A cellular protein that competes with SV40 T antigen for binding to the retinoblastoma gene product

Tumour-suppressor genes, such as the human retinoblastoma susceptibility gene (Rb), are widely recognized as being vital in the control of cell growth and tumour formation. This role is indicated, in part, by the suppression of tumorigenicity of human tumour cells after retrovirus-mediated Rb replacement. How Rb acts to bring about this suppression is not clear but one clue is that the Rb protein forms complexes with the transforming oncoproteins of several DNA tumour viruses, and that two regions of Rb essential for such binding frequently contain mutations in tumour cells. These observations suggest that endogenous cellular proteins might exist that bind to the same regions of Rb and thereby mediate its function. We report here the identification of one such human cellular Rb-associated protein of relative molecular mass 46,000 (46K) (RbAP46). Two lines of evidence support the notion that RbAP46 and simian virus 40 T antigen have homologous Rb-binding properties: first, several mutated Rb proteins that failed to bind to T also did not associate with RbAP46; and second, both T antigen and T peptide (amino acids 101-118) were able to compete with RbAP46 for binding to Rb. The apparent targeting of the RbAP46-Rb interaction by oncoproteins of DNA tumour viruses strongly suggests that formation of this complex is functionally important.     

Cellular immortalization by a cDNA clone encoding the transformation-associated phosphoprotein p53

Malignant transformation of primary cells requires at least two distinct and characteristic alterations in cellular behaviour. The first, cellular immortality, can be induced by chemical carcinogens or by cloned oncogenes such as polyoma large T (ref. 4), adenovirus early region 1A (E1A) or the oncogene from avian (MC29) myelocytomatosis virus, v-myc. Cells whose in vitro life-span has been extended by these procedures can be fully transformed by transfection with oncogenes belonging to a different complementation group, including genes of the ras family, adenovirus E1b and polyoma virus middle T (refs 4, 5). The unstable cellular phosphoprotein p53 is frequently present at elevated levels in transformed cells and is stabilized by the formation of complexes with simian virus 40 (SV40) large T or adenovirus E1b 57K protein. Although several reports have associated p53 with cell proliferation, its role remains obscure. We have cloned complementary DNA sequences encoding murine p53 and report here that transfection of p53 expression constructs into cells of finite lifespan in vitro results in cellular immortality and susceptibility to transformation by a ras oncogene.     

A region of SV40 large T antigen can substitute for a transforming domain of the adenovirus E1A products

SV40 large T antigen contains a small region of amino acid sequence, conserved among the papovaviruses, that shows considerable similarity to conserved domain 2 of the adenovirus E1A oncogene, a domain which plays an important role in the E1A transforming functions. To learn whether the analogous SV40 T antigen sequences could substitute functionally for E1A domain 2, a chimaeric gene was constructed, coding for T antigen amino acid residues 101 to 118 in place of E1A domain 2. The resulting product showed much of the activity of the wild-type E1A products. It induced proliferation of primary BRK cells and cooperated with the ras oncogene to transform these cells fully. In addition, the chimaeric protein coprecipitated two cellular proteins whose specific binding to the E1A products depends on the presence of domain 2. The activity of the chimaeric product suggests that a similar functional unit exists in the transforming proteins of both SV40 and adenovirus, and that these proteins may exert their cell growth regulating effects through similar mechanisms.     

转录因子E2F—1的DNA结合结构域与GST的融合表达和纯化

通过聚合酶链式反应方法扩增转录因子Ｅ２Ｆ－１中ＤＮＡ结合结构域的基因片段,并将其克隆到ｐＧＥＸ－２Ｔ表达载体中,转化ＢＬ２１菌株．经ＩＰＴＧ诱导,目的蛋白在大肠杆菌中得到高效表达,其表达量达１５％．经ＧＳＴ－Ａｇａｒｏｓｅ亲合层析,目的蛋白得到了高度纯化．经胶迁移率改变实验（ｇｅｌｓｈｉｆｔｍｏｂｉｌｉｔｙａｓａｙ）证明目的蛋白具有与腺病毒Ｅ２启动子ＤＮＡ片段结合的能力．     

Adenovirus oncoproteins inactivate the Mre11-Rad50-NBS1 DNA repair complex

In mammalian cells, a conserved multiprotein complex of Mre11, Rad50 and NBS1 (also known as nibrin and p95) is important for double-strand break repair, meiotic recombination and telomere maintenance. This complex forms nuclear foci and may be a sensor of double-strand breaks. In the absence of the early region E4, the double-stranded DNA genome of adenovirus is joined into concatemers too large to be packaged. We have investigated the cellular proteins involved in this concatemer formation and how they are inactivated by E4 products during a wild-type infection. Here we show that concatemerization requires functional Mre11 and NBS1, and that these proteins are found at foci adjacent to viral replication centres. Infection with wild-type virus results in both reorganization and degradation of members of the Mre11-Rad50-NBS1 complex. These activities are mediated by three viral oncoproteins that prevent concatemerization. This targeting of cellular proteins involved in genomic stability suggests a mechanism for 'hit-and-run' transformation observed for these viral oncoproteins.     

Stimulation of protein-directed strand exchange by a DNA helicase

The protein-mediated exchange of strands between a DNA double helix and a homologous DNA single strand involves both synapsis and branch migration, which are two important aspects of any general recombination reaction. Purified DNA-dependent ATPases from Escherichia coli (recA protein), Ustilago (rec 1 protein) and phage T4 (uvsX protein) have been shown to drive both synapsis and branch migration in vitro. The T4 gene 32 protein is a helix-destabilizing protein that greatly stimulates uvsX-protein-catalysed synapsis, and the E. coli SSB (single-strand binding) protein stimulates the analogous recA-protein-mediated reaction to a lesser degree. One suspects that several other proteins also play a role in the strand exchange process. For example, a DNA helicase could in principle accelerate branch migration rates by helping to melt the helix at the branch point. The T4 dda protein is a DNA helicase that is required to move the T4 replication fork past DNA template-bound proteins in vitro. Previously, we have shown that the dda protein binds to a column that contains immobilized T4 uvsX protein. We show here that this helicase specifically stimulates the branch migration reaction that the uvsX protein catalyses as a central part of the genetic recombination process in a T4 bacteriophage-infected cell.     

Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product

The proteins encoded by the myc gene family are involved in the control of cell proliferation and differentiation, and aberrant expression of myc proteins has been implicated in the genesis of a variety of neoplasms. In the carboxyl terminus, myc proteins have two domains that encode a basic domain/helix-loop-helix and a leucine zipper motif, respectively. These motifs are involved both in DNA binding and in protein dimerization. In addition, myc protein family members share several regions of highly conserved amino acids in their amino termini that are essential for transformation. We report here that an N-terminal domain present in both the c-myc and N-myc proteins mediates binding to the retinoblastoma gene product, pRb. We show that the human papilloma virus E7 protein competes with c-myc for binding to pRb, indicating that these proteins share overlapping binding sites on pRb. Furthermore, a mutant Rb protein from a human tumour cell line that carried a 35-amino-acid deletion in its C terminus failed to bind to c-myc. Our results suggest that c-myc and pRb cooperate through direct binding to control cell proliferation.     

p53 and DNA polymerase alpha compete for binding to SV40 T antigen

The large T antigen (T) of simian virus 40 is a multifunctional protein required for both viral DNA replication and cellular transformation. T antigen forms specific protein complexes with the host protein p53 in both virus-infected and transformed cells. p53 has recently been shown to be an oncogene, but its normal function is not clear. We previously established a radioimmunoassay to study the newly described complex between T antigen and DNA polymerase alpha, and have noted a similarity between the antigenic changes induced in T by the binding of both p53 and polymerase. We now extend this analysis to a larger collection of anti-T antibodies and formally establish that p53 and DNA polymerase alpha can compete for binding to the SV40 T antigen. At a critical concentration of the three components it is possible to detect a trimeric complex of T, p53 and DNA polymerase alpha. Our observations have important implications for the control by these nuclear oncogenes of viral and cellular DNA synthesis and viral host range in both normal and transformed cells. We present a model for the action of p53 in growth control.