mRNA Cap Formation
Eukaryotic mRNAs contain the 5'-cap structure m7GpppN. Our long-term goal is to understand the mechanism of cap synthesis and the role of the cap in cellular RNA metabolism. We've shown that cap formation in the budding yeast Saccharomyces cerevisiae is mediated by the sequential action of 3 enzymes — RNA triphosphatase (Cet1p), RNA guanylyltransferase (Ceg1p), and RNA (guanine-7) methyltransferase (Abd1p) — which are encoded by 3 separate genes. Cet1p and Ceg1p form a heteromeric complex. The mammalian capping apparatus consists of 2 gene products: a bifunctional triphosphatase-guanylyltransferase (Mce1p) and a separate cap methyltransferase (Hcm1p).
Our current research focuses on the first 2 enzymes in the cap synthetic pathway: RNA triphosphatase and RNA guanylyltransferase. The guanylyltransferases of fungi and metazoans are structurally homologous proteins that catalyze the transfer of GMP from GTP to a 5'-diphosphate RNA terminus via a covalent enzyme-GMP intermediate. We have elucidated the mechanism of covalent nucleotidyl transfer by a combination of mutagenesis and crystallography. Guanylyltransferase activity is essential for cell growth; failure to cap newly made transcripts results in their accelerated 5'-exonucleolytic decay.
RNA triphosphatase hydrolyzes the gamma phosphate of mRNA. The triphosphatase component of the capping apparatus has diverged in structure and mechanism during the transition from fungal to metazoan species. The metazoan triphosphatases belong to a superfamily of phosphatases (which includes protein tyrosine phosphatases and dual-specificity protein phosphatases) that act via formation and hydrolysis of a cysteinyl phosphate intermediate; the reaction requires no metal cofactor. The yeast and poxvirus RNA triphosphatases comprise a novel family of metal-requiring phosphohydrolases that share several sequence motifs implicated in catalysis. We have solved the structure of yeast RNA triphosphatase by x-ray crystallography, and have defined the active site in detail by site-directed mutagenesis.
Cap formation in vivo is targeted specifically to cellular transcripts made by RNA polymerase II. Our studies suggest that mammalian and yeast guanylyltransferases are directed to nascent pre-mRNAs by direct binding to the phosphorylated carboxyl-terminal domain (CTD) of the largest subunit of polymerase II. We hypothesize that the RNA triphosphatase component is targeted to the pol II transcription elongation complex by virtue of its association with the guanylyltransferase.
Topoisomerase I Structure and Mechanism
The eukaryotic type IB topoisomerase family includes nuclear topo I and the topoisomerases encoded by vaccinia and other cytoplasmic poxviruses. These enzymes relax DNA supercoils by transiently breaking and rejoining 1 strand of the DNA duplex. They act via a common mechanism, involving a covalent DNA-(3'-phosphotyrosyl)-topo intermediate. The participation of type IB topoisomerases in DNA replication, genetic recombination, and transcription, plus the fact that nuclear topo I is the target of the camptothecin anti-tumor drugs, mandates a thorough understanding of their mechanism of action.
Nuclear type IB topos vary in size from 765 to 1019 amino acids. The poxvirus topos (314 to 333 amino acids) are considerably smaller than the cellular counterparts and likely constitute the minimal functional unit of a type IB enzyme. A distinctive feature of the poxvirus topos is their sequence specificity in strand cleavage. Vaccinia topo binds duplex DNA and forms a covalent adduct at sites, containing a pentapyrimidine element 5'(C/T)CCTT in the scissile strand. The T residue is linked via a 3'-phosphodiester bond to Tyr274 of the enzyme.
We've shown that the catalytic repertoire of eukaryotic topo I extends well beyond the relaxation of DNA supercoils. For example, vaccinia topo can function as
(i) a Holliday junction resolvase,
(ii) an endoribonuclease,
(iii) a polynucleotide ligase for joining 2',3' cyclic phosphate and 5' OH termini, and
(iv) a DNA endonuclease.
The spectrum of topo-catalyzed reactions can be altered by amino acid substitutions at the enzyme's active site. These findings have broad implications for the evolution of phosphoryl and nucleotidyl transfer enzymes. They also engender predictions for the role topo might play during poxvirus infection in vivo, particularly with respect to DNA recombination.
We are now engaged in biochemical and genetic experiments that will advance our understanding of the topo reaction chemistry and the essential role of topo in vaccinia biology.
In our hands, vaccinia topo has proved to be an instructive model system for mechanistic studies of the type IB family. The essential functional groups at the active site and the circumferential topo-DNA interface were correctly surmised by our mutational and footprint analysis of the vaccinia enzyme in advance of structure determination by x-ray crystallography. The crystal structure reveals that the catalytic domains of type IB topoisomerases and site-specific recombinases derive from a common ancestral strand transferase. A constellation of conserved amino acids catalyzes attack of the tyrosine nucleophile on the scissile phosphate. Topo IB and recombinases consist of 2 domains that form a C-shaped clamp around duplex DNA. Our work indicates that domain dynamics and DNA-induced conformational changes within the catalytic domain play a role in triggering strand scission and coordinating the strand passage or strand exchange steps.
DNA Ligase and DNA Repair
ATP-dependent DNA ligases catalyze the joining of a 5'-phosphate-terminated strand to a 3'-hydroxyl-terminated strand via 3 sequential nucleotidyl transfer reactions. In the first step, ligase attacks the alpha-phosphorus of ATP to release PPi and form a covalent intermediate (ligase-adenylate) in which AMP is linked via a phosphoamide (P-N) bond to a lysine side chain on the enzyme. In the second step, the AMP is transferred to the 5'-end of the 5'-phosphate-terminated DNA strand to form a DNA-adenylate intermediate, A(5')pp(5')N. In the third step, ligase catalyzes attack by the 3'-OH of the nick on DNA-adenylate to join the 2 polynucleotides and release AMP.
ATP-dependent ligases are ubiquitous in eukaryotes; they are also encoded by certain eubacteria, bacteriophages, and eukaryotic DNA viruses. Sequence comparisons suggest that a catalytic domain common to all ATP-dependent ligases is embellished by additional isozyme-specific protein segments at the amino or carboxyl termini. The catalytic domain includes a set of 6 collinear motifs (I, III, IIIa, IV, V, and VI) that define a superfamily of covalent nucleotidyl transferases, encompassing the ATP-dependent polynucleotide ligases and GTP-dependent mRNA capping enzymes.
The nucleotide binding pocket of DNA ligase is composed of motifs I, III, IIIa, IV, and V. The lysine in motif I (KxDGxR) is the site of covalent attachment of AMP to the enzyme. Crystallography and mutagenesis have illuminated several of the enzymic functional groups that are involved in forming the ligase-adenylate intermediate. What remains obscure is the structural basis for DNA nick recognition by DNA ligases and the basis for the chemistry of 5' end-activation (step 2) and phoshodiester bond formation (step 3).
Conversion of nicks into phosphodiester bonds is the common final step in the DNA repair and replication pathways. Nicks are potentially deleterious DNA lesions that, if not corrected, may give rise to lethal double-strand breaks. One imagines a mechanism to ensure that ligases are directed to sites where their action is required, either via the interaction of ligase with other replication or repair proteins assembled at a nick or via an intrinsic capacity to discriminate nicks from other DNA structures.
We are using vaccinia virus DNA ligase and Chlorella virus DNA ligase as model systems
(i) to dissect the the structural basis for catalysis of each of the 3 steps of the DNA ligation pathway and
(ii) to determine how ligase specifically recognizes DNA nicks.