Unraveling DNA helicases from plant cells*

Plant Molecular Biology -     

Structure and function of RecQ DNA helicases

RecQ family helicases play important roles in coordinating genome maintenance pathways in living cells. In the absence of functional RecQ proteins, cells exhibit a variety of phenotypes, including increased mitotic recombination, elevated chromosome missegregation, hypersensitivity to DNA-damaging agents, and defects in meiosis. Mutations in three of the five human RecQ family members give rise to genetic disorders associated with a predisposition to cancer and premature aging, highlighting the importance of RecQ proteins and their cellular activities for human health. Current evidence suggests that RecQ proteins act at multiple steps in DNA replication, including stabilization of replication forks and removal of DNA recombination intermediates, in order to maintain genome integrity. The cellular basis of RecQ helicase function may be explained through interactions with multiple components of the DNA replication and recombination machinery. This review focuses on biochemical and structural aspects of the RecQ helicases and how these features relate to their known cellular function, specifically in preventing excessive recombination.     

Studies on three E. coli DEAD-box helicases point to an unwinding mechanism different from that of model DNA helicases

DEAD-box proteins participate in various aspects of RNA metabolism in all organisms. These RNA-dependent ATPases are usually regarded as double-stranded RNA unwinding enzymes, though in vitro this activity has only been demonstrated for a subset of them. Given their high biological specificity, their equivocal unwinding activity may reflect the noncognate character of the substrates used in vitro. Here, we pinpoint other reasons for this elusiveness. We have compared the ATPase and helicase activities of three E. coli DEAD-box proteins, CsdA, RhlE and SrmB. Whereas the ATPase activity of all proteins is stimulated (albeit to various degree) by long RNAs, only RhlE is stimulated by short oligoribonucleotides. Consistently, all three proteins can unwind RNA duplexes with long single-stranded extensions, but only RhlE is effective when extensions are short or absent. Another critical constraint concerns the length of the duplex region: in the case of RhlE, the ratio (duplex unwound)/(ATP hydrolyzed) drops 1000-fold upon going from 11 to 14 base pairs, indicating a low processivity. Remarkably, allowing for these constraints, all three proteins can unwind substrates with either 5' or 3' extensions (or no extension in the case of RhlE). This behavior, which contrasts with that of well studied SF1 DNA helicases, is discussed in the light of available structural and biochemical data.     

Influence of DNA end structure on the mechanism of initiation of DNA unwinding by the Escherichia coli RecBCD and RecBC helicases

Escherichiacoli RecBCD is a bipolar DNA helicase possessing two motor subunits (RecB, a 3′-to-5′ translocase, and RecD, a 5′-to-3′ translocase) that is involved in the major pathway of recombinational repair. Previous studies indicated that the minimal kinetic mechanism needed to describe the ATP-dependent unwinding of blunt-ended DNA by RecBCD in vitro is a sequential n-step mechanism with two to three additional kinetic steps prior to initiating DNA unwinding. Since RecBCD can “melt out” ∼ 6 bp upon binding to the end of a blunt-ended DNA duplex in a Mg²⁺-dependent but ATP-independent reaction, we investigated the effects of noncomplementary single-stranded (ss) DNA tails [3′-(dT)₆ and 5′-(dT)₆ or 5′-(dT)₁₀] on the mechanism of RecBCD and RecBC unwinding of duplex DNA using rapid kinetic methods. As with blunt-ended DNA, RecBCD unwinding of DNA possessing 3′-(dT)₆ and 5′-(dT)₆ noncomplementary ssDNA tails is well described by a sequential n-step mechanism with the same unwinding rate (mk_U = 774 ± 16 bp s^− 1) and kinetic step size (m = 3.3 ± 1.3 bp), yet two to three additional kinetic steps are still required prior to initiation of DNA unwinding (k_C = 45 ± 2 s^− 1). However, when the noncomplementary 5′ ssDNA tail is extended to 10 nt [5′-(dT)₁₀ and 3′-(dT)₆], the DNA end structure for which RecBCD displays optimal binding affinity, the additional kinetic steps are no longer needed, although a slightly slower unwinding rate (mk_U = 538 ± 24 bp s^− 1) is observed with a similar kinetic step size (m = 3.9 ± 0.5 bp). The RecBC DNA helicase (without the RecD subunit) does not initiate unwinding efficiently from a blunt DNA end. However, RecBC does initiate well from a DNA end possessing noncomplementary twin 5′-(dT)₆ and 3′-(dT)₆ tails, and unwinding can be described by a simple uniform n-step sequential scheme, without the need for the additional k_C initiation steps, with a similar kinetic step size (m = 4.4 ± 1.7 bp) and unwinding rate (mk_obs = 396 ± 15 bp s^− 1). These results suggest that the additional kinetic steps with rate constant k_C required for RecBCD to initiate unwinding of blunt-ended and twin (dT)₆-tailed DNA reflect processes needed to engage the RecD motor with the 5′ ssDNA.     

Unraveling the complex network of cuticular structure and function

A hydrophobic cuticle is deposited at the outermost extracellular matrix of the epidermis in primary tissues of terrestrial plants. Besides forming a protective shield against the environment, the cuticle is potentially involved in several developmental processes during plant growth. A high degree of variation in cuticle composition and structure exists between different plant species and tissues. Lots of progress has been made recently in understanding the different steps of biosynthesis, transport, and deposition of cuticular components. However, the molecular mechanisms that underlie cuticular function remain largely elusive.     

5-Aminolaevulinic acid dehydratase: structure, function, and mechanism.

delta-Aminolaevulinic acid dehydratase catalyses the synthesis of porphobilinogen. The enzyme has a molecular mass of 285000 and is composed of eight similar subunits of molecular mass 35000. The N-terminal amino acid is acylated, and the number of peptides found on tryptic digestion equals the number of lysine and arginine residues per mass of 35000. The eight subunits are apparently arranged at the corners of a cube and therefore have dihedral (D4) symmetry. The bovine liver enzyme which has been cystallized contains 4--6 atoms of zinc per mole of enzyme. The apo-enzyme obtained on prolonged hydrolysis can be reactivated by the addition of zinc or cadmium ions. The dialysed enzyme must be first treated with dithiothreitol. There are two very active SH groups in a total of 6--7-SH groups per subunit. The substrate forms a Schiff base with the epsilon-amino group of a lysine residue. Reduction of the Schiff base with NaBH4 should reveal the number of active sites per mole of enzyme. It appears that only four of the eight subunits form a Schiff base with the substrate indicating that the enzyme exhibits the phenomenon of either half-site reactivity or negative cooperativity. The enzyme appears to have a strong subunit-subunit interaction for an immobilized preparation remained stable for at least a month. An immobilized enzyme preparation was treated in a manner so that it dissociated into tetramers. Both the eluate and protein still attached to the Sepharose on a column were enzymically active. The bound enzyme could not reassociate under assay conditions but still contained about 50% of the original enzyme activity. It would seem that the enzyme is active when composed with less than eight subunits.     

DNA gyrase: structure and function.

DNA gyrase is an essential bacterial enzyme that catalyzes the ATP-dependent negative super-coiling of double-stranded closed-circular DNA. Gyrase belongs to a class of enzymes known as topoisomerases that are involved in the control of topological transitions of DNA. The mechanism by which gyrase is able to influence the topological state of DNA molecules is of inherent interest from an enzymological standpoint. In addition, much attention has been focused on DNA gyrase as the intracellular target of a number of antibacterial agents as a paradigm for other DNA topoisomerases. In this review we summarize the current knowledge concerning DNA gyrase by addressing a wide range of aspects of the study of this enzyme.     

Human premature aging, DNA repair and RecQ helicases

Genomic instability leads to mutations, cellular dysfunction and aberrant phenotypes at the tissue and organism levels. A number of mechanisms have evolved to cope with endogenous or exogenous stress to prevent chromosomal instability and maintain cellular homeostasis. DNA helicases play important roles in the DNA damage response. The RecQ family of DNA helicases is of particular interest since several human RecQ helicases are defective in diseases associated with premature aging and cancer. In this review, we will provide an update on our understanding of the specific roles of human RecQ helicases in the maintenance of genomic stability through their catalytic activities and protein interactions in various pathways of cellular nucleic acid metabolism with an emphasis on DNA replication and repair. We will also discuss the clinical features of the premature aging disorders associated with RecQ helicase deficiencies and how they relate to the molecular defects.     

Crawling and wiggling on DNA: structural insights to the mechanism of DNA unwinding by helicases

Crystal structures have recently been solved of the monomeric DNA helicase PcrA bound to forked DNA, and of the hexameric helicase domain of the bacteriophage T7 gene 4 protein, a replication fork DNA helicase/primase. These structures have led to the elaboration of the first molecular models to describe DNA helicase action.     

Structure, function and regulation of spliceosomal RNA helicases

Pre-mRNA splicing requires the activities of several ATPases from the DEAH-box, DEAD-box and Ski2-like helicase families to control conformational rearrangements within the spliceosome. Recent findings indicate that several spliceosomal helicases can act at multiple stages of the splicing reaction, and information on how those multiple actions are controlled are emerging. The recently solved crystal structure of the DEAH-box helicase Prp43 provides novel insights into the similarities and differences between the three helicase families. Here we discuss the potential family-specific mechanisms of spliceosomal RNA helicases and their regulation.     

Four different DNA helicases from calf thymus.

Using a strand displacement assay we have followed DNA helicase activities during the simultaneous isolation of several enzymes from calf thymus such as DNA polymerases alpha, delta, and epsilon, proliferating cell nuclear antigen, and replication factor A. Thus we were able to discriminate and isolate four different DNA helicases called A, B, C, and D. DNA helicase A is identical with the enzyme described earlier (Th?mmes, P., and Hübscher, U. (1990) J. Biol. Chem. 265, 14347-14354). The four enzymes can be distinguished by (i) their putative molecular weights after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (ii) glycerol gradient sedimentation under low and high salt conditions, (iii) sensitivity to salt, (iv) binding to DNA, (v) nucleoside- and deoxynucleoside 5'-triphosphate requirements, and (vi) by their direction of movement. DNA helicase A unwinds in the 3'----5' direction on the DNA it was bound to, while DNA helicases B, C, and D do so in the 5'----3' direction. DNA helicase D, and to some extent DNA helicases B and C, are able to unwind long substrates of more than 400 nucleotides. Replication factor A, a single-stranded heterotrimeric DNA binding protein involved in cellular DNA replication and DNA repair stimulates the DNA helicases. The stimulatory effect is most pronounced on DNA helicase A, where replication factor A enables this helicase to unwind longer substrates. DNA helicases B, C, and D are also stimulated by replication factor A. The effect of replication factor A appears to be specific since corresponding single-stranded DNA binding proteins from Escherichia coli and bacteriophage T4 have no or even a negative effect on the four DNA helicases. Heterologous human replication factor A has no stimulatory effect on any of the four DNA helicases suggesting a species specificity of these interactions. Thus it appears that mammalian cells possess, as does E. coli, a variety of different enzymes that can transiently abolish the double helical DNA structure in the cell.     

Biochemical, biophysical, and proteomic approaches to study DNA helicases

Helicases are a family of enzymes that play an essential role in nearly all DNA metabolic processes, catalyzing the transient opening of DNA duplexes. These motor proteins couple the chemical energy of ATP binding and hydrolysis to the separation of the complementary strands of a DNA or RNA duplex substrate. A full understanding of their mechanism of DNA unwinding can be achieved only through careful investigation of the thermodynamic and kinetic parameters that control this ATP-driven process, as well as through analysis of the helicases' tertiary and quaternary structures associated with nucleic acids and/or nucleotide recognition. This review describes the various biochemical, biophysical, and, more recently, proteomic techniques that have been developed to shed light on the still controversial, and in some aspects elusive, helicase-catalyzed mechanism of DNA unwinding.     

Structure,function, and mechanism of HhaI DNA methyltransferases

A vast amount of literature has accumulated on the characterization of DNA methyltransferases. The HhaI DNA methyltransferase, a C5-cytosine methyltransferase, has been the subject of investigation for the last 2 decades. Biochemical and kinetic characterization have led to an understanding of the catalytic and kinetic mechanism of the methyltransfer reaction. The HhaI methyltransferase has also been subjected to extensive structural analysis, with the availability of 12 structures with or without a cofactor and a variety of DNA substrates. The mechanism of base flipping, first described for the HhaI methyltransferase, is conserved among all DNA methyltransferases and is also found to occur in numerous DNA repair enzymes. Studies with other methyltransferase reveal a significant structural and functional similarity among different types of methyltransferases. This review aims to summarize the available information on the HhaI DNA methyltransferase.     

Structure, function and mechanism of exocyclic DNA methyltransferases

DNA MTases (methyltransferases) catalyse the transfer of methyl groups to DNA from AdoMet (S-adenosyl-L-methionine) producing AdoHcy (S-adenosyl-L-homocysteine) and methylated DNA. The C5 and N4 positions of cytosine and N6 position of adenine are the target sites for methylation. All three methylation patterns are found in prokaryotes, whereas cytosine at the C5 position is the only methylation reaction that is known to occur in eukaryotes. In general, MTases are two-domain proteins comprising one large and one small domain with the DNA-binding cleft located at the domain interface. The striking feature of all the structurally characterized DNA MTases is that they share a common core structure referred to as an 'AdoMet-dependent MTase fold'. DNA methylation has been reported to be essential for bacterial virulence, and it has been suggested that DNA adenine MTases (Dams) could be potential targets for both vaccines and antimicrobials. Drugs that block Dam could slow down bacterial growth and therefore drug-design initiatives could result in a whole new generation of antibiotics. The transfer of larger chemical entities in a MTase-catalysed reaction has been reported and this represents an interesting challenge for bio-organic chemists. In general, amino MTases could therefore be used as delivery systems for fluorescent or other reporter groups on to DNA. This is one of the potential applications of DNA MTases towards developing non-radioactive DNA probes and these could have interesting applications in molecular biology. Being nucleotide-sequence-specific, DNA MTases provide excellent model systems for studies on protein-DNA interactions. The focus of this review is on the chemistry, enzymology and structural aspects of exocyclic amino MTases.     

A skipping rope translocation mechanism in a widespread family of DNA repair helicases

Mitomycin repair factor A represents a family of DNA helicases that harbor a domain of unknown function (DUF1998) and support repair of mitomycin C-induced DNA damage by presently unknown molecular mechanisms. We determined crystal structures of Bacillus subtilis Mitomycin repair factor A alone and in complex with an ATP analog and/or DNA and conducted structure-informed functional analyses. Our results reveal a unique set of auxiliary domains appended to a dual-RecA domain core. Upon DNA binding, a Zn²⁺-binding domain, encompassing the domain of unknown function, acts like a drum that rolls out a canopy of helicase-associated domains, entrapping the substrate and tautening an inter-domain linker across the loading strand. Quantification of DNA binding, stimulated ATPase and helicase activities in the wild type and mutant enzyme variants in conjunction with the mode of coordination of the ATP analog suggest that Mitomycin repair factor A employs similar ATPase-driven conformational changes to translocate on DNA, with the linker ratcheting through the nucleotides like a ‘skipping rope’. The electrostatic surface topology outlines a likely path for the displaced DNA strand. Our results reveal unique molecular mechanisms in a widespread family of DNA repair helicases linked to bacterial antibiotics resistance.     

Unraveling the catalytic mechanism of lactoperoxidase and myeloperoxidase.

Although belonging to the widely investigated peroxidase superfamily, lactoperoxidase (LPO) and myeloperoxidase (MPO) share structural and functional features that make them peculiar with respect to other enzymes of the same group. A survey of the available literature on their catalytic intermediates enabled us to ask some questions that remained unanswered. These questions concern controversial features of the LPO and MPO catalytic cycle, such as the existence of Compound I and Compound II isomers and the identification of their spectroscopic properties. After addressing each of these questions, we formulated a hypothesis that describes an integrated vision of the catalytic mechanism of both enzymes. The main points are: (a) a re-evaluation of the role of superoxide as a reductant in the catalytic cycle; (b) the existence of Cpd I isomers; (c) reciprocal interactions between catalytic intermediates and (d) a mechanistic explanation for catalase activity in both enzymes.