Roles of PCNA-binding and ubiquitin-binding domains in human DNA polymerase η in translesion DNA synthesis

Treatment of yeast and human cells with DNA-damaging agents elicits Rad6–Rad18-mediated monoubiquitination of proliferating cell nuclear antigen (PCNA) at its Lys-164 residue [ubiquitin (Ub)-PCNA], and this PCNA modification is indispensable for promoting the access of translesion synthesis (TLS) polymerases (Pols) to PCNA. However, the means by which K164-linked Ub modulates the proficiency of TLS Pols to bind PCNA and take over synthesis from the replicative Pol has remained unclear. One model that has gained considerable credence is that the TLS Pols bind PCNA at 2 sites, to the interdomain connector loop via their PCNA-interacting protein (PIP) domain and to the K164-linked Ub moiety via their Ub-binding domain (UBD). Specifically, this model postulates that the UBD-mediated binding of TLS Pols to the Ub moiety on PCNA is necessary for TLS. To test the validity of this model, we examine the contributions that the PIP and Ub-binding zinc finger (UBZ) domains of human Polη make to its functional interaction with PCNA, its colocalization with PCNA in replication foci, and its role in TLS in vivo. We conclude from these studies that the binding to PCNA via its PIP domain is a prerequisite for Polη''s ability to function in TLS in human cells and that the direct binding of the Ub moiety on PCNA via its UBZ domain is not required. We discuss the possible role of the Ub moiety on PCNA in TLS.     

Dominant thalassemia-like phenotypes associated with mutations in exon 3 of the beta-globin gene.

Mutations producing beta-thalassemia reach individual gene frequencies greater than .01 in malarial-endemic regions because beta-thalassemia trait individuals have increased genetic fitness over that of normal individuals. Exon 3 of the beta-globin gene has been relatively spared as a site of common beta-thalassemia mutations. Frameshifts caused by the loss of a single nucleotide and nonsense mutations produce beta-thalassemia trait when they occur in exons 1 and 2. In contrast, they usually produce chronic hemolytic anemia when present in exon 3. Certain missense mutations in exon 3 produce unstable globins and thalassemia intermedia with hemolysis in heterozygotes. Here we report two new mutations in exon 3 of the beta-globin gene. One is a single nucleotide deletion in codon 109 in a 78-year-old Lithuanian with chronic hemolytic anemia and features of thalassemia. It leads to an abnormal globin (beta Manhattan) that is elongated to 156 amino acids. The second is a CAG-CGG missense mutation at codon 127 that causes a Gln----Pro substitution (beta Houston) and a thalassemia intermedia with hemolysis in three generations of a British-American family. Although the clinical phenotypes of these two patients differed little, differences in globin-synthetic ratios were significant, presumably reflecting differences in the ability of each abnormal beta-globin to form alpha beta dimers. The paucity of high-frequency exon 3 mutations and their worldwide distribution is likely attributable to their phenotypic severity and loss of increased genetic fitness vis-a-vis malaria.     

Electrocardiographic responses to atrial pacing and multistage treadmill exercise testing. Correlation with coronary arteriography

Atrial pacing was compared with multistage treadmill exercise testing in 50 patients undergoing diagnostic cardiac catheterization to determine the diagnostic sensitivity of atrial pacing. Coronary artery disease was considered significant if luminal narrowing greater than 75 percent was present. Twenty-one subjects (Group I) had no significant coronary artery disease with vessel narrowing of less than 50 percent. Twelve (Group II) had single vessel disease and 17 (Group III) had disease of two or more vessels.The mean maximal heart rate during atrial pacing was 140/min and during exercise testing was 131/min. A positive atrial pacing test result was obtained in 5 percent of patients in Group I, 17 percent of patients in Group II and 24 percent of patients in Group III. A positive multistage treadmill exercise test result was obtained in 10 percent of patients in Group I, 67 percent of patients in Group II and 94 percent of patients in Group III. These differences are statistically significant (P < 0.001). The sensitivity of atrial pacing was 20 percent compared with 83 percent for multistage treadmill exercise testing. The specificity of atrial pacing was 95 percent compared with 90 percent for multistage treadmill exercise testing. Thus, atrial pacing is an insensitive test in the diagnosis of ischemic heart disease and does not improve the diagnostic value of multistage treadmill exercise testing.     

Discriminant scorecard for diagnosis of invasive pulmonary aspergillosis in patients with acute leukemia

Invasive pulmonary aspergillosis, a serious opportunistic infection in adult patients with acute leukemia, is difficult to diagnose antemortem. To identify patients with invasive pulmonary aspergillosis without reliance on invasive diagnostic procedures, a discriminant scorecard for invasive pulmonary aspergillosis based on clinical parameters was evaluated in a three-phase study. In phase I, the records of 62 patients, including 15 with invasive pulmonary aspergillosis, were reviewed. Eleven clinical parameters distinguished patients with invasive pulmonary aspergillosis from control subjects. These parameters were combined into a discriminant scorecard. In phase II, the discriminant scorecard was validated by a blinded, retrospective review of 94 consecutive admissions. The discriminant scorecard score was highly associated with the clinical outcome (p less than 0.0005). The sensitivity of the discriminant scorecard was calculated as a range from 62.9 to 92.8 percent and the specificity as a range from 87.5 to 98.3 percent. In phase III, the clinical utility of the discriminant scorecard was determined by its prospective application to 49 consecutive patient admissions. The discriminant scorecard identified patients with invasive pulmonary aspergillosis at an average of 4.1 days prior to clinical recognition of the disease and initiation of amphotericin B therapy. The discriminant scorecard outperformed a complex function based on multiple linear regressions, was easy to use, and did not require difficult calculations. Thus, for this patient population, the discriminant scorecard was an accurate, useful noninvasive screening test for invasive pulmonary aspergillosis. The scorecard allows more rapid clinical identification of patients with this infection and could lead to improved patient survival through earlier diagnostic and therapeutic intervention.     

Evidence that a second tumor antigen coded by adenovirus early gene region E1a is required for efficient cell transformation.

The expression of the adenovirus (Ad) early coding region 1a (E1a) is required for virus-induced cell transformation and for the activation of other viral early genes and some cellular genes. Two overlapping early mRNAs of 13S and 12S that are transcribed from this region code for a 289-amino acid protein and a 243-amino acid protein, respectively. Earlier studies have shown that the 289-amino acid protein is essential for cell transformation. We have constructed an Ad type 2 (Ad2) deletion mutant (dl231) in which the intervening sequence for the 13S mRNA is precisely removed. Mutant dl231 is completely viable in human KB cells and produces normal amounts of 13S mRNA but much reduced amounts of a defective 12S mRNA. Mutant dl231 induces focal transformation of established rat embryo fibroblasts at a frequency one-fifth to one-half that of wild-type virus. However, the transformed cells are defective in their ability to form anchorage-independent colonies on semisolid medium. Therefore, our results demonstrate that the 243-amino acid protein is required for full transformation of rat embryo cells.     

Conversion of ?X174 and fd Single-Stranded DNA to Replicative Forms in Extracts of Escherichia coli

varphiX174 and M13 (fd) single-stranded circular DNAs are converted to their replicative forms by extracts of E. coli pol A1 cells. We find that the varphiX174 DNA-dependent reaction requires Mg(++), ATP, and all four deoxynucleoside triphosphates, but not CTP, UTP, or GTP. This reaction also involves the products of the dnaC, dnaD, dnaE (DNA polymerase III), and dnaG genes, but not that of dnaF (ribonucleotide reductase). The in vitro conversion of fd single-stranded DNA to the replicative form requires all four ribonucleoside triphosphates, Mg(++), and all four deoxynucleoside triphosphates. The reaction involves the product of gene dnaE but not those of genes dnaC, dnaD, dnaF, or dnaG. The reaction with fd DNA is inhibited by rifampicin or antibody to RNA polymerase, while the reaction with varphiX174 DNA is not affected by either. With the varphiX174 DNA-dependent reaction, activities have been detected that specifically complement extracts of dnaA, dnaB, dnaC, dnaD, or dnaG mutants.     

Evidence for an altered adenovirus DNA polymerase in cells infected with the mutant H5ts149

The N complementation group of adenovirus (Ad) serotype 5 mutants, which are temperature sensitive for viral DNA synthesis in vivo, has been used to study a 140,000-dalton DNA polymerase (Pol) that copurified with the 80,000-dalton terminal protein precursor (pTP). Extracts prepared from HeLa cells infected with the N group mutant H5ts149 at nonpermissive temperature were unable to synthesize viral DNA. The defect in these extracts was specifically reversed by addition of the Pol purified from wild-type Ad-infected cytosol. Addition of the pTP, free of the Pol, did not restore replicative activity to H5ts149 extracts. The reactions studied depend on the presence of the DNA template and include the initiation reaction (the covalent attachment of dCMP to the pTP) and the selective replication of Ad DNA restriction endonuclease fragments containing the origin sequences. Glycerol gradient sedimentation showed that a replicative activity representing the pTP-Pol complex was greatly reduced in H5ts149 extracts as compared with wild-type extracts, suggesting some alteration in the mutant. A pool of pTP free of Pol was detected on these gradients in extracts from both wild-type and H5ts149-infected cells. In addition, the initiation and elongation of Ad DNA catalyzed by H5ts149 extracts prepared from cells grown at permissive temperatures was more labile to urea inactivation than extracts prepared from cells infected with wild-type virus. These results, considered together with the mapping of the H5ts149 mutation within an open reading frame approximately large enough to code for the 140,000-dalton DNA polymerase [Gingeras, T. R., Sciaky, D., Gelinas, R. E., Bing-Dong, J., Yen, C. E., Kelly, M. M., Bullock, P. A., Parsons, B. L., O'Neill, K. E. & Roberts, R. J. (1982) J. Biol. Chem. 257, 13475-13491; Alestrom, P., Akusjarui, G., Pettersson, M. & Pettersson, U. (1982) J. Biol. Chem. 257, 13492-13498], suggest that the Pol is a virally encoded protein, as is the pTP.     

Association of DNA-dependent and -independent Ribonucleoside Triphosphatase Activities with dnaB Gene Product of Escherichia coli

Preparations of E. coli dnaB gene product contain ribonucleoside triphosphatase activity that is stimulated 10-fold by DNA. The products of the triphosphatase activity are nucleoside diphosphates and P(i). The dnaB complementing activity in the varphiX174 DNA-dependent system and these triphosphatase activities copurify over the last 20-fold of an extensive (about 40,000-fold) purification procedure. Acrylamide gel electrophoresis of the purified material shows a single band of protein coincident with eluted dnaB complementing and DNA-dependent and -independent nucleoside triphosphatase activities.