Mutations in the XPD helicase gene result in XP and TTD phenotypes, preventing interaction between XPD and the p44 subunit of TFIIH.

In most cases, xeroderma pigmentosum group D (XP-D) and trichothiodystrophy (TTD) patients carry mutations in the carboxy-terminal domain of the evolutionarily conserved helicase XPD, which is one of the subunits of the transcription/repair factor TFIIH (refs 1,2). In this study, we demonstrate that XPD interacts specifically with p44, another subunit of TFIIH, and that this interaction results in the stimulation of 5'-->3' helicase activity. Mutations in the XPD C-terminal domain, as found in most patients, prevent the interaction with p44, thus explaining the decrease in XPD helicase activity and the nucleotide excision repair (NER) defect.

Dissecting the interaction network of multiprotein complexes by pairwise coexpression of subunits in E. coli.

Using the human basal transcription factors TFIID and TFIIH as examples, we show that pairwise coexpression of polypeptides in Escherichia coli can be used as a tool for the identification of specifically interacting subunits within multiprotein complexes. We find that coexpression of appropriate combinations generally leads to an increase in the solubility and stability of the polypeptides involved, which means that large amounts of the resulting complexes can immediately be obtained for subsequent biochemical and structural analysis. Furthermore, we demonstrate that the solubilization and/or the proper folding of a protein by its natural partner can be used as a monitor for deletion mapping to determine precise interaction domains. Coexpression can be used as an alternative or complementary approach to conventional techniques for interaction studies such as yeast two-hybrid analysis, GST pulldown and immunoprecipitation.

Mutations in the amino-terminal domain of the human poly(ADP-ribose) polymerase that affect its catalytic activity but not its DNA binding capacity.

Poly-ADP ribosylation of nuclear proteins is activated when poly(ADP-ribose) polymerase (PARP), a nuclear zinc-finger enzyme, binds to single-strand DNA breaks. To understand how the signal emerging from its DNA-binding domain (DBD) bound to such breaks is transduced to its catalytic domain, the structure-function relationship of the DBD was investigated. We have used mutagenesis by the polymerase chain reaction (PCR) to generate a random library of PARP mutants. In this work, we describe the identification of catalytically inactive mutants bearing single point mutations, located outside the two zinc fingers in the DBD, that have conserved their full capacity to bind DNA. The results obtained demonstrate that the DNA-dependent activation of PARP requires not only a capacity to bind DNA but also a number of crucial residues to maintain a conformation of the domain necessary to transfer an 'activation signal' to the catalytic domain.

Poly(ADP-ribose) polymerase: structure-function relationship.

Dissection of the human poly(ADP-ribose) polymerase (PARP) molecule in terms of its structure-function relationship has proved to be an essential step towards understanding the biological role of poly(ADP-ribosylation) as a cellular response to DNA damage in eukaryotes. Current approaches aimed at elucidating the implication of this multifunctional enzyme in the maintenance of the genomic integrity will be presented.

Inflammatory disease with use of IUD.

PIP: Drs. Edelman and Bergers' report "Contraceptive practice and tuboovarian abscess (Am. J. Obstet. Gynecol. 138:541, 1980) may produce the impression in the medical profession that the IUD does not predispose to salpingitis, salpingo-oophoritis, and tubo-ovarian abscess, as published data and clinical experience would suggest. Also, the diagnostic criteria for diagnosing 'acute pelvic inflammatory disease' stated in the report, and published studies of Jacobson and Westrom and Chaparro et al question a diagnosis of pelvic inflammatory disease that is not confirmed endoscopically or by some direct visualization obtained surgically. 35% of patients who had laparoscopy by Jacobson and Westrom and who were suspected of having salpingitis, or pelvic inflammatory disease, and 54% of laparoscoped patients suspected by Chaparoo et.al. of having pelvic inflammatory disease were found not to have either salpingitis or pelvic inflammatory disease of gynecologic etiology. As pelvic inflammation may be caused by a variety of disorders, such as appendicitis, colitis, diverticulitis, and others, the term pelvic inflammatory disease is an imprecise diagnostic term. Edelman and Berger's results can also be questioned on the ground that numerous reports (e.g., Second Report on Intrauterine Contraceptive Devices, Food and Drug Administration, 1978; Population Reports, Series B, No. 3, May 1979, the Johns Hopkins University) indicate an increased incidence of salpingitis with its attendant pelvic crippling pain and infertility that is many times more common in IUD users than in nonIUD users. Available published data therefore strongly suggest that an IUD user is at far greater risk of developing inflammatory disease of infectious etiology in her reproductive tract with its attendant pain, morbidity, infertility, and even death than nonIUD users.^ieng

Sex education for teens debated.

PIP: Letter emphasizing the importance of sex education for unmarried teenagers, stressing findings that the risk of pregnancy soon after beginning intercourse may be greater than that reported in an earlier article, and that unmarried teenagers may be more sexually active than married couples. The writer also notes the importance of including males in sex education efforts, and that education of adults about the need for sex education for children seems called for.^ieng

Structural basis for the recognition of a nucleoporin FG repeat by the NTF2-like domain of the TAP/p15 mRNA nuclear export factor.

TAP-p15 heterodimers have been implicated in the export of mRNAs through nuclear pore complexes (NPCs). We report a structural analysis of the interaction domains of TAP and p15 in a ternary complex with a Phe-Gly (FG) repeat of an NPC component. The TAP-p15 heterodimer is structurally similar to the homodimeric transport factor NTF2, but unlike NTF2, it is incompatible with either homodimerization or Ran binding. The NTF2-like heterodimer functions as a single structural unit in recognizing an FG repeat at a hydrophobic pocket present only on TAP and not on p15. This FG binding site interacts synergistically with a second site at the C terminus of TAP to mediate mRNA transport through the pore. In general, our findings suggest that FG repeats bind with a similar conformation to different classes of transport factors.

Molecular structure of human TFIIH.

TFIIH is a multiprotein complex required for both transcription and DNA repair. Single particles of human TFIIH were revealed by electron microscopy and image processing at a resolution of 3.8 nm. TFIIH is 16 x 12.5 x 7.5 nm in size and is organized into a ring-like structure from which a large protein domain protrudes out. A subcomplex assembled from five recombinant core subunits also forms a circular architecture that can be superimposed on the ring found in human TFIIH. Immunolabeling experiments localize several subunits: p44, within the ring structure, forms the base of the protruding protein density which includes the cdk7 kinase, cyclin H, and MAT1. Within the ring structure, p44 was flanked on either side by the XPB and XPD helicases. These observations provide us with a quartenary organizational model of TFIIH.