Ky Lowenhaupt

Crystal structure of a junction between B-DNA and Z-DNA reveals two extruded bases

Left-handed Z-DNA is a higher-energy form of the double helix, stabilized by negative supercoiling generated by transcription or unwrapping nucleosomes. Regions near the transcription start site frequently contain sequence motifs favourable for forming Z-DNA, and formation of Z-DNA near the promoter region stimulates transcription. Z-DNA is also stabilized by specific protein binding; several proteins have been identified with low nanomolar binding constants. Z-DNA occurs in a dynamic state, forming as a result of physiological processes then relaxing to the right-handed B-DNA. Each time a DNA segment turns into Z-DNA, two B–Z junctions form. These have been examined extensively, but their structure was unknown. Here we describe the structure of a B–Z junction as revealed by X-ray crystallography at 2.6 Å resolution. A 15-base-pair segment of DNA is stabilized at one end in the Z conformation by Z-DNA binding proteins, while the other end remains B-DNA. Continuous stacking of bases between B-DNA and Z-DNA segments is found, with the breaking of one base pair at the junction and extrusion of the bases on each side (Fig. 1). These extruded bases may be sites for DNA modification.

Structure of the DLM-1-Z-DNA complex reveals a conserved family of Z-DNA-binding proteins.

The first crystal structure of a protein, the Zα high affinity binding domain of the RNA editing enzyme ADAR1, bound to left-handed Z-DNA was recently described. The essential set of residues determined from this structure to be critical for Z-DNA recognition was used to search the database for other proteins with the potential for Z-DNA binding. We found that the tumor-associated protein DLM-1 contains a domain with remarkable sequence similarities to ZαADAR. Here we report the crystal structure of this DLM-1 domain bound to left-handed Z-DNA at 1.85 Å resolution. Comparison of Z-DNA binding by DLM-1 and ADAR1 reveals a common structure-specific recognition core within the binding domain. However, the domains differ in certain residues peripheral to the protein–DNA interface. These structures reveal a general mechanism of Z-DNA recognition, suggesting the existence of a family of winged-helix proteins sharing a common Z-DNA binding motif.

A role for Z-DNA binding in vaccinia virus pathogenesis.

The N-terminal domain of the E3L protein of vaccinia virus has sequence similarity to a family of Z-DNA binding proteins of defined three-dimensional structure and it is necessary for pathogenicity in mice. When other Z-DNA-binding domains are substituted for the similar E3L domain, the virus retains its lethality after intracranial inoculation. Mutations decreasing Z-DNA binding in the chimera correlate with decreases in viral pathogenicity, as do analogous mutations in wild-type E3L. A chimeric virus incorporating a related protein that does not bind Z-DNA is not pathogenic, but a mutation that creates Z-DNA binding makes a lethal virus. The ability to bind the Z conformation is thus essential to E3L activity. This finding may allow the design of a class of antiviral agents, including agents against variola (smallpox), which has an almost identical E3L.

Rational cytokine design for increased lifetime and enhanced potency using pH-activated "histidine switching"

We describe a method for the rational design of more effective therapeutic proteins using amino acid substitutions that reduce receptor binding affinity in intracellular endosomal compartments, thereby leading to increased recycling in the ligand-sorting process and consequently resulting in longer half-life in extracellular medium. We demonstrate this approach for granulocyte colony-stimulating factor by using computationally predicted histidine substitutions that switch protonation states between cell-surface and endosomal pH. Molecular modeling of binding electrostatics indicates two different single-histidine mutants that fulfill our design requirements; experimental assays demonstrate that each mutant indeed exhibits an order-of-magnitude increase in medium half-life along with enhanced potency due to increased endocytic recycling.

Drosophila telomeres: new views on chromosome evolution

In Drosophila, chromosome ends (telomeres) are composed of telomere-specific transposable elements (the retroposons HeT-A and TART). These elements are a bona fide part of the cellular machinery yet have many of the hallmarks of retrotransposable elements and retroviruses, raising the possibility that parasitic transposable elements and viruses might have evolved from mechanisms that the cell uses to maintain its chromosomes. It is striking that Drosophila, the model organism for many discoveries in genetics, development and molecular biology (including the classical concept of telomeres), should prove to have chromosome ends different from the generally accepted model. Studies of these telomere-specific retrotransposable elements raise questions about conventional wisdom concerning not only telomeres, but also transposable elements and heterochromatin.

(dC―dA)n•(dG―dT)n sequences have evolutionarily conserved chromosomal locations in Drosophila with implications for roles in chromosome structure and function

In situ hybridization of (dC‐dA)n.(dG‐dT)n to the polytene chromosomes of Drosophila melanogaster reveals a clearly non‐random distribution of chromosomal sites for this sequence. Sites are distributed over most euchromatic regions but the density of sites along the X chromosome is significantly higher than the density over the autosomes. All autosomes show approximately equal levels of hybridization except chromosome 4 which has no detectable stretches of (dC‐dA)n.(dG‐dT)n. Another striking feature is the lack of hybridization of the beta‐heterochromatin of the chromocenter. The specific sites are conserved between different strains of D. melanogaster. The same overall chromosomal pattern of hybridization is seen for the other Drosophila species studied, including D. simulans, a sibling species with a much lower content of middle repetitive DNA, and D. virilis, a distantly related species. The evolutionary conservation of the distribution of (dC‐dA)n.(dG‐dT)n suggests that these sequences are of functional importance. The distribution patterns seen for D. pseudoobscura and D. miranda raise interesting speculations about function. In these species a chromosome equivalent to an autosomal arm of D. melanogaster has been translocated onto the X chromosome and acquired dosage compensation. In each species the new arm of the X also has a higher density of (dC‐dA)n.(dG‐dT)n similar to that seen on other X chromosomes. In addition to correlations with dosage compensation, the depletion of (dC‐dA)n.(dG‐dT)n in beta‐heterochromatin and chromosome 4 may also be related to the fact that these regions do not normally undergo meiotic recombination.

Evolutionary links between telomeres and transposable elements

Transposable elements are abundant in the genomes of higher organisms but are usually thought to affect cells only incidentally, by transposing in or near a gene and influencing its expression. Telomeres of Drosophila chromosomes are maintained by two non-LTR retrotransposons, HeT-A and TART. These are the first transposable elements with identified roles in chromosome structure. We suggest that these elements may be evolutionarily related to telomerase; in both cases an enzyme extends the end of a chromosome by adding DNA copied from an RNA template. The evolution of transposable elements from chromosomal replication mechanisms may have occurred multiple times, although in other organisms the new products have not replaced the endogenous telomerase, as they have in Drosophila. This is somewhat reminiscent of the oncogenes that have arisen from cellular genes. Perhaps the viruses that carry oncogenes have also arisen from cellular genetic systems.

Nonrandom distribution of long mono- and dinucleotide repeats in Drosophila chromosomes: correlations with dosage compensation, heterochromatin, and recombination.

Long stretches of (dC-dA)n.(dT-dG)n, abbreviated CA/TG, have a distinctive distribution on Drosophila chromosomes (M.L. Pardue, K. Lowenhaupt, A. Rich, and A. Nordheim, EMBO J. 6:1781-1789, 1987). The distribution of CA/TG suggests a correlation with the overall transcriptional activity of chromosomal regions and with the ability to undergo meiotic recombination. These correlations are conserved among Drosophila species and may indicate one or more chromosomal functions. To test the generality of these findings, we analyzed the distribution of the rest of the six possible mono- and dinucleotide repeats (A/T, C/G, AT/AT, CA/TG, CT/AG, and CG/CG). All but CG/CG were present at significant levels in the genomes of the six Drosophila species studied; however, A/T levels were an order of magnitude lower than those of the other sequences. Data base analyses suggested that the same sequences are present in other eucaryotes. Like CA/TG, both CT/AG and C/G showed increased levels on dosage-compensating chromosomes; however, the individual sites clearly differed for each sequence. In contrast, A/T and AT/AT, although present in Drosophila DNA, could not be detected in situ in polytene chromosomes. We also used in situ hybridization to analyze the neo-Y chromosome of Drosophila miranda, an ancestral autosome that has become attached to the Y chromosome and is now partially heterochromatic. The neo-Y has acquired repeated DNA sequences; we found that the added sequences are as devoid of mono- and dinucleotide repeats as other heterochromatin. The distribution and function of these sequences are likely to result from both their repetitious nature and base contents.

Proteolytic dissection of Zab, the Z-DNA-binding domain of human ADAR1

Zα is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zα is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1) , a candidate enzyme for nuclear pre-mRNA editing in mammals. Zα is conserved in ADAR1 from many species; in each case, there is a second similar motif,Zβ, separated from Zα by a more divergent linker. To investigate the structure-function relationship ofZα, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zα is part of a domain, Zab, of 229 amino acids (residues 133–361 in human ADAR1). This domain contains both Zα and Zβas well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133–209), containing only Zα, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zβ, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zαand Zβ act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.

A change in the stability of globin mRNA during the induction of murine erythroleukemia cells

The stability of globin mRNA in murine erythroleukemia cells (Friend cells) before and during DMSO-induced differentiation was investigated. Cells were exposed to 3H-uridine for 2 hr and then transferred to medium without the radioactive precursor. The loss of radioactivity in total RNA, poly(A)-containing RNA and globin mRNA was followed. The globin mRNA was isolated using a highly specific globin cDNA column. In uninduced cells and cells early in differentiation, the globin mRNA decays with a half-life of less than 50 hr. After 4 days of induction, the globin mRNA decays with a half-life of 17 hr, demonstrating a change in stability during the induction process. Although the stability of globin mRNA changes during induction, this is not true for total poly(A)-containing RNA. At all times of induction, the poly(A)-containing RNA decays as two populations, one with a half-life of 6 hr and the other with a half-life of 36 hr. The half-life of the rRNA also remains unchanged during differentiation.