Crystal Structure of the Vaccinia Virus DNA Polymerase Holoenzyme Subunit D4 in Complex with the A20 N-Terminal Domain

Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase E9, the uracil-DNA glycosylase D4 and A20, a protein with no known enzymatic activity. The D4/A20 heterodimer is the DNA polymerase co-factor whose function is essential for processive DNA synthesis. Genetic and biochemical data have established that residues located in the N-terminus of A20 are critical for binding to D4. However, no information regarding the residues of D4 involved in A20 binding is yet available. We expressed and purified the complex formed by D4 and the first 50 amino acids of A20 (D4/A20_1–50). We showed that whereas D4 forms homodimers in solution when expressed alone, D4/A20_1–50 clearly behaves as a heterodimer. The crystal structure of D4/A20_1–50 solved at 1.85 Å resolution reveals that the D4/A20 interface (including residues 167 to 180 and 191 to 206 of D4) partially overlaps the previously described D4/D4 dimer interface. A20_1–50 binding to D4 is mediated by an α-helical domain with important leucine residues located at the very N-terminal end of A20 and a second stretch of residues containing Trp43 involved in stacking interactions with Arg167 and Pro173 of D4. Point mutations of the latter residues disturb D4/A20_1–50 formation and reduce significantly thermal stability of the complex. Interestingly, small molecule docking with anti-poxvirus inhibitors selected to interfere with D4/A20 binding could reproduce several key features of the D4/A20_1–50 interaction. Finally, we propose a model of D4/A20_1–50 in complex with DNA and discuss a number of mutants described in the literature, which affect DNA synthesis. Overall, our data give new insights into the assembly of the poxvirus DNA polymerase cofactor and may be useful for the design and rational improvement of antivirals targeting the D4/A20 interface.     

Rpb4, a Subunit of RNA Polymerase II, Enables the Enzyme To Transcribe at Temperature Extremes In Vitro

Rpb4 is a subunit of Saccharomyces cerevisiae RNA polymerase II (Pol II). It associates with the polymerase preferentially in stationary phase and is essential for some stress responses. Using the promoter-independent initiation and chain elongation assay, we monitored Pol II enzymatic activity in cell extracts. We show here that Rpb4 is required for the polymerase activity at temperature extremes (10 and 35°C). In contrast, at moderate temperature (23°C) Pol II activity is independent of Rpb4. These results are consistent with the role previously attributed to Rpb4 as a subunit whose association with Pol II helps Pol II to transcribe during extreme temperatures. The enzymatic inactivation of Pol II lacking Rpb4 at the nonoptimal temperature was prevented by the addition of recombinant Rpb4 produced in Escherichia coli prior to the in vitro reaction assay. This finding suggests that modification of Rpb4 is not required for its functional association with the other Pol II subunits. Sucrose gradient and immunoprecipitation experiments demonstrated that Rpb4 is present in the cell in excess over the Pol II complex during all growth phases. Nevertheless, the rescue of Pol II activity at the nonoptimal temperature by Rpb4 is possible only when cell extracts are obtained from postlogarithmic cells, not from logarithmically growing cells. This result suggests that Pol II molecules should be modified in order to recruit Rpb4; the portion of the modified Pol II molecules is small during logarithmic phase and becomes predominant in stationary phase.     

The Molecular Chaperone Calnexin Interacts with the NSP4 Enterotoxin of Rotavirus In Vivo and In Vitro

Calnexin is an endoplasmic reticulum (ER)-associated molecular chaperone proposed to promote folding and assembly of glycoproteins that traverse the secretory pathway in eukaryotic cells. In this study we examined if calnexin interacts with the ER-associated luminal (VP7) and transmembrane (NSP4) proteins of rotavirus. Only glycosylated NSP4 interacted with calnexin and did so in a time-dependent manner (half-life, 20 min). In vitro translation experiments programmed with gene 10 of rhesus rotavirus confirmed that calnexin recognizes only glycosylated NSP4. Castanospermine (a glucosidase I and II inhibitor) experiments established that calnexin associates only with partly deglucosylated (di- or monoglucosylated) NSP4. Furthermore, enzymatic removal of the remaining glucose residues on the N-linked glycan units was essential to disengage the NSP4-calnexin complex. Novel experiments with castanospermine revealed that glucose trimming and the calnexin-NSP4 interaction were not critical for the assembly of infectious virus.     

A Complex Containing RNA Polymerase II, Paf1p, Cdc73p, Hpr1p, and Ccr4p Plays a Role in Protein Kinase C Signaling

Yeast contains at least two complex forms of RNA polymerase II (Pol II), one including the Srbps and a second biochemically distinct form defined by the presence of Paf1p and Cdc73p (X. Shi et al., Mol. Cell. Biol. 17:1160–1169, 1997). In this work we demonstrate that Ccr4p and Hpr1p are components of the Paf1p-Cdc73p-Pol II complex. We have found many synthetic genetic interactions between factors within the Paf1p-Cdc73p complex, including the lethality of paf1Δ ccr4Δ, paf1Δ hpr1Δ, ccr4Δ hpr1Δ, and ccr4Δ gal11Δ double mutants. In addition, paf1Δ and ccr4Δ are lethal in combination with srb5Δ, indicating that the factors within and between the two RNA polymerase II complexes have overlapping essential functions. We have used differential display to identify several genes whose expression is affected by mutations in components of the Paf1p-Cdc73p-Pol II complex. Additionally, as previously observed for hpr1Δ, deleting PAF1 or CDC73 leads to elevated recombination between direct repeats. The paf1Δ and ccr4Δ mutations, as well as gal11Δ, demonstrate sensitivity to cell wall-damaging agents, rescue of the temperature-sensitive phenotype by sorbitol, and reduced expression of genes involved in cell wall biosynthesis. This unusual combination of effects on recombination and cell wall integrity has also been observed for mutations in genes in the Pkc1p-Mpk1p kinase cascade. Consistent with a role for this novel form of RNA polymerase II in the Pkc1p-Mpk1p signaling pathway, we find that paf1Δ mpk1Δ and paf1Δ pkc1Δ double mutants do not demonstrate an enhanced phenotype relative to the single mutants. Our observation that the Mpk1p kinase is fully active in a paf1Δ strain indicates that the Paf1p-Cdc73p complex may function downstream of the Pkc1p-Mpk1p cascade to regulate the expression of a subset of yeast genes.     

RNA聚合酶II中的CTD结构在mRNA转录和加工偶联过程中的重要作用

RNA聚合酶II最大亚基末端的羧基端结构域（CTD）是由以七氨基酸（-Tyr-Ser-Pro-Thr-Ser- Pro-Ser-）为重复单位的高度保守的重复序列组成的。因其结构与功能上的特殊性,它在近年来受到学术界的广泛关注。诸多的实验表明,该结构可通过与mRNA加工因子的相互作用而对其加工过程产生直接或间接的影响,在mRNA的转录和加工的偶联过程中具有重要的作用。本文即是根据近年来的实验结果对其结构和功能做出的综述性介绍。     

The C-Terminal Domain of Sin1 Interacts with the SWI-SNF Complex in Yeast

In the yeast Saccharomyces cerevisiae, the SWI-SNF complex has been proposed to antagonize the repressive effects of chromatin by disrupting nucleosomes. The SIN genes were identified as suppressors of defects in the SWI-SNF complex, and the SIN1 gene encodes an HMG1-like protein that has been proposed to be a component of chromatin. Specific mutations (sin mutations) in both histone H3 and H4 genes produce the same phenotypic effects as do mutations in the SIN1 gene. In this study, we demonstrate that Sin1 and the H3 and H4 histones interact genetically and that the C terminus of Sin1 physically associates with components of the SWI-SNF complex. In addition, we demonstrate that this interaction is blocked in the full-length Sin1 protein by the N-terminal half of the protein. Based on these and additional results, we propose that Sin1 acts as a regulatable bridge between the SWI-SNF complex and the nucleosome.     

Connecting Mutations of the RNA Polymerase II C-Terminal Domain to Complex Phenotypic Changes Using Combined Gene Expression and Network Analyses

The C-terminal domain (CTD) of the largest subunit in DNA-dependent RNA polymerase II (RNAP II) is essential for mRNA synthesis and processing, through coordination of an astounding array of protein-protein interactions. Not surprisingly, CTD mutations can have complex, pleiotropic impacts on phenotype. For example, insertions of five alanine residues between CTD diheptads in yeast, which alter the CTD''s overall tandem structure and physically separate core functional units, dramatically reduce growth rate and result in abnormally large cells that accumulate increased DNA content over time. Patterns by which specific CTD-protein interactions are disrupted by changes in CTD structure, as well as how downstream metabolic pathways are impacted, are difficult to target for direct experimental analyses. In an effort to connect an altered CTD to complex but quantifiable phenotypic changes, we applied network analyses of genes that are differentially expressed in our five alanine CTD mutant, combined with established genetic interactions from the Saccharomyces cerevisiae Genome Database (SGD). We were able to identify candidate genetic pathways, and several key genes, that could explain how this change in CTD structure leads to the specific phenotypic changes observed. These hypothetical networks identify links between CTD-associated proteins and mitotic function, control of cell cycle checkpoint mechanisms, and expression of cell wall and membrane components. Such results can help to direct future genetic and biochemical investigations that tie together the complex impacts of the CTD on global cellular metabolism.     

The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

The protein p21(Cip1, Waf1, Sdi1) is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase delta. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.     

FERM Domain Containing Protein 7 Interacts with the Rho GDP Dissociation Inhibitor and Specifically Activates Rac1 Signaling

The FERM domain containing protein 7 gene (FRMD7) associated with the X-linked disorder idiopathic congenital nystagmus (ICN) is involved in the regulation of neurite elongation during neuronal development. Members of the Rho family of small G-proteins (Rho GTPases) are key regulators of the actin cytoskeleton and are implicated in the control of neuronal morphology. The Rho GDP dissociation inhibitor alpha, RhoGDIα, the main regulator of Rho GTPases, can form a complex with the GDP-bound form of Rho GTPases and inhibit their activation. Here, we demonstrate that the full length of the mouse FRMD7, rather than the N-terminus or the C-terminus alone, directly interacts with RhoGDIα and specifically initiates Rac1 signaling in mouse neuroblastoma cell line (neuro-2a). Moreover, we show that wild-type human FRMD7 protein is able to activate Rac1 signaling by interacting with RhoGDIα and releasing Rac1 from Rac1-RhoGDIα complex. However, two missense mutations (c.781C>G and c.886G>C) of human FRMD7 proteins weaken the ability to interact with RhoGDIα and release less Rac1, that induce the activation of Rac1 to a lesser degree; while an additional mutant, c.1003C>T, which results in a C-terminal truncated protein, almost fails to interact with RhoGDIα and to activate Rac1 signaling. Collectively, these results suggest that FRMD7 interacts with one of the Rho GTPase regulators, RhoGDIα, and activates the Rho subfamily member Rac1, which regulates reorganization of actin filaments and controls neuronal outgrowth. We predict that human mutant FRMD7 thus influences Rac1 signaling activation, which can lead to abnormal neuronal outgrowth and cause the X-linked ICN.