Analysis of E1A-mediated growth regulation functions: binding of the 300-kilodalton cellular product correlates with E1A enhancer repression function and DNA synthesis-inducing activity.

Adenovirus E1A transforming function requires two distinct regions of the protein. Transforming activity is closely linked with the presence of a region designated conserved domain 2 and the ability of this region to bind the product of the cellular retinoblastoma tumor suppressor gene. We have investigated the biological properties of the second transforming region of E1A, which is located near the N terminus. Transformation-defective mutants containing deletions in the N terminus (deletion of residues between amino acids 2 and 36) were deficient in the ability to induce DNA synthesis and repress insulin enhancer-stimulated activity. The function of the N-terminal region correlated closely with binding of the 300-kilodalton E1A-associated protein and not with binding of the retinoblastoma protein. These results indicate that transformation by E1A is mediated by two functionally independent regions of the protein which interact with different specific cellular proteins and suggest that the 300-kilodalton E1A-associated protein plays a major role in E1A-mediated cell growth control mechanisms.     

Urea-induced binding of histone 1 to nucleosomes lacking linker DNA.

The binding of H1 (and H5) to nucleosome core particles was demonstrated by separating mononucleosomes according to their DNA size on acrylamide gels containing high molarity urea. The presence of urea causes a redistribution of H1 so that it associates with some particles of all linker lengths, including no linker. When the urea is removed the H1 remains associated with particles of all DNA sizes if the different size classes are not mixed with each other. Therefore, urea can effect the transfer of H1 from particles with linker to particles with no linker. When nucleosomes of uniform DNA fragment length, some containing and some lacking H1, are re-electrophoresed under native conditions, they migrate as two widely separated bands. The mobilities of these variants do not depend on linker length and are identical to the mobilities of native H1-containing and H1-lacking particles. When the same collection of particles is electrophoresed in the presence of high molarity urea they migrate with a uniform mobility. These results suggest that H1-containing nucleosomes are conformationally different from H1-lacking particles, but that this difference is eliminated when histone-histone interactions are disrupted by urea.     

Limited temperature-sensitive transactivation by mutant adenovirus type 2 E1a proteins.

A series of linker-scanning and deletion mutations was generated in the transactivating domain of the larger, 289-amino-acid-residue E1a protein of adenovirus type 2. Mutant genes were recombined into virus to assay the ability of the variant E1a proteins to activate expression of an E1a-dependent viral gene during infection. Results of assays performed at 32, 37, and 40 degrees C indicated that at least 2 of the 10 mutants tested showed limited temperature sensitivity for transactivation.     

Characterization of an iron sensitive Mud1 mutant in E. coli lacking the ribonucleotide reductase subunit B2

The mutant, generated by a Mud1 insertion, formed long non-viable filaments in the presence of iron and air. Under anaerobic conditions normal growth in the presence of iron was observed. The mutation was mapped by P1 transductions at 48 min on the genetic map of Escherichia coli. By Southern blotting the insertion point was determined to be in nrdB, the structural gene for the ribonucleotide reductase subunit B2. The mutation could be complemented by the cloned nrdB gene. Up to now it was assumed that E. coli possesses only one enzyme for the synthesis of deoxyribunucleotides and only conditional lethal (temperature sensitive) mutants were isolated in nrdB. The insertion of Mud1 in nrdB should lead to a complete loss of the essential B2 subunit. Since the strain was able to grow under anaerobic conditions on minimal medium lacking deoxyribonucleotides an additional pathway for the synthesis of deoxyribonucleotides is postulated.     

E2F-1 regulation by an unusual DNA damage-responsive DP partner subunit

E2F activity is negatively regulated by retinoblastoma protein (pRb) through binding to the E2F-1 subunit. Within the E2F heterodimer, DP proteins are E2F partner subunits that allow proper cell cycle progression. In contrast to the other DP proteins, the newest member of the family, DP-4, downregulates E2F activity. In this study we report an unexpected role for DP-4 in regulating E2F-1 activity during the DNA damage response. Specifically, DP-4 is induced in DNA-damaged cells, upon which it binds to E2F-1 as a non-DNA-binding E2F-1/DP-4 complex. Consequently, depleting DP-4 in cells re-instates E2F-1 activity that coincides with increased levels of chromatin-bound E2F-1, E2F-1 target gene expression and associated apoptosis. Mutational analysis of DP-4 highlighted a C-terminal region, outside the DNA-binding domain, required for the negative control of E2F-1 activity. Our results define a new pathway, which acts independently of pRb and through a biochemically distinct mechanism, involved in negative regulation of E2F-1 activity.     

Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product.

The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.