Cloning the human and mouse MMS19 genes and functional complementation of a yeast mms19 deletion mutant

The MMS19 gene of the yeast Saccharomyces cerevisiae encodes a polypeptide of unknown function which is required for both nucleotide excision repair (NER) and RNA polymerase II (RNAP II) transcription. Here we report the molecular cloning of human and mouse orthologs of the yeast MMS19 gene. Both human and Drosophila MMS19 cDNAs correct thermosensitive growth and sensitivity to killing by UV radiation in a yeast mutant deleted for the MMS19 gene, indicating functional conservation between the yeast and mammalian gene products. Alignment of the translated sequences of MMS19 from multiple eukaryotes, including mouse and human, revealed the presence of several conserved regions, including a HEAT repeat domain near the C-terminus. The presence of HEAT repeats, coupled with functional complementation of yeast mutant phenotypes by the orthologous protein from higher eukaryotes, suggests a role of Mms19 protein in the assembly of a multiprotein complex(es) required for NER and RNAP II transcription. Both the mouse and human genes are ubiquitously expressed as multiple transcripts, some of which appear to derive from alternative splicing. The ratio of different transcripts varies in several different tissue types.

Critical role for the docking-protein FRS2α in FGF receptor-mediated signal transduction pathways

The docking protein FRS2α has been implicated as a mediator of signaling via fibroblast growth factor receptors (FGFRs). We have demonstrated that targeted disruption of FRS2α gene causes severe impairment in mouse development resulting in embryonal lethality at E7.0–E7.5. Experiments with FRS2α-deficient fibroblasts demonstrate that FRS2α plays a critical role in FGF-induced mitogen-activated protein (MAP) kinase stimulation, phosphatidylinositol-3 (PI-3) kinase activation, chemotactic response, and cell proliferation. Following FGF stimulation, tyrosine phosphorylated FRS2α functions as a site for coordinated assembly of a multiprotein complex that includes Gab1 and the effector proteins that are recruited by this docking protein. Furthermore, we demonstrate that different tyrosine phosphorylation sites on FRS2α are responsible for mediating different FGF-induced biological responses. These experiments establish the central role of FRS2α in signaling via FGFRs and demonstrate that FRS2α mediates multiple FGFR-dependent signaling pathways critical for embryonic development.

Interaction between human tRNA synthetases involves repeated sequence elements.

Aminoacyl-tRNA synthetases (tRNA synthetases) of higher eukaryotes form a multiprotein complex. Sequence elements that are responsible for the protein assembly were searched by using a yeast two-hybrid system. Human cytoplasmic isoleucyl-tRNA synthetase is a component of the multi-tRNA synthetase complex and it contains a unique C-terminal appendix. This part of the protein was used as bait to identify an interacting protein from a HeLa cDNA library. The selected sequence represented the internal 317 amino acids of human bifunctional (glutamyl- and prolyl-) tRNA synthetase, which is also known to be a component of the complex. Both the C-terminal appendix of the isoleucyl-tRNA synthetase and the internal region of bifunctional tRNA synthetase comprise repeating sequence units, two repeats of about 90 amino acids, and three repeats of 57 amino acids, respectively. Each repeated motif of the two proteins was responsible for the interaction, but the stronger interaction was shown by the native structures containing multiple motifs. Interestingly, the N-terminal extension of human glycyl-tRNA synthetase containing a single motif homologous to those in the bifunctional tRNA synthetase also interacted with the C-terminal motif of the isoleucyl-tRNA synthetase although the enzyme is not a component of the complex. The data indicate that the multiplicity of the binding motif in the tRNA synthetases is necessary for enhancing the interaction strength and may be one of the determining factors for the tRNA synthetases to be involved in the formation of the multi-tRNA synthetase complex.

XMCM7, a novel member of the Xenopus MCM family, interacts with XMCM3 and colocalizes with it throughout replication.

A minichromosome maintenance (MCM) protein complex has been implicated in restricting DNA replication to once per cell cycle in Xenopus egg extracts, based on the behavior of a single protein, XMCM3. Using a two-hybrid screen with XMCM3, we have identified a novel member of the MCM family in Xenopus that is essential for DNA replication. The protein shows strong homology to Saccharomyces cerevisiae MCM7 (CDC47) and has thus been named XMCM7. XMCM7 is present in a multiprotein complex with other MCM proteins. It binds to chromatin and is displaced from chromatin by the act of replication. XMCM7 does not preferentially colocalize with sites of DNA replication but colocalizes with XMCM3 throughout replication. Immunodepletion of the MCM complex from Xenopus egg extract by anti-XMCM7 antibodies inhibits DNA replication of sperm and permeable HeLa G2 nuclei but not permeable HeLa G1 nuclei. Replication capacity of the Xenopus egg extract immunodepleted of the MCM complex by anti-XMCM7 antibody can be rescued by MCM proteins eluted from anti-XMCM3 antibody. We conclude that both proteins are present in the same complex in Xenopus egg extract throughout the cell cycle, that they remain together after binding to chromatin and during DNA replication, and that they perform similar functions.

In vivo and in vitro characterization of the B1 and B2 zinc-binding domains from the acute promyelocytic leukemia protooncoprotein PML.

Acute promyelocytic leukemia (APL) has been ascribed to a chromosomal translocation event which results in a fusion protein comprising the PML protein and retinoic acid receptor alpha. PML is normally a component of a nuclear multiprotein complex which is disrupted in the APL disease state. Here, two newly defined cysteine/histidine-rich protein motifs called the B-box (B1 and B2) from PML have been characterized in terms of their effect on PML nuclear body formation, their dimerization, and their biophysical properties. We have shown that both peptides bind Zn2+, which induces changes in the peptides' structures. We demonstrate that mutants in both B1 and B2 do not form PML nuclear bodies in vivo and have a phenotype that is different from that observed in the APL disease state. Interestingly, these mutations do not affect the ability of wild-type PML to dimerize with mutant proteins in vitro, suggesting that the B1 and B2 domains are involved in an additional interaction central to PML nuclear body formation. This report in conjunction with our previous work demonstrates that the PML RING-Bl/B2 motif plays a fundamental role in formation of a large multiprotein complex, a function that may be common to those unrelated proteins which contain the motif.

Analysis of homeodomain function by structure-based design of a transcription factor.

The homeodomain is a 60-amino acid module which mediates critical protein-DNA and protein-protein interactions for a large family of regulatory proteins. We have used structure-based design to analyze the ability of the Oct-1 homeodomain to nucleate an enhancer complex. The Oct-1 protein regulates herpes simplex virus (HSV) gene expression by participating in the formation of a multiprotein complex (C1 complex) which regulates alpha (immediate early) genes. We recently described the design of ZFHD1, a chimeric transcription factor containing zinc fingers 1 and 2 of Zif268, a four-residue linker, and the Oct-1 homeodomain. In the presence of alpha-transinduction factor and C1 factor, ZFHD1 efficiently nucleates formation of the C1 complex in vitro and specifically activates gene expression in vivo. The sequence specificity of ZFHD1 recruits C1 complex formation to an enhancer element which is not efficiently recognized by Oct-1. ZFHD1 function depends on the recognition of the Oct-1 homeodomain surface. These results prove that the Oct-1 homeodomain mediates all the protein-protein interactions that are required to efficiently recruit alpha-transinduction factor and C1 factor into a C1 complex. The structure-based design of transcription factors should provide valuable tools for dissecting the interactions of DNA-bound domains in other regulatory circuits.

Elongin BC complex prevents degradation of von Hippel-Lindau tumor suppressor gene products

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene causes the familial cancer syndrome, VHL disease, characterized by a predisposition to renal cell carcinoma and other tumor types. Loss of VHL gene function also is found in a majority of sporadic renal carcinomas. A preponderance of the tumor-disposing inherited missense mutations detected in VHL disease are within the elongin-binding domain of VHL. This region mediates the formation of a multiprotein VHL complex containing elongin B, elongin C, cul-2, and Rbx1. This VHL complex is thought to function as an E3 ubiquitin ligase. Here, we report that VHL proteins harboring mutations which disrupt elongin binding are unstable and rapidly degraded by the proteasome. In contrast, wild-type VHL proteins are directly stabilized by associating with both elongins B and C. In addition, elongins B and C are stabilized through their interactions with each other and VHL. Thus, the entire VHL/elongin complex is resistant to proteasomal degradation. Because the elongin-binding domain of VHL is frequently mutated in cancers, these results suggest that loss of elongin binding causes tumorigenesis by compromising VHL protein stability and/or potential VHL ubiquitination functions.

Transcription regulation by inflexibility of promoter DNA in a looped complex.

The gal operon of Escherichia coli is negatively regulated by repressor binding to bipartite operators separated by 11 helical turns of DNA. Synergistic binding of repressor to separate sites on DNA results in looping, with the intervening DNA as a topologically closed domain containing the two promoters. A closed DNA loop of 11 helical turns, which is in-flexible to torsional changes, disables the promoters either by resisting DNA unwinding needed for open complex formation or by impeding the processive DNA contacts by an RNA polymerase in flux during transcription initiation. Interaction between two proteins bound to different sites on DNA modulating the activity of the intervening segment toward other proteins by allostery may be a common mechanism of regulation in DNA-multiprotein complexes.

Characterization of the VHL tumor suppressor gene product: localization, complex formation, and the effect of natural inactivating mutations.

The human VHL tumor suppressor gene has been implicated in the inherited disorder von Hippel-Lindau disease and in sporadic renal carcinoma. The homologous rat gene encodes a 185-amino acid protein that is 88% sequence identical to the aligned 213-amino acid human VHL gene product. When expressed in COS-7 cells, both the human and the rat VHL proteins showed predominant nuclear, nuclear and cytosolic, or predominant cytosolic VHL staining by immunofluorescence. A complicated pattern of cellular proteins was seen that could be specifically coimmunoprecipitated with the introduced VHL protein. A complex containing VHL and proteins of apparent molecular masses 16 and 9 kDa was the most consistently observed. Certain naturally occurring VHL missense mutations demonstrated either complete or partial loss of the p16-p9 complex. Thus, the VHL tumor suppressor gene product is a nuclear protein, perhaps capable of specifically translocating between the nucleus and the cytosol. It is likely that VHL executes its functions via formation of specific multiprotein complexes. Identification of these VHL-associated proteins will likely clarify the physiology of this tumor suppressor gene.