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The Arthur Kornberg Papers: The Synthesis of DNA, 1953 …

Cells often spend much more energy repairing DNA than synthesizing it.

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Arthur Kornberg's Discovery of DNA Polymerase I

DNA synthesis requires a primer strand with a free 3’-hydroxyl terminus annealed to a DNA template strand and the deoxynucleotide triphosphates form base pairs with the template. Addition is in the 5’ to 3’ direction with release of pyrophosphate. The enzyme is active with DNAs containing single stranded gaps and also with DNAs with single-strand breaks or nicks. Under some conditions, RNA-DNA hybrids and an RNA duplex may serve as template-primer (Setlow 1972).

The Synthesis of DNA, 1953-1959 ..

Our goal is to elucidate the fundamental basis of gene regulation. We study the control of transcription, the first step in the pathway of gene expression. Current work focuses on discovery of the molecular machines involved in transcription, reconstitution of the process with purified components, structure determination of the transcription machinery, and structure-function relationships in chromatin, the natural DNA template for transcription.

Highlights of work from the past three years include:

1. Discovery of a human homolog of the 20-protein yeast Mediator complex. Mediator is the central processing unit of gene regulation, receiving both positive and negative inputs and transducing the information to the transcription machinery.

2. Structure determination of 10-subunit, half million Dalton RNA polymerase II, in the act of transcription, with template DNA and protduct RNA, by X-ray crystallography at atomic resolution.

3. Structure determination of the entire transcription initiation complex by two-dimensional protein crystallography.

4. Discovery of a stably altered nucleosome produced by a purified chromatin-remodeling complex.

Current work is directed towards the structure of the entire transcription apparatus at atomic resolution and the mechanism of transcription control in living cells.

Enzymatic synthesis of DNA by Arthur Kornberg ..

In humans, DNA polymerase α is composed of 4 subunits, two of which begin the primer by synthesizing RNA complementary to the template DNA.

RSC and SWI/SNF chromatin-remodeling complexes were previously reported to generate a stably altered nucleosome. We now describe the formation of hybrids between nucleosomes of different sizes, showing that the stably altered structure is a noncovalent dimer. A basis for dimer formation is suggested by an effect of RSC on the supercoiling of closed, circular arrays of nucleosomes. The effect may be explained by the interaction of RSC with DNA at the ends of the nucleosome, which could lead to the release 60--80 bp or more from the ends. DNA released in this way may be trapped in the stable dimer or lead to alternative fates such as histone octamer transfer to another DNA or sliding along the same DNA molecule.

The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 A resolution. Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site. Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3' end of the RNA in the nucleotide addition site. The 3' end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking. The 5'-most residue of the RNA is close to the point of entry to an exit groove. Changes in protein structure between the transcribing complex and free enzyme include closure of a clamp over the DNA and RNA and ordering of a series of "switches" at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid. Protein-nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, "abortive cycling" during transcription initiation, and RNA and DNA translocation during transcription elongation.

Biochem synthesis of dna - SlideShare

starts the synthesis of DNA

DNA polymerase I is the predominant polymerizing enzyme found in E. coli. It contains a single disulfide bond and one sulfhydryl group (Jovin et al. 1969b). Five distinct DNA polymerases have been isolated from E. coli and have been designated I, II, III, IV, and V. DNA polymerase I functions to fill DNA gaps that arise during DNA replication, repair, and recombination. DNA polymerase II also functions in editing and proofreading mainly in the lagging strand (Kim et al. 1997, Wagner and Nohmi 2000). DNA polymerase III is the main replicative enzyme. DNA polymerase IV and V have large active sites that allow for more base misincorporation, and are therefore more error-prone. They also lack proofreading-exonuclease subunits to correct misincorporations (Nohmi 2006, and Hastings et al. 2010). DNA polymerase V is present at significant levels only in SOS-induced cells and over-expression restricts DNA synthesis (Marsh and Walker 1985).

This enzyme needs the 4 deoxynucleoside 5'-triphosphates, primer DNA, and template DNA and directs the synthesis of a DNA molecule following the sequence of the template strand.

Then, it is thought that DNA polymerase δ or ε takes over to synthesize most of the Okazaki fragment.
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  • Enzymatic Synthesis of DNA. - [PDF Document]

    Kornberg's success in unraveling the process of coenzyme synthesis established him as a biochemist by the early 1950s

  • Kornberg, A.: Enzymatic Synthesis of DNA

    ARTHUR KORNBERG, shared (with Severo Ochoa) the Nobel Prize in Medicine in 1959 for his laboratory synthesis of DNA

  • He was the first to accomplish the cell-free synthesis of DNA

    The synthesis of DNA.

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His writings include Enzymatic Synthesis of DNA (1961)

During transcription in E. coli, the DNA-dependent RNA polymerase locates specific promoter sequences in the DNA template, melts a small region containing the transcription start site, initiates RNA synthesis, processively elongates the transcript, and finally terminates and releases the RNA product. Each step is regulated by interactions between the polymerase, the DNA, the nascent RNA, and a variety of regulatory proteins and ligands. The E. coli enzyme contains a catalytic core of two alpha-subunits, one beta- and one beta'-subunit, with relative molecular masses (Mr) of 36,512, 150,619 and 155,162, respectively. The holoenzyme has an additional regulatory subunit, normally sigma, of Mr 70,236. Preparations may also contain the omega-subunit (Mr approximately 10,000), which can be removed without affecting any known properties of the enzyme. Because the amino-acid sequences of the beta- and beta'-subunits are homologous to those of the largest subunits of the yeast, Drosophila and murine RNA polymerases, it seems likely that essential features of the three-dimensional structure and catalytic mechanism of RNA polymerase are also conserved across species. Crystals of RNA polymerase suitable for X-ray analysis have not yet been obtained, but two-dimensional crystals of E. coli RNA polymerase holoenzyme can be grown on positively charged lipid layers. Electron microscopy of these crystals in negative stain shows the enzyme in projection as an irregularly shaped complex approximately 100 x 100 x 160 A in size. We have now determined the three-dimensional structure by electron microscopy of negatively stained, two-dimensional crystals tilted at various angles to the incident electron beam. We find a structure in RNA polymerase similar to the active-site cleft of DNA polymerase I. In the light of functional similarities between these two enzymes, together with other evidence, this probably identifies the active-site region of RNA polymerase.

he had elucidated all of the basic features of DNA synthesis that ..

ABFI (ARS-binding protein I) is a yeast protein that binds specific DNA sequences associated with several autonomously replicating sequences (ARSs). ABFI also binds sequences located in promoter regions of some yeast genes, including DED1, an essential gene of unknown function that is transcribed constitutively at a high level. ABFI was purified by specific binding to the DED1 upstream activating sequence (UAS) and was found to recognize related sequences at several other promoters, at an ARS (ARS1), and at a transcriptional silencer (HMR E). All ABFI-binding sites, regardless of origin, provided weak UAS function in vivo when examined in test plasmids. UAS function was abolished by point mutations that reduced ABFI binding in vitro. Analysis of the DED1 promoter showed that two ABFI-binding sites combine synergistically with an adjacent T-rich sequence to form a strong constitutive activator. The DED1 T-rich element acted synergistically with all other ABFI-binding sites and with binding sites for other multifunctional yeast activators. An examination of the properties of sequences surrounding ARS1 left open the possibility that ABFI enhances the initiation of DNA replication at ARS1 by transcriptional activation.

Kornberg: RNA Priming of DNA Replication

Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4 gene was on a high-copy plasmid. Purification of GAL4 by a procedure including affinity chromatography on a GAL4-binding DNA column yielded not only GAL4 but also a second protein, shown to be GAL80 by its reaction with an antipeptide antibody. Sequence comparisons of GAL4 and other members of a family of proteins sharing homologous cysteine finger motifs identified an additional region of homology in the middle of these proteins shown by genetic analysis to be important for GAL4 function. GAL4 could be cleaved proteolytically at the boundary of the conserved region, defining internal and carboxy-terminal folded domains.

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