Alison Burgess Hickman

Transposition of hAT elements links transposable elements and V(D)J recombination

Transposons are DNA sequences that encode functions that promote their movement to new locations in the genome. If unregulated, such movement could potentially insert additional DNA into genes, thereby disrupting gene expression and compromising an organisms viability. Transposable elements are classified by their transposition mechanisms and by the transposases that mediate their movement. The mechanism of movement of the eukaryotic hAT superfamily elements was previously unknown, but the divergent sequence of hAT transposases from other elements suggested that these elements might use a distinct mechanism. Here we have analysed transposition of the insect hAT element Hermes in vitro. Like other transposons, Hermes excises from DNA via double-strand breaks between the donor-site DNA and the transposon ends, and the newly exposed transposon ends join to the target DNA. Interestingly, the ends of the donor double-strand breaks form hairpin intermediates, as observed during V(D)J recombination, the process which underlies the combinatorial formation of antigen receptor genes. Significant similarities exist in the catalytic amino acids of Hermes transposase, the V(D)J recombinase RAG, and retroviral integrase superfamily transposases, thereby linking the movement of transposable elements and V(D)J recombination.

Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis.

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC 2.3.1.87). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN → RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the Km for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.

MOLECULAR ORGANIZATION IN SITE-SPECIFIC RECOMBINATION : THE CATALYTIC DOMAIN OF BACTERIOPHAGE HP1 INTEGRASE AT 2.7 A RESOLUTION

HP1 integrase promotes site-specific recombination of the HP1 genome into that of Haemophilus influenzae. The isolated C-terminal domain (residues 165-337) of the protein interacts with the recombination site and contains the four catalytic residues conserved in the integrase family. This domain represents a novel fold consisting principally of well-packed alpha helices, a surface beta sheet, and an ordered 17-residue C-terminal tail. The conserved triad of basic residues and the active-site tyrosine are contributed by a single monomer and occupy fixed positions in a defined active-site cleft. Dimers are formed by mutual interactions of the tail of one monomer with an adjacent monomer; this orients active-site clefts antiparallel to each other.

Molecular Pathology of the MEN1 Gene

Abstract: Multiple endocrine neoplasia type 1 (MEN1), among all syndromes, causes tumors in the highest number of tissue types. Most of the tumors are hormone producing (e.g., parathyroid, enteropancreatic endocrine, anterior pituitary) but some are not (e.g., angiofibroma). MEN1 tumors are multiple for organ type, for regions of a discontinuous organ, and for subregions of a continuous organ. Cancer contributes to late mortality; there is no effective prevention or cure for MEN1 cancers. Morbidities are more frequent from benign than malignant tumor, and both are indicators for screening. Onset age is usually earlier in a tumor type of MEN1 than of nonhereditary cases. Broad trends contrast with those in nonneoplastic excess of hormones (e.g., persistent hyperinsulinemic hypoglycemia of infancy). Most germline or somatic mutations in the MEN1 gene predict truncation or absence of encoded menin. Similarly, 11q13 loss of heterozygosity in tumors predicts inactivation of the other MEN1 copy. MEN1 somatic mutation is prevalent in nonhereditary, MEN1‐like tumor types. Compiled germline and somatic mutations show almost no genotype/phenotype relation. Normal menin is 67 kDa, widespread, and mainly nuclear. It may partner with junD, NF‐kB, PEM, SMAD3, RPA2, FANCD2, NM23β, nonmuscle myosin heavy chain II‐A, GFAP, and/or vimentin. These partners have not clarified menins pathways in normal or tumor tissues. Animal models have opened approaches to menin pathways. Local overexpression of menin in Drosophila reveals its interaction with the jun‐kinase pathway. The Men1+/− mouse has robust MEN1; its most important difference from human MEN1 is marked hyperplasia of pancreatic islets, a tumor precursor stage.

The structural basis of ordered substrate binding by serotonin N-acetyltransferase: enzyme complex at 1.8 A resolution with a bisubstrate analog.

Serotonin N-acetyltransferase, a member of the GNAT acetyltransferase superfamily, is the penultimate enzyme in the conversion of serotonin to melatonin, the circadian neurohormone. Comparison of the structures of the substrate-free enzyme and the complex with a bisubstrate analog, coenzyme A-S-acetyltryptamine, demonstrates that acetyl coenzyme A (AcCoA) binding is accompanied by a large conformational change that in turn leads to the formation of the serotonin-binding site. The structure of the complex also provides insight into how the enzyme may facilitate acetyl transfer. A water-filled channel leading from the active site to the surface provides a pathway for proton removal following amine deprotonation. Furthermore, structural and mutagenesis results indicate an important role for Tyr-168 in catalysis.

The 32-Kilodalton Subunit of Replication Protein A Interacts with Menin, the Product of the MEN1 Tumor Suppressor Gene

ABSTRACT Menin is a 70-kDa protein encoded by MEN1, the tumor suppressor gene disrupted in multiple endocrine neoplasia type 1. In a yeast two-hybrid system based on reconstitution of Ras signaling, menin was found to interact with the 32-kDa subunit (RPA2) of replication protein A (RPA), a heterotrimeric protein required for DNA replication, recombination, and repair. The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins. Menin-RPA2 binding was inhibited by a number of menin missense mutations found in individuals with multiple endocrine neoplasia type 1, and the interacting regions were mapped to the N-terminal portion of menin and amino acids 43 to 171 of RPA2. This region of RPA2 contains a weak single-stranded DNA-binding domain, but menin had no detectable effect on RPA-DNA binding in vitro. Menin bound preferentially in vitro to free RPA2 rather than the RPA heterotrimer or a subcomplex consisting of RPA2 bound to the 14-kDa subunit (RPA3). However, the 70-kDa subunit (RPA1) was coprecipitated from HeLa cell extracts along with RPA2 by menin-specific antibodies, suggesting that menin binds to the RPA heterotrimer or a novel RPA1-RPA2-containing complex in vivo. This finding was consistent with the extensive overlap in the nuclear localization patterns of endogenous menin, RPA2, and RPA1 observed by immunofluorescence.

Breaking and joining single-stranded DNA: the HUH endonuclease superfamily

HUH endonucleases are numerous and widespread in all three domains of life. The major function of these enzymes is processing a range of mobile genetic elements by catalysing cleavage and rejoining of single-stranded DNA using an active-site Tyr residue to make a transient 5′-phosphotyrosine bond with the DNA substrate. These enzymes have a key role in rolling-circle replication of plasmids and bacteriophages, in plasmid transfer, in the replication of several eukaryotic viruses and in various types of transposition. They have also been appropriated for cellular processes such as intron homing and the processing of bacterial repeated extragenic palindromes. Here, we provide an overview of these fascinating enzymes and their functions, using well-characterized examples of Rep proteins, relaxases and transposases, and we explore the molecular mechanisms used in their diverse activities.

Structural Unity among Viral Origin Binding Proteins: Crystal Structure of the Nuclease Domain of Adeno-Associated Virus Rep

Adeno-associated virus (AAV), unique among animal viruses in its ability to integrate into a specific chromosomal location, is a promising vector for human gene therapy. AAV Replication (Rep) protein is essential for viral replication and integration, and its amino terminal domain possesses site-specific DNA binding and endonuclease activities required for replication initiation and integration. This domain displays a novel endonuclease fold and demonstrates an unexpected structural relationship to other viral origin binding proteins such as the papillomavirus E1 protein and the SV40 T antigen. The active site, located at the bottom of a positively charged cleft, is formed by the spatial convergence of a divalent metal ion and two conserved sequence motifs that define the rolling circle replication superfamily.

Molecular architecture of a eukaryotic DNA transposase

Mobile elements and their inactive remnants account for large proportions of most eukaryotic genomes, where they have had central roles in genome evolution. Over 50 years ago, McClintock reported a form of stress-induced genome instability in maize in which discrete DNA segments move between chromosomal locations. Our current mechanistic understanding of enzymes catalyzing transposition is largely limited to prokaryotic transposases. The Hermes transposon from the housefly is part of the eukaryotic hAT superfamily that includes hobo from Drosophila, McClintocks maize Activator and Tam3 from snapdragon. We report here the three-dimensional structure of a functionally active form of the transposase from Hermes at 2.1-Å resolution. The Hermes protein has some structural features of prokaryotic transposases, including a domain with a retroviral integrase fold. However, this domain is disrupted by the insertion of an additional domain. Finally, transposition is observed only when Hermes assembles into a hexamer.

Melatonin biosynthesis: the structure of serotonin N-acetyltransferase at 2.5 A resolution suggests a catalytic mechanism.

Conversion of serotonin to N-acetylserotonin, the precursor of the circadian neurohormone melatonin, is catalyzed by serotonin N-acetyltransferase (AANAT) in a reaction requiring acetyl coenzyme A (AcCoA). AANAT is a globular protein consisting of an eight-stranded beta sheet flanked by five alpha helices; a conserved motif in the center of the beta sheet forms the cofactor binding site. Three polypeptide loops converge above the AcCoA binding site, creating a hydrophobic funnel leading toward the cofactor and serotonin binding sites in the protein interior. Two conserved histidines not found in other NATs are located at the bottom of the funnel in the active site, suggesting a catalytic mechanism for acetylation involving imidazole groups acting as general acid/base catalysts.