Molecular Analysis of (R)-(+)-Mandelonitrile Lyase Microheterogeneity in Black Cherry1

The flavoprotein (R)-(+)-mandelonitrile lyase (MDL; EC 4.1.2.10), which plays a key role in cyanogenesis in rosaceous stone fruits, occurs in black cherry (Prunus serotina Ehrh.) homogenates as several closely related isoforms. Biochemical and molecular biological methods were used to investigate MDL microheterogeneity and function in this species. Three novel MDL cDNAs of high sequence identity (designated MDL2, MDL4, and MDL5) were isolated. Like MDL1 and MDL3 cDNAs (Z. Hu, J.E. Poulton [1997] Plant Physiol 115: 1359–1369), they had open reading frames that predicted a flavin adenine dinucleotide-binding site, multiple N-glycosylation sites, and an N-terminal signal sequence. The N terminus of an MDL isoform purified from seedlings matched the derived amino acid sequence of the MDL4 cDNA. Genomic sequences corresponding to the MDL1, MDL2, and MDL4 cDNAs were obtained by polymerase chain reaction amplification of genomic DNA. Like the previously reported mdl3 gene, these genes are interrupted at identical positions by three short, conserved introns. Given their overall similarity, we conclude that the genes mdl1, mdl2, mdl3, mdl4, and mdl5 are derived from a common ancestral gene and constitute members of a gene family. Genomic Southern-blot analysis showed that this family has approximately eight members. Northern-blot analysis using gene-specific probes revealed differential expression of the genes mdl1, mdl2, mdl3, mdl4, and mdl5.

ADP-Dependent Phosphorylation Regulates Association of a DNA-Binding Complex with the Barley Chloroplast psbD Blue-Light-Responsive Promoter1

The chloroplast gene psbD encodes D2, a chlorophyll-binding protein located in the photosystem II reaction center. Transcription of psbD in higher plants involves at least three promoters, one of which is regulated by blue light. The psbD blue-light-regulated promoter (BLRP) consists of a −10 promoter element and an activating complex, AGF, that binds immediately upstream of −35. A second sequence-specific DNA-binding complex, PGTF, binds upstream of AGF between −71 and −100 in the barley (Hordeum vulgare) psbD BLRP. In this study we report that ADP-dependent phosphorylation selectively inhibits the binding of PGTF to the barley psbD BLRP. ATP at high concentrations (1–5 mm) inhibits PGTF binding, but in the presence of phosphocreatine and phosphocreatine kinase, this capacity is lost, presumably due to scavenging of ADP. ADP inhibits PGTF binding at relatively low concentrations (0.1 mm), whereas other nucleotides are unable to mediate this response. ADP-mediated inhibition of PGTF binding is reduced in the presence of the protein kinase inhibitor K252a. This and other results suggest that ADP-dependent phosphorylation of PGTF (or some associated protein) inhibits binding of PGTF to the psbD BLRP and reduces transcription. ADP-dependent phosphorylation is expected to increase in darkness in parallel with the rise in ADP levels in chloroplasts. ADP-dependent phosphorylation in chloroplasts may, therefore, in coordination, inactivate enzymes involved in carbon assimilation, protein synthesis, and transcription during diurnal light/dark cycles.

An essential amino acid residue in the protein translocation channel revealed by targeted random mutagenesis of SecY

The SecY/Sec61α family of membrane proteins are the central subunits of the putative protein translocation channel. We introduced random mutations into a segment of Escherichia coli SecY within its cytoplasmic domain 5, which was shown previously to be important for the SecA-dependent translocation activity. Mutations were classified into those retaining function and those gaining a dominant-interfering ability caused by a loss of function. These analyses showed that Arg-357, Pro-358, Gly-359, and Thr-362 are functionally important; Arg-357, conserved in almost all organisms, was identified as an indispensable residue.

Non-Arrhenius kinetics for the loop closure of a DNA hairpin

Intramolecular chain diffusion is an elementary process in the conformational fluctuations of the DNA hairpin-loop. We have studied the temperature and viscosity dependence of a model DNA hairpin-loop by FRET (fluorescence resonance energy transfer) fluctuation spectroscopy (FRETfs). Apparent thermodynamic parameters were obtained by analyzing the correlation amplitude through a two-state model and are consistent with steady-state fluorescence measurements. The kinetics of closing the loop show non-Arrhenius behavior, in agreement with theoretical prediction and other experimental measurements on peptide folding. The fluctuation rates show a fractional power dependence (β = 0.83) on the solution viscosity. A much slower intrachain diffusion coefficient in comparison to that of polypeptides was derived based on the first passage time theory of SSS [Szabo, A., Schulten, K. & Schulten, Z. (1980) J. Chem. Phys. 72, 4350–4357], suggesting that intrachain interactions, especially stacking interaction in the loop, might increase the roughness of the free energy surface of the DNA hairpin-loop.

The Evaluation of Forensic DNA Evidence*

The following is an excerpt from the Executive Summary of the National Research Council Report.

High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides

Homologous DNA recombination is a fundamental, regenerative process within living organisms. However, in most organisms, homologous recombination is a rare event, requiring a complex set of reactions and extensive homology. We demonstrate in this paper that Beta protein of phage λ generates recombinants in chromosomal DNA by using synthetic single-stranded DNAs (ssDNA) as short as 30 bases long. This ssDNA recombination can be used to mutagenize or repair the chromosome with efficiencies that generate up to 6% recombinants among treated cells. Mechanistically, it appears that Beta protein, a Rad52-like protein, binds and anneals the ssDNA donor to a complementary single-strand near the DNA replication fork to generate the recombinant. This type of homologous recombination with ssDNA provides new avenues for studying and modifying genomes ranging from bacterial pathogens to eukaryotes. Beta protein and ssDNA may prove generally applicable for repairing DNA in many organisms.

Systematic Reverse Genetics of Transfer-DNA-Tagged Lines of Arabidopsis1 : Isolation of Mutations in the Cytochrome P450 Gene Superfamily

We have developed an efficient reverse-genetics protocol that uses expedient pooling and hybridization strategies to identify individual transfer-DNA insertion lines from a collection of 6000 independently transformed lines in as few as 36 polymerase chain reactions. We have used this protocol to systematically isolate Arabidopsis lines containing insertional mutations in individual cytochrome P450 genes. In higher plants P450 genes encode enzymes that perform an exceptionally wide range of functions, including the biosynthesis of primary metabolites necessary for normal growth and development, the biosynthesis of secondary products, and the catabolism of xenobiotics. Despite their importance, progress in assigning enzymatic function to individual P450 gene products has been slow. Here we report the isolation of the first 12 such lines, including one (CYP83B1-1) that displays a runt phenotype (small plants with hooked leaves), and three insertions in abundantly expressed genes. The DNAs used in this study are publicly available and can be used to systematically isolate mutants in Arabidopsis.

A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA

Werner syndrome (WS) is a premature aging disorder where the affected individuals appear much older than their chronological age. The single gene that is defective in WS encodes a protein (WRN) that has ATPase, helicase and 3′→5′ exonuclease activities. Our laboratory has recently uncovered a physical and functional interaction between WRN and the Ku heterodimer complex that functions in double-strand break repair and V(D)J recombination. Importantly, Ku specifically stimulates the exonuclease activity of WRN. We now report that Ku enables the Werner exonuclease to digest through regions of DNA containing 8-oxoadenine and 8-oxoguanine modifications, lesions that have previously been shown to block the exonuclease activity of WRN alone. These results indicate that Ku significantly alters the exonuclease function of WRN and suggest that the two proteins function concomitantly in a DNA damage processing pathway. In support of this notion we also observed co-localization of WRN and Ku, particularly after DNA damaging treatments.

Condensation by DNA looping facilitates transfer of large DNA molecules into mammalian cells

Experimental studies of complete mammalian genes and other genetic domains are impeded by the difficulty of introducing large DNA molecules into cells in culture. Previously we have shown that GST–Z2, a protein that contains three zinc fingers and a proline-rich multimerization domain from the polydactyl zinc finger protein RIP60 fused to glutathione S-transferase (GST), mediates DNA binding and looping in vitro. Atomic force microscopy showed that GST–Z2 is able to condense 130–150 kb bacterial artificial chromosomes (BACs) into protein–DNA complexes containing multiple DNA loops. Condensation of the DNA loops onto the Z2 protein–BAC DNA core complexes with cationic lipid resulted in particles that were readily transferred into multiple cell types in culture. Transfer of total genomic linear DNA containing amplified DHFR genes into DHFR– cells by GST–Z2 resulted in a 10-fold higher transformation rate than calcium phosphate co-precipitation. Chinese hamster ovarian cells transfected with a BAC containing the human TP53 gene locus expressed p53, showing native promoter elements are active after GST–Z2-mediated gene transfer. Because DNA condensation by GST–Z2 does not require the introduction of specific recognition sequences into the DNA substrate, condensation by the Z2 domain of RIP60 may be used in conjunction with a variety of other agents to provide a flexible and efficient non-viral platform for the delivery of large genes into mammalian cells.

Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled

Replication forks formed at bacterial origins often encounter template roadblocks in the form of DNA adducts and frozen protein–DNA complexes, leading to replication-fork stalling and inactivation. Subsequent correction of the corrupting template lesion and origin-independent assembly of a new replisome therefore are required for survival of the bacterium. A number of models for replication-fork restart under these conditions posit that nascent strand regression at the stalled fork generates a Holliday junction that is a substrate for subsequent processing by recombination and repair enzymes. We show here that early replication intermediates containing replication forks stalled in vitro by the accumulation of excess positive supercoils could be cleaved by the Holliday junction resolvases RusA and RuvC. Cleavage by RusA was inhibited by the presence of RuvA and was stimulated by RecG, confirming the presence of Holliday junctions in the replication intermediate and supporting the previous proposal that RecG could catalyze nascent strand regression at stalled replication forks. Furthermore, RecG promoted Holliday junction formation when replication intermediates in which the replisome had been inactivated were negatively supercoiled, suggesting that under intracellular conditions, the action of RecG, or helicases with similar activities, is necessary for the catalysis of nascent strand regression.

Instability of repetitive DNA sequences: The role of replication in multiple mechanisms

Rearrangements between tandem sequence homologies of various lengths are a major source of genomic change and can be deleterious to the organism. These rearrangements can result in either deletion or duplication of genetic material flanked by direct sequence repeats. Molecular genetic analysis of repetitive sequence instability in Escherichia coli has provided several clues to the underlying mechanisms of these rearrangements. We present evidence for three mechanisms of RecA-independent sequence rearrangements: simple replication slippage, sister-chromosome exchange-associated slippage, and single-strand annealing. We discuss the constraints of these mechanisms and contrast their properties with RecA-dependent homologous recombination. Replication plays a critical role in the two slipped misalignment mechanisms, and difficulties in replication appear to trigger rearrangements via all these mechanisms.

Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material

Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes. A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins. In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology. Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2′ methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and single-stranded DNA during the formation of heteroduplex joints. The pivotal role of the 2′ methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes. Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling). These properties may give an extensive evolutionary advantage to DNA.