ABSTRACT
The SUN proteins are a conserved family of proteins in eukaryotes. Human UNC84A (Sun1) is a homolog of Caenorhabditis elegans UNC-84, a protein involved in nuclear anchorage and migration. We have analyzed targeting of UNC84A to the nuclear envelope (NE) and show that the N-terminal 300 amino acids are crucial for efficient NE localization of UNC84A whereas the conserved C-terminal SUN domain is not required. Furthermore, we demonstrate by combining RNA interference with immunofluorescence and fluorescence recovery after photobleaching analysis that localization and anchoring of UNC84A is not dependent on the lamin proteins, in contrast to what had been observed for C. elegans UNC-84.
Subject(s)
Lamins/physiology , Microtubule-Associated Proteins/metabolism , Nuclear Envelope/metabolism , Amino Acid Sequence , Carrier Proteins/metabolism , HeLa Cells , Humans , Membrane Proteins , Nuclear Envelope/chemistry , Nuclear Lamina/physiology , Nuclear Proteins , Protein Transport , RNA, Small Interfering/pharmacology , TransfectionABSTRACT
We describe a novel regulatory mechanism for DNA polymerase beta (Polbeta), a protein involved in DNA base excision repair (BER). Polbeta colocalized in vivo and formed a complex with the transcriptional coactivator p300. p300 interacted with Polbeta through distinct domains and acetylated Polbeta in vitro. Polbeta acetylation was furthermore observed in vivo. Lysine 72 of Polbeta was identified as the main target for acetylation by p300. Interestingly, acetylated Polbeta showed a severely reduced ability to participate in a reconstituted BER assay. This was due to an impairment of the dRP-lyase activity of Polbeta. Acetylation of Polbeta thus acts as an intranuclear regulatory mechanism and implies that p300 plays a critical regulatory role in BER.
Subject(s)
Base Pair Mismatch , DNA Polymerase beta/metabolism , DNA Repair , DNA/chemistry , Nuclear Proteins/metabolism , Trans-Activators/metabolism , Transcriptional Activation , Acetylation , Cell Line , DNA/metabolism , DNA-Directed DNA Polymerase/metabolism , Humans , Immunohistochemistry , Lysine/chemistry , Microscopy, Fluorescence , Precipitin Tests , Protein Binding , Transcription, GeneticABSTRACT
In eukaryotic cells DNA is associated with proteins to form a complex known as chromatin. The dominant proteins within this chromatin complex are the histones, which are subject to a wide variety of covalent and reversible posttranslational modifications such as acetylation. A specialized family of enzymes, the histone acetyl transferases, catalyzes the transfer of acetyl groups from their cosubstrate acetyl-coenzyme A to lysine residues of histones. Acetylation of histone N-terminal lysine residues induces chromosomal changes and results in the loss of chromosomal repression that allows the successful transcription of the underlying genes. Analogously, in DNA repair and also DNA replication the chromosomal repression is thought to be relieved by such mechanisms. Recently several publications have provided evidence that histone acetyl transferases also modify nonhistone proteins and thereby regulate their activities. This review discusses various aspects of histone acetyl transferases and summarizes recent findings which suggest a role for histone acetyl transferases in DNA repair and DNA replication.
Subject(s)
Acetyltransferases/physiology , Chromatin/physiology , DNA Repair/physiology , DNA Replication/physiology , Saccharomyces cerevisiae Proteins/physiology , Animals , Histone Acetyltransferases , Histones/metabolism , HumansABSTRACT
The Epstein-Barr virus nuclear antigen 3C (EBNA3C), encoded by Epstein-Barr virus (EBV), is essential for mediating transformation of human B lymphocytes. Previous studies demonstrated that EBNA3C interacts with a small, nonhistone, highly acidic, high-mobility group-like nuclear protein prothymosin alpha (ProT(alpha)) and the transcriptional coactivator p300 in complexes from EBV-infected cells. These complexes were shown to be associated with histone acetyltransferase (HAT) activity in that they were able to acetylate crude histones in vitro. In this report we show that ProT(alpha) interacts with p300 similarly to p53 and other known oncoproteins at the CH1 amino-terminal domain as well as at a second domain downstream of the bromodomain which includes the CH3 region and HAT domain. Similarly, EBNA3C also interacts with p300 at regions which include the CH1 and CH3/HAT domains, suggesting that ProT(alpha) and EBNAC3C may interact in a complex with p300. We also show that ProT(alpha) activates transcription when targeted to promoters by fusion to the GAL4 DNA binding domain and that this activation is enhanced by the addition of an exogenous source of p300 under the control of a heterologous promoter. This overall activity is down-modulated in the presence of EBNA3C. These results further establish the interaction of cellular coactivator p300 with ProT(alpha) and demonstrate that the associated activities resulting from this interaction, which plays a role in acetylation of histones and coactivation, can be regulated by EBNA3C. Furthermore, this study establishes for the first time a transcriptional role for ProT(alpha) in recruitment or stabilization of coactivator p300, as well as other basal transcription factors, at the nucleosomes for regulation of transcription.
