Mutations in the middle domain of yeast poly(A) polymerase affect interactions with RNA but not ATP

The eukaryotic poly(A) polymerase (PAP) is responsible for the posttranscriptional extension of mRNA 3′ ends by the addition of a poly(A) tract. The recently published three-dimensional structures of yeast and bovine PAPs have made a more directed biochemical analysis of this enzyme possible. Based on these structures, the middle domain of PAP was predicted to interact with ATP. However, in this study, we show that mutations of conserved residues in this domain of yeast PAP, Pap1, do not affect interaction with ATP, but instead disrupt the interaction with RNA and affect the enzyme’s ability to process substrate lacking 2′ hydroxyls at the 3′ end. These results are most consistent with a model in which the middle domain of PAP interacts directly with the recently extended RNA and pyrophosphate byproduct

Novel interactions at the essential N-terminus of poly(A) polymerase that could regulate poly(A) addition in Saccharomyces cerevisiae

AbstractAddition of poly(A) to the 3′ ends of cleaved pre-mRNA is essential for mRNA maturation and is catalyzed by Pap1 in yeast. We have previously shown that a non-viable Pap1 mutant lacking the first 18 amino acids is fully active for polyadenylation of oligoA, but defective for pre-mRNA polyadenylation, suggesting that interactions at the N-terminus are important for enzyme function in the processing complex. We have now identified proteins that interact specifically with this region. Cft1 and Pta1 are subunits of the cleavage/polyadenylation factor, in which Pap1 resides, and Nab6 and Sub1 are nucleic-acid binding proteins with known links to 3′ end processing. Our results suggest a novel mechanism for controlling Pap1 activity, and possible models invoking these newly-discovered interactions are discussed.Structured summary of protein interactionsPAP1 binds to Fip1 by anti bait coimmunoprecipitation (View interaction)PAP1 binds to Fip1 by pull down (View interaction)PAP1 physically interacts with PTA1 by two hybrid (View interaction)PAP1 binds to Sub1 by pull down (View interaction)PAP1 physically interacts with Fip1 by two hybrid (View Interaction: 1, 2)PAP1 binds to Nab6 by pull down (View interaction)Nab6 physically interacts with PAP1 by two hybrid (View interaction)Cft1 binds to PAP1 by pull down (View interaction)PTA1 binds to PAP1 by pull down (View interaction

Comparative analysis of the end-joining activity of several DNA ligases

<div><p>DNA ligases catalyze the repair of phosphate backbone breaks in DNA, acting with highest activity on breaks in one strand of duplex DNA. Some DNA ligases have also been observed to ligate two DNA fragments with short complementary overhangs or blunt-ended termini. In this study, several wild-type DNA ligases (phage T3, T4, and T7 DNA ligases, <i>Paramecium bursaria</i> chlorella virus 1 (PBCV1) DNA ligase, human DNA ligase 3, and <i>Escherichia coli</i> DNA ligase) were tested for their ability to ligate DNA fragments with several difficult to ligate end structures (blunt-ended termini, 3′- and 5′- single base overhangs, and 5′-two base overhangs). This analysis revealed that T4 DNA ligase, the most common enzyme utilized for <i>in vitro</i> ligation, had its greatest activity on blunt- and 2-base overhangs, and poorest on 5′-single base overhangs. Other ligases had different substrate specificity: T3 DNA ligase ligated only blunt ends well; PBCV1 DNA ligase joined 3′-single base overhangs and 2-base overhangs effectively with little blunt or 5′- single base overhang activity; and human ligase 3 had highest activity on blunt ends and 5′-single base overhangs. There is no correlation of activity among ligases on blunt DNA ends with their activity on single base overhangs. In addition, DNA binding domains (Sso7d, hLig3 zinc finger, and T4 DNA ligase N-terminal domain) were fused to PBCV1 DNA ligase to explore whether modified binding to DNA would lead to greater activity on these difficult to ligate substrates. These engineered ligases showed both an increased binding affinity for DNA and increased activity, but did not alter the relative substrate preferences of PBCV1 DNA ligase, indicating active site structure plays a role in determining substrate preference.</p></div

Schematic representation of DNA ligase fusions.

<p>All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.</p

Wild type DNA ligase λ DNA digest ligation assay.

<p>Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, <b>1</b>), NruI (G/C Blunt, <b>2</b>), BstNI (5′ SBO, <b>3</b>), Hpy188I (3′SBO, <b>4</b>), NdeI (2 BO, <b>5</b>) and BamHI (4 BO, <b>6</b>), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl₂) or NEBNext^® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.</p

Measured binding constants.

<p>Measured binding constants.</p