James m. Berger

DNA topoisomerases are a diverse set of essential enzymes responsible for maintaining chromosomes in an appropriate topological state. Although they vary considerably in structure and mechanism, the partnership between topoisomerases and DNA has engendered commonalities in how these enzymes engage nucleic acid substrates and control DNA strand manipulations. All topoisomerases can harness the free energy stored in supercoiled DNA to drive their reactions; some further use the energy of ATP to alter the topology of DNA away from an enzyme-free equilibrium ground state. In the cell, topoisomerases regulate DNA supercoiling and unlink tangled nucleic acid strands to actively maintain chromosomes in a topological state commensurate with particular replicative and transcriptional needs. To carry out these reactions, topoisomerases rely on dynamic macromolecular contacts that alternate between associated and dissociated states throughout the catalytic cycle. In this review, we describe how structural and biochemical studies have furthered our understanding of DNA topoisomerases, with an emphasis on how these complex molecular machines use interfacial interactions to harness and constrain the energy required to manage DNA topology.

Cyclic dinucleotides (CDNs) play central roles in bacterial pathogenesis and innate immunity. The mammalian enzyme cGAS synthesizes a unique cyclic dinucleotide (cGAMP) containing a 2'-5' phosphodiester linkage essential for optimal immune stimulation, but the molecular basis for linkage specificity is unknown. Here, we show that the Vibrio cholerae pathogenicity factor DncV is a prokaryotic cGAS-like enzyme whose activity provides a mechanistic rationale for the unique ability of cGAS to produce 2'-5' cGAMP. Three high-resolution crystal structures show that DncV and human cGAS generate CDNs in sequential reactions that proceed in opposing directions. We explain 2' and 3' linkage specificity and test this model by reprogramming the human cGAS active site to produce 3'-5' cGAMP, leading to selective stimulation of alternative STING adaptor alleles in cells. These results demonstrate mechanistic homology between bacterial signaling and mammalian innate...

ABSTRACT The presence of γ-carboxamide linkages in poly(γ-glutamylcysteinyl)glycines suggests that these polypeptides are likely to be the products of a biosynthetic pathway. However, the in vivo deamidation of proteins can result in the introduction of γ-carboxamide linkages. Synthetic oligodeoxynucleotide sequences were therefore used as probes to hydridize to any mRNA sequences which could encode such proteins. These probes did not hybridize to any mRNA sequences from cells grown either in the presence or absence of Cd, confirming that these metal-binding polypeptides are not directly encoded by structural genes. The kinetics of induction of synthesis of G following exposure to Cd, was studied using reverse phase HPLC of extracts from cells grown in the presence of [35S]cysteine. These data indicate that the shorter polypeptides are substrates for the synthesis of longer forms. Induced synthesis of (γEC)2G and (γEC)3G was detected 5 min after exposure of cells to Cd. This rapid response implies that the pathway is regulated, at least initially, at a post-translational level. Observed insensitivity of this response to cycloheximide further supports this conclusion.

DNA replication of the papillomaviruses is specified by cooperative binding of two proteins to the ori site: the enhancer E2 and the viral initiator E1, a distant member of the AAA+ family of proteins. Formation of this prereplication complex is an essential step toward the construction of a functional, multimeric E1 helicase and DNA melting. To understand how E2 interacts with E1 to regulate this process, we have solved the X-ray structure of a complex containing the HPV18 E2 activation domain bound to the helicase domain of E1. Modeling the monomers of E1 to a hexameric helicase shows that E2 blocks hexamerization of E1 by shielding a region of the E1 oligomerization surface and stabilizing a conformation of E1 that is incompatible with ATP binding. Further biochemical experiments and structural analysis show that ATP is an allosteric effector of the dissociation of E2 from E1. Our data provide the first molecular insights into how a protein can regulate the assembly of an oligomeric AAA+ complex and explain at a structural level why E2, after playing a matchmaker role by guiding E1 to the DNA, must dissociate for subsequent steps of initiation to occur. Building on previously proposed ideas, we discuss how our data advance current models for the conversion of E1 in the prereplication complex to a hexameric helicase assembly.

Ring-shaped nucleic acid translocases and helicases catalyze the directed and processive movement of nucleic acid strands to support essential transactions such as replication, transcription, and chromosome partitioning. Assembled typically as hexamers, ring helicase/translocase systems use coordinated cycles of nucleoside triphosphate (NTP) hydrolysis to translocate extended DNA or RNA substrates through a central pore. Ring formation presents a topological challenge to the engagement of substrate oligonucleotides, and is frequently overcome by distinct loading strategies for shepherding specific motors onto their respective substrates. Recent structural studies that capture different loading intermediates have begun to reveal how different helicase/translocase rings either assemble around substrates or crack open to allow DNA or RNA strand entry, and how dedicated chaperones facilitate these events in some instances. Both prevailing mechanistic models and remaining knowledge gaps are discussed.