells. These results suggest that inhibition of HIV IN may contribute to the antiviral activity of the ATA monomers and monomer analogs in cell culture.