Claudia Gruss

Phosphorylation by Protein Kinase CK2 Changes the DNA Binding Properties of the Human Chromatin Protein DEK

ABSTRACT We have examined the posttranslational modification of the human chromatin protein DEK and found that DEK is phosphorylated by the protein kinase CK2 in vitro and in vivo. Phosphorylation sites were mapped by quadrupole ion trap mass spectrometry and found to be clustered in the C-terminal region of the DEK protein. Phosphorylation fluctuates during the cell cycle with a moderate peak during G1 phase. Filter binding assays, as well as Southwestern analysis, demonstrate that phosphorylation weakens the binding of DEK to DNA. In vivo, however, phosphorylated DEK remains on chromatin. We present evidence that phosphorylated DEK is tethered to chromatin throughout the cell cycle by the un- or underphosphorylated form of DEK.

Large T-Antigen Double Hexamers Imaged at the Simian Virus 40 Origin of Replication

ABSTRACT The initial step of simian virus 40 (SV40) DNA replication is the binding of the large tumor antigen (T-Ag) to the SV40 core origin. In the presence of Mg2+ and ATP, T-Ag forms a double-hexamer complex covering the complete core origin. By using electron microscopy and negative staining, we visualized for the first time T-Ag double hexamers bound to the SV40 origin. Image processing of side views of these nucleoprotein complexes revealed bilobed particles 24 nm long and 8 to 12 nm wide, which indicates that the two T-Ag hexamers are oriented head to head. Taking into account all of the biochemical data known on the T-Ag–DNA interactions at the replication origin, we present a model in which the DNA passes through the inner channel of both hexamers. In addition, we describe a previously undetected structural domain of the T-Ag hexamer and thereby amend the previously published dimensions of the T-Ag hexamer. This domain we have determined to be the DNA-binding domain of T-Ag.

In vitro chromatin remodelling by chromatin accessibility complex (CHRAC) at the SV40 origin of DNA replication

DNA replication is initiated by binding of initiation factors to the origin of replication. Nucleosomes are known to inhibit the access of the replication machinery to origin sequences. Recently, nucleosome remodelling factors have been identified that increase the accessibility of nucleosomal DNA to transcription regulators. To test whether the initiation of DNA replication from an origin covered by nucleosomes would also benefit from the action of nucleosome remodelling factors, we reconstituted SV40 DNA into chromatin in Drosophila embryo extracts. In the presence of T‐antigen and ATP, a chromatin‐associated cofactor allowed efficient replication from a nucleosomal origin in vitro. In search of the energy‐dependent cofactor responsible we found that purified ‘chromatin accessibility complex’ (CHRAC) was able to alter the nucleosomal structure at the origin allowing the binding of T‐antigen and efficient initiation of replication. These experiments provide evidence for the involvement of a nucleosome remodelling machine in structural changes at the SV40 origin of DNA replication in vitro.

Functional Domains of the Ubiquitous Chromatin Protein DEK

ABSTRACT DEK was originally described as a proto-oncogene protein and is now known to be a major component of metazoan chromatin. DEK is able to modify the structure of DNA by introducing supercoils. In order to find interaction partners and functional domains of DEK, we performed yeast two-hybrid screens and mutational analyses. Two-hybrid screening yielded C-terminal fragments of DEK, suggesting that DEK is able to multimerize. We could localize the domain to amino acids 270 to 350 and show that multimerization is dependent on phosphorylation by CK2 kinase in vitro. We also found two DNA binding domains of DEK, one on a fragment including amino acids 87 to 187 and containing the SAF-box DNA binding motif, which is located between amino acids 149 and 187. This region is sufficient to introduce supercoils into DNA. The second DNA binding domain is located between amino acids 270 and 350 and thus overlaps the multimerization domain. We show that the two DNA-interacting domains differ in their binding properties and in their abilities to respond to CK2 phosphorylation.

Large T antigen on the simian virus 40 origin of replication: a 3D snapshot prior to DNA replication

Large T antigen is the replicative helicase of simian virus 40. Its specific binding to the origin of replication and oligomerization into a double hexamer distorts and unwinds dsDNA. In viral replication, T antigen acts as a functional homolog of the eukaryotic minichromosome maintenance factor MCM. T antigen is also an oncoprotein involved in transformation through interaction with p53 and pRb. We obtained the three‐dimensional structure of the full‐length T antigen double hexamer assembled at its origin of replication by cryoelectron microscopy and single‐particle reconstruction techniques. The double hexamer shows different degrees of bending along the DNA axis. The two hexamers are differentiated entities rotated relative to each other. Isolated strands of density, putatively assigned to ssDNA, protrude from the hexamer–hexamer junction mainly at two opposite sites. The structure of the T antigen at the origin of replication can be understood as a snapshot of the dynamic events leading to DNA unwinding. Based on these results a model for the initiation of simian virus 40 DNA replication is proposed.

Prothymosin α associates with the oncoprotein SET and is involved in chromatin decondensation

Prothymosin α (ProTα) is a histone H1‐binding protein that interacts with the transcription coactivator CREB‐binding protein and potentiates transcription. Based on coimmunoprecipitation and mammalian two‐hybrid assays, we show here that ProTα forms a complex with the oncoprotein SET. ProTα efficiently decondenses human sperm chromatin, while overexpression of GFP–ProTα in mammalian cells results in global chromatin decondensation. These results indicate that decondensation of compacted chromatin fibers is an important step in the mechanism of ProTα function.

INFLUENCE OF CORE HISTONE ACETYLATION ON SV40 MINICHROMOSOME REPLICATION IN VITRO

We have used the SV40 in vitro replication system to analyze the replication efficiencies of SV40 minichromosomes associated with normal or hyperacetylated histones. We found that elongation of replication occurs with higher efficiency in hyperacetylated minichromosomes in comparison with normal minichromosomes. Our results indicate that the movement of the replication machinery through nucleosomal DNA is facilitated by charge neutralization due to acetylation of the histone tails.

Structure of replicating chromatin.

Publisher Summary This chapter summarizes the structural changes occurring in chromatin as replication-dependent events. The large genome of a eukaryotic cell is organized in the nucleus as a complex nucleoprotein structure, chromatin. The most dramatic changes in chromatin structure occur during genome replication, when advancing replication forks invade the parental chromatin and when new chromatin is assembled on emerging progeny DNA strands. Several methods are particularly popular among investigators studying the replication of chromatin. A technically simple and widely used method is the supercoiling assay. The formation of chromatin on replicated DNA depends on two different processes—namely, (1) the transmission of “old” prefork nucleosomes from unreplicated to replicated DNA and (2) the assembly of new nucleosomes from newly synthesized histones. The structure of parental nucleosome core particles most probably changes when invaded by the approaching replication fork because experiments reveal the presence of old histone H3/H4 tetramers on nascent DNA closest to the replication fork.

High-affinity SV40 T-antigen binding sites in the human genome

We describe two different approaches to isolate human genomic sequences possessing high-affinity binding sites for the simian virus 40 (SV40) large T antigen. First, SV40 T antigen was added to Sau3A-restricted human DNA; the resulting T-antigen-DNA complexes were collected after repeated passages through nitrocellulose filters. The second approach involves the specific immunoprecipitation of chromatin fragments, generated by Sau3A treatment of nuclear chromatin from SV40-transformed human cells. The DNA fragments obtained were cloned in plasmid vectors for further investigation. Using the filter binding approach we isolated four different fragments with high-affinity binding sites. The binding site in one fragment was related to the strong T-antigen binding site I in the SV40 genome. The other three fragments contained multiple recognition pentamers, GA(G)GGC. Only one fragment with a high-affinity binding site was identified among the immunoprecipitable chromatin fragments. This DNA fragment belongs to the L1 family of human repetitive DNA. We present evidence suggesting that a significant fraction of human L1 elements possesses T-antigen binding sites. L1-related sequences appear as extrachromosomal elements in an SV40-transformed human cell line, and the amount of extrachromosomal L1 DNA was found to increase after fusion of transformed cells to permissive monkey cells.

[7] The SV40 minichromosome

Publisher Summary Simian virus 40 (SV40) has served as a useful model system for numerous studies in the fields of eukaryotic gene transcription, DNA replication, and cell transformation. A reason for the popularity of SV40 as a model system is that the viral genome is small enough to be handled in standard biochemical procedures without much danger of damage or breakage, and yet it contains several interesting sequence elements that make it a eukaryotic genome in a nutshell. Thus, the viral genome contains promoters with complex arrays of regulatory elements as typically found in regulated cellular genes. The viral genome is organized as chromatin, which has many structural features in common with cellular chromatin. Therefore, the viral chromatin complex is frequently referred to as the “SV40 minichromosome.” The chapter summarizes the current methods to isolate and characterize minichromosomes from infected cells. SV40 chromatin is a densely packed nucleoprotein particle when extracted from nuclei of infected cells under low-salt conditions. These particles contain the four core histones, H2A, H2B, H3, and H4, in stoichiometric amounts as well as the outer histone H1 and a variety of nonhistone chromatin proteins. SV40 minichromosomes have served as excellent models for the study of chromatin assembly during replication.