Dale B. Wigley

Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks

RecBCD is a multi-functional enzyme complex that processes DNA ends resulting from a double-strand break. RecBCD is a bipolar helicase that splits the duplex into its component strands and digests them until encountering a recombinational hotspot (Chi site). The nuclease activity is then attenuated and RecBCD loads RecA onto the 3′ tail of the DNA. Here we present the crystal structure of RecBCD bound to a DNA substrate. In this initiation complex, the DNA duplex has been split across the RecC subunit to create a fork with the separated strands each heading towards different helicase motor subunits. The strands pass along tunnels within the complex, both emerging adjacent to the nuclease domain of RecB. Passage of the 3′ tail through one of these tunnels provides a mechanism for the recognition of a Chi sequence by RecC within the context of double-stranded DNA. Gating of this tunnel suggests how nuclease activity might be regulated.

The nature of inhibition of DNA gyrase by the coumarins and the cyclothialidines revealed by X-ray crystallography.

This study describes the first crystal structures of a complex between a DNA topoisomerase and a drug. We present the structures of a 24 kDa N‐terminal fragment of the Escherichia coli DNA gyrase B protein in complexes with two different inhibitors of the ATPase activity of DNA gyrase, namely the coumarin antibiotic, novobiocin, and GR122222X, a member of the cyclothialidine family. These structures are compared with the crystal structure of the complex with an ATP analogue, adenylyl‐beta‐gamma‐imidodiphosphate (ADPNP). The likely mechanism, by which mutant gyrase B proteins become resistant to inhibition by novobiocin are discussed in light of these comparisons. The three ligands are quite dissimilar in chemical structure and bind to the protein in very different ways, but their binding is competitive because of a small degree of overlap of their binding sites. These crystal structures consequently describe a chemically well characterized ligand binding surface and provide useful information to assist in the design of novel ligands.

Structural Analysis of DNA Replication Fork Reversal by RecG

The stalling of DNA replication forks that occurs as a consequence of encountering DNA damage is a critical problem for cells. RecG protein is involved in the processing of stalled replication forks, and acts by reversing the fork past the damage to create a four-way junction that allows template switching and lesion bypass. We have determined the crystal structure of RecG bound to a DNA substrate that mimics a stalled replication fork. The structure not only reveals the elegant mechanism used by the protein to recognize junctions but has also trapped the protein in the initial stage of fork reversal. We propose a mechanism for how forks are processed by RecG to facilitate replication fork restart. In addition, this structure suggests that the mechanism and function of the two largest helicase superfamilies are distinct.

X-Ray Crystallography Reveals a Large Conformational Change during Guanyl Transfer by mRNA Capping Enzymes

We have solved the crystal structure of an mRNA capping enzyme at 2.5 A resolution. The enzyme comprises two domains with a deep, but narrow, cleft between them. The two molecules in the crystallographic asymmetric unit adopt very different conformations; both contain a bound GTP, but one protein molecule is in an open conformation while the other is in a closed conformation. Only in the closed conformation is the enzyme able to bind manganese ions and undergo catalysis within the crystals to yield the covalent guanylated enzyme intermediate. These structures provide direct evidence for a mechanism that involves a significant conformational change in the enzyme during catalysis.

Crystal structure of the site-specific recombinase, XerD.

The structure of the site‐specific recombinase, XerD, that functions in circular chromosome separation, has been solved at 2.5 Å resolution and reveals that the protein comprises two domains. The C‐terminal domain contains two conserved sequence motifs that are located in similar positions in the structures of XerD, λ and HP1 integrases. However, the extreme C‐terminal regions of the three proteins, containing the active site tyrosine, are very different. In XerD, the arrangement of active site residues supports a cis cleavage mechanism. Biochemical evidence for DNA bending is encompassed in a model that accommodates extensive biochemical and genetic data, and in which the DNA is wrapped around an α‐helix in a manner similar to that observed for CAP complexed with DNA.

Crystal Structure of an ATP-Dependent DNA Ligase from Bacteriophage T7

The crystal structure of the ATP-dependent DNA ligase from bacteriophage T7 has been solved at 2.6 A resolution. The protein comprises two domains with a deep cleft running between them. The structure of a complex with ATP reveals that the nucleotide binding pocket is situated on the larger N-terminal domain, at the base of the cleft between the two domains of the enzyme. Comparison of the overall domain structure with that of DNA methyltransferases, coupled with other evidence, suggests that DNA also binds in this cleft. Since this structure is the first of the nucleotidyltransferase superfamily, which includes the eukaryotic mRNA capping enzymes, the relationship between the structure of DNA ligase and that of other nucleotidyltransferases is also discussed.

Modularity and Specialization in Superfamily 1 and 2 Helicases

The family of nucleic acid (NA) strand separation enzymes known as helicases are found in all organisms and participate in a wide variety of cellular processes. The central reaction catalyzed is always the same: hydrolysis of a nucleoside triphosphate (NTP; usually ATP) is coupled to the separation of an NA duplex, be it DNA-DNA, DNA-RNA, or RNA-RNA. This central process is required in almost every aspect of NA metabolism in the cell, including chromosomal and plasmid replication, transcription, translation, RNA processing, and DNA recombination and repair (30, 46). This widespread usage may be seen by examining the cellular complement of helicases; for example, at least 12 putative DNA helicases have been identified in the genome of Escherichia coli, while it has been estimated that more than 2% of the Saccharomyces cerevisiae genome encodes helicase-related proteins (48).

HELICASES : A UNIFYING STRUCTURAL THEME ?

The recent structure determinations of PcrA DNA helicase, NS3 RNA helicase, and Rep DNA helicase have revealed similarities between their folds. When these data are examined with sequence and biochemical analyses, as well as microscopy studies of hexameric helicases, a picture of a unifying structure and mechanism for all helicases is beginning to emerge.

Uncoupling DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism

DNA footprinting and nuclease protection studies of PcrA helicase complexed with a 3′‐tailed DNA duplex reveal a contact region that covers a significant region of the substrate both in the presence and absence of a non‐hydrolysable analogue of ATP, ADPNP. However, details of the interactions of the enzyme with the duplex region are altered upon binding of nucleotide. By combining this information with that obtained from crystal structures of PcrA complexed with a similar DNA substrate, we have designed mutant proteins that are defective in helicase activity but that leave the ATPase and single‐stranded DNA translocation activities intact. These mutants are all located in domains 1B and 2B, which interact with the duplex portion of the DNA substrate. Taken together with the crystal structures, these data support an ‘active’ mechanism for PcrA that involves two distinct ATP‐dependent processes: destabilization of the duplex DNA ahead of the enzyme that is coupled to DNA translocation along the single strand product.

The high-resolution crystal structure of a 24-kDa gyrase B fragment from E. coli complexed with one of the most potent coumarin inhibitors, clorobiocin

Coumarin antibiotics, such as clorobiocin, novobiocin, and coumermycin A1, inhibit the supercoiling activity of gyrase by binding to the gyrase B (GyrB) subunit. Previous crystallographic studies of a 24‐kDa N‐terminal domain of GyrB from E. coli complexed with novobiocin and a cyclothialidine analogue have shown that both ligands act by binding at the ATP‐binding site. Clorobiocin is a natural antibiotic isolated from several Streptomyces strains and differs from novobiocin in that the methyl group at the 8 position in the coumarin ring of novobiocin is replaced by a chlorine atom, and the carbamoyl at the 3′ position of the noviose sugar is substituted by a 5‐methyl‐2‐pyrrolylcarbonyl group. To understand the difference in affinity, in order that this information might be exploited in rational drug design, the crystal structure of the 24‐kDa GyrB fragment in complex with clorobiocin was determined to high resolution. This structure was determined independently in two laboratories, which allowed the validation of equivalent interpretations. The clorobiocin complex structure is compared with the crystal structures of gyrase complexes with novobiocin and 5′‐adenylyl‐β,γ‐imidodiphosphate, and with information on the bound conformation of novobiocin in the p24‐novobiocin complex obtained by heteronuclear isotope‐filtered NMR experiments in solution. Moreover, to understand the differences in energetics of binding of clorobiocin and novobiocin to the protein, the results from isothermal titration calorimetry are also presented.