Historical perspective: Arthur Kornberg, a giant of 20th century biochemistry.

For physics, the period from the beginning to the middle of the 20th century was one of great scientific excitement and revolutionary discovery. The analogous era for biochemistry, and its offspring, molecular biology, was the second half of the 20th century. One of the most important and influential leaders of this scientific revolution was Arthur Kornberg. The DNA polymerase, which he discovered in 1955 and showed to have the remarkable capacity to catalyze the template-directed synthesis of DNA, contributed in major ways to the present-day understanding of how DNA is replicated and repaired, and how it is transcribed. The discovery of DNA polymerase also permitted the development of PCR and DNA sequencing, upon which much of modern biotechnology is based. Kornberg's studies of DNA replication, which spanned a period of nearly 30 years, culminated in a detailed biochemical description of the mechanism by which a chromosome is replicated. The final years of Kornberg's life were devoted to the study of polyphosphate, which he was convinced had a crucial role in cellular function.

Endonuclease G: a role for the enzyme in recombination and cellular proliferation.

Our earlier studies had suggested that endonuclease G (EndoG), a member of the evolutionarily conserved DNA/RNA nonspecific betabetaalpha-Me-finger nuclease family, functioned in the a sequence-mediated segment inversion observed during herpes simplex virus 1 replication. To test this hypothesis, we used RNA interference to reduce the level of EndoG in mammalian cells in culture. Reduction of EndoG produced a small but statistically significant decrease in a sequence-mediated recombination, suggesting that EndoG does play a role in this process. We also observed that reduction in the level of EndoG resulted in a deficiency in cell proliferation. Cells with a reduced level of EndoG also showed changes in cell distribution in the cell cycle, producing a pattern characteristic of cells that have been arrested in the G(2) phase. These findings suggest that EndoG is required for normal cellular proliferation.

Wanderings of a DNA enzymologist: from DNA polymerase to viral latency.

I am a member of what has been called, perhaps too grandiosely, "The Greatest Generation." I grew up during the Great Depression and served in the U.S. Army during World War II. Because of my military service and the benefits of the GI Bill, I was able to attend college and, later, graduate school. Early in my graduate studies, I became fascinated with enzymes and the biochemical reactions that they catalyze. This fascination has never left me during the 50 years I have been a "DNA enzymologist." I was fortunate to have had as a mentor Arthur Kornberg, one of the great biochemists of the twentieth century, and a splendid group of postdocs and graduate students. I have studied DNA polymerases, DNA nucleases, DNA ligases, and DNA recombinases, enzymes that are critical to our understanding of DNA replication, repair, and recombination. Most recently, I have been studying herpes virus replication and inadvertently wandered into an entirely new area-viral latency.

The neural F-box protein NFB42 mediates the nuclear export of the herpes simplex virus type 1 replication initiator protein (UL9 protein) after viral infection.

The neural F-box 42-kDa protein (NFB42) is a component of the SCF(NFB42) E3 ubiquitin ligase that is expressed in all major areas of the brain; it is not detected in nonneuronal tissues. We previously identified NFB42 as a binding partner for the herpes simplex virus 1 (HSV-1) UL9 protein, the viral replication-initiator, and showed that coexpression of NFB42 and UL9 in human embryonic kidney (293T) cells led to a significant decrease in the level of UL9 protein. We have now found that HSV-1 infection promotes the shuttling of NFB42 between the cytosol and the nucleus in both 293T cells and primary hippocampal neurons, permitting NFB42 to bind to the phosphorylated UL9 protein, which is localized in the nucleus. This interaction mediates the export of the UL9 protein from the nucleus to the cytosol, leading to its ubiquitination and degradation via the 26S proteasome. Because the intranuclear localization of the UL9 protein, along with other viral and cellular factors, is an essential step in viral DNA replication, degradation of the UL9 protein in neurons by means of nuclear export through its specific interaction with NFB42 may prevent active replication and promote neuronal latency of HSV-1.

Replication-initiator protein (UL9) of the herpes simplex virus 1 binds NFB42 and is degraded via the ubiquitin-proteasome pathway.

The ubiquitin-proteasome pathway plays a critical role in the degradation of short-lived and regulatory proteins in a variety of cellular processes. The F-box proteins are part of the ubiquitin-ligase complexes, which mediate ubiquitination and proteasome-dependent degradation of phosphorylated proteins. We previously identified NFB42, an F-box protein that is highly enriched in the nervous system, as a binding partner for the herpes simplex virus 1 UL9 protein, the viral replication-initiator protein, in a yeast two-hybrid screen. In the present work, we find that coexpression of NFB42 and UL9 genes in 293T cells leads to a significant decrease in the level of UL9 protein. Treatment with the 26S-proteasome inhibitor MG132 restores the UL9 protein to normal levels. We have observed also that the UL9 protein is polyubiquitinated in vivo and that the interaction between NFB42 and the UL9 protein is dependent upon phosphorylation of the UL9 protein. These results suggest that the interaction of the UL9 protein with NFB42 results in its polyubiquitination and subsequent degradation by the 26S proteasome. They suggest further a mechanism by which latency of herpes simplex virus 1 can be established in neuronal cells.

Origin-specific unwinding of herpes simplex virus 1 DNA by the viral UL9 and ICP8 proteins: visualization of a specific preunwinding complex.

Herpes simplex virus 1 contains three origins of replication; two copies of oriS and one of a similar sequence, oriL. Here, the combined action of multiple factors known or thought to influence the opening of oriS are examined. These include the viral origin-binding protein, UL9, and single-strand binding protein ICP8, host cell topoisomerase I, and superhelicity of the DNA template. By using electron microscopy, it was observed that when ICP8 and UL9 proteins were added together to oriS-containing supertwisted DNA, a discrete preunwinding complex was formed at oriS on 40% of the molecules, which was shown by double immunolabeling electron microscopy to contain both proteins. This complex was relatively stable to extreme dilution. Addition of ATP led to the efficient unwinding of approximately 50% of the DNA templates. Unwinding proceeded until the acquisition of a high level of positive supertwists in the remaining duplex DNA inhibited further unwinding. Addition of topoisomerase I allowed further unwinding, opening >1 kb of DNA around oriS.

Endonuclease G, a candidate human enzyme for the initiation of genomic inversion in herpes simplex type 1 virus.

The herpes simplex virus type 1 (HSV-1) a sequence is present as a direct repeat at the two termini of the 152-kilobase viral genome and as an inverted repeat at the junction of the two unique components L and S. During replication, the HSV-1 genome undergoes inversion of L and S, producing an equimolar mixture of the four possible isomers. Isomerization is believed to result from recombination triggered by breakage at the a sequence, a recombinational hot spot. We have identified an enzyme in HeLa cell extracts that preferentially cleaves the a sequence and have purified it to near homogeneity. Microsequencing showed it to be human endonuclease G, an enzyme with a strong preference for G+C-rich sequences. Endonuclease G appears to be the only cellular enzyme that can specifically cleave the a sequence. Endonuclease G also showed the predicted recombination properties in an in vitro recombination assay. Based on these findings, we propose that endonuclease G initiates the a sequence-mediated inversion of the L and S components during HSV-1 DNA replication.

The human DnaJ protein, hTid-1, enhances binding of a multimer of the herpes simplex virus type 1 UL9 protein to oris, an origin of viral DNA replication.

We have identified cellular proteins that interact with the herpes simplex virus type 1 (HSV-1) origin-binding protein (UL9 protein) by screening a HeLa cell complementary DNA library by using the yeast two-hybrid system. Approximately 7 x 10(5) colonies were screened. Five of the 48 positive clones contained cDNAs that encoded the p150(Glued) component of the dynactin complex, three contained cDNAs for the neural F Box 42-kDa protein (NFB42), which is highly enriched in neural tissue, and three contained hTid-1, a human homologue of the bacterial DnaJ protein. We have focused in this report on the interaction of the viral UL9 protein with the cellular hTid-1. In vitro immunoprecipitation experiments confirmed that hTid-1 interacts with the UL9 protein. Electrophoretic mobility-shift assays indicated that the hTid-1 enhances the binding of UL9 protein to an HSV-1 origin, ori(s), and facilitates formation of the multimer from the dimeric UL9 protein. hTid-1 had no effect on the DNA-dependent ATPase or helicase activities associated with the UL9 protein. These findings implicate hTid-1 in HSV-1 DNA replication, and suggest that this cellular protein may provide a chaperone function analogous to the DnaJ protein in Escherichia coli DNA replication.