Structure of yeast poly(A) polymerase alone and in complex with 3'-dATP.

Polyadenylate [poly(A)] polymerase (PAP) catalyzes the addition of a polyadenosine tail to almost all eukaryotic messenger RNAs (mRNAs). The crystal structure of the PAP from Saccharomyces cerevisiae (Pap1) has been solved to 2.6 angstroms, both alone and in complex with 3'-deoxyadenosine triphosphate (3'-dATP). Like other nucleic acid polymerases, Pap1 is composed of three domains that encircle the active site. The arrangement of these domains, however, is quite different from that seen in polymerases that use a template to select and position their incoming nucleotides. The first two domains are functionally analogous to polymerase palm and fingers domains. The third domain is attached to the fingers domain and is known to interact with the single-stranded RNA primer. In the nucleotide complex, two molecules of 3'-dATP are bound to Pap1. One occupies the position of the incoming base, prior to its addition to the mRNA chain. The other is believed to occupy the position of the 3' end of the mRNA primer.

Structure-function relationships in the Saccharomyces cerevisiae poly(A) polymerase. Identification of a novel RNA binding site and a domain that interacts with specificity factor(s).

We have constructed deletions in the nonconserved regions at the amino and carboxyl ends of the poly(A) polymerase (PAP) of Saccharomyces cerevisiae and examined the effects of these truncations on function of the enzyme. PAP synthesizes a poly(A) tail onto the 3'-end of RNA without any primer specificity but, in the presence of cellular factors, is directed specifically to the cleaved ends of mRNA precursors. The last 31 amino acids of PAP are dispensable for both nonspecific and specific activities. Removal of the next 36 amino acids affects an RNA binding domain, which is essential for the activity of the enzyme and for cell viability. This novel RNA binding site was further localized using additional deletions, cyanogen bromide cleavage of PAP cross-linked with RNA or 8-azido-ATP, and a monoclonal antibody against a COOH-terminal PAP epitope. A deletion that partially disrupts this domain has reduced nonspecific activity but functions in specific polyadenylation. In contrast, deletion of the first 18 amino acids of PAP has no effect on nonspecific polyadenylation but completely eliminates specific activity. This region is essential for enzyme function in vivo and is probably involved in the interaction of PAP with other protein(s) of the polyadenylation machinery.

Monoclonal antibodies to yeast poly(A) polymerase (PAP) provide evidence for association of PAP with cleavage factor I.

Purified yeast poly(A) polymerase (PAP) was used to produce monoclonal antibodies which recognize the enzyme in immunoblots. Epitope mapping using truncated forms of PAP and cyanogen bromide cleavage products revealed two classes of antibodies. One class (N-term) recognizes an epitope in the first 100 amino acids, and a second class (C-term) is specific for a determinant located in the last 20 amino acids of PAP. These C-terminal 20 amino acids can be removed without affecting the nonspecific poly(A) addition activity of the purified enzyme. Neither antibody inhibits the nonspecific poly(A) polymerase activity or the sequence-specific activity observed in processing extracts. The antibodies show species specificity and cannot recognize mammalian, Xenopus, or vaccinia PAP. The C-term antibodies can deplete PAP from yeast whole cell extracts, resulting in loss of poly(A) addition activity. This immunodepletion also causes a reduction in the cleavage activity which can be restored by addition of yeast cleavage factor I [CF I; Chen, J., & Moore, C. (1992) Mol. Cell Biol. 12, 3470-3481], a factor needed for both the cleavage and poly(A) addition reactions. This demonstrates that a complex of PAP and CF I exists in extracts in the absence of ATP or exogenous RNA substrate. The monoclonal antibodies against yeast PAP will be a useful tool for further study of factors required for yeast mRNA 3' end processing.

Overexpression of human DNA topoisomerase I in insect cells using a baculovirus vector.

The 3645-bp human DNA topoisomerase I cDNA isolated by D'Arpa et al. (Proc. Natl. Acad. Sci. USA 85, 1988, 2543-2547) was integrated into the Autographa californica multiple nuclear polyhedrosis virus genome. The recombinant protein was expressed by infecting the SF9 insect cell line with this baculovirus and resulted in a 100-fold overexpression of human DNA topoisomerase I compared to the level found in human cell lines. This 100-kDa recombinant protein has the same electrophoretic mobility as the human DNA topoisomerase I from HeLa cells and is recognized by topoisomerase I-specific monoclonal antibody. The recombinant DNA topoisomerase I was isolated and purified to homogeneity with a two-step fractionation protocol and has a specific activity of 2 x 10(6) U/mg. Enzymatic properties such as stimulation by magnesium and inhibition by camptothecin resemble properties of the enzyme purified from human cell lines.

Regulation of poly(ADP-ribose) transferase activity by 2',5'-oligoadenylates.

pppA2'pA2'pA appears to be a potent natural noncompetitive inhibitor of poly (ADP-ribose) transferase activity in the histone dependent reaction of ADP-ribosylation with Ki=5 microM. Moreover, it is a noncompetitive inhibitor of the Mg2+ dependent reaction of autoADPRT-ribosylation with Ki=20 microM. The activity of ADPRT falls down abruptly both in the cytoplasm and nuclei of mouse L-cells treated with interferon. In contrast, the activities of 2',5'-oligo (A) polymerase and 2'-phosphodiesterase remain virtually unchanged after the treatment with ADPRT preparation. The regulation of ADPRT activity and active form of ADPRT by 2',5-oligoadenylates is presumed to be one of the factors responsible for inducing the antiviral and/or antiproliferative effects of interferon.