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Outline for DNA Polymerase I article:

i) Introduction/Quick Summary

ii) History

-mention Nobel Prize of Arthur Kornberg; use: https://www.nobelprize.org/nobel_prizes/medicine/laureates/1959/

iii) Discovery

-by Arthur Kornberg in the Department of Microbiology at the Washington University School of Medicine in December 1955.

-use this journal for more details: http://www.jbc.org/content/278/37/34733

-include picture of Arthur Kornberg using this link: http://www.nature.com/nrm/journal/v7/n2/pdf/nrm1787.pdf

A) General Structure

-use structure and function paragraphs from this journal: http://www.nature.com/nrg/journal/v9/n8/full/nrg2345.html

-also use picture from the same journal

B) Primary Function

-talk about how DNA Polymerase I is not used for DNA replication, and thus, DNA Polymerase II and III were discovered.

-use this journal to talk about function: http://www.nature.com/nrg/journal/v9/n8/full/nrg2345.html

iv) Mechanism

A)Catalytic

-Two fundamental properties that all DNA polymerases share

-include picture of reaction catalyzed by DNA Polymerase using this journal: https://www.ncbi.nlm.nih.gov/books/NBK9940/

v)Eukaryotic vs Prokaryotic OR Talk about Regulation.....

vi) Research Applications (already on the page); should I leave it alone?

DNA Polymerase I
DNA Polymerase I (or Pol I) is an enzyme that participates in the process of prokaryotic DNA replication. Discovered by Arthur Kornberg in 1956,[1] it was the first known DNA polymerase (and, indeed, the first known of any kind of polymerase). It was initially characterized in E. coli and is ubiquitous in prokaryotes. In E. coli and many other bacteria, the gene that encodes Pol I is known as polA. The E. coli form of the enzyme is composed of 928 amino acids, and is an example of a processive enzyme—it can sequentially catalyze multiple polymerisations.

Discovery


Arthur Kornberg never meant to discover DNA Polymerase I. In fact, he never even aspired to become a biochemist. As a medical student at the University of Rochester Medical School, Arthur published a research paper on latent liver disease in medical students. Little did he know that this paper would interest the National Institute of Health (NIH) in his research so much that he was removed him from World War II as soon as he enlisted in the navy in 1942. In 1956, Kornberg and his colleagues used E. coli extracts to develop a DNA synthesis assay. The scientists added [14C]Thymidine so that a radioactive polymer of DNA, not RNA, could be retrieved. To initiate the purification of DNA Polymerase, the scientists added streptomycin sulfate to the E. coli extract which created a precipitate that consisted of nucleic acid-free supernatant (S-fraction) and nucleic acid-containing precipitate (P-fraction). It was discovered that the P-fraction contained heat-stable factors that were essential for the DNA synthesis reactions. These factors were identified as nucleoside triphosphate, the building blocks of nucleic acids. The S-fraction contained multiple deoxynucleoside kinases. In 1959, the Nobel Prize in Physiology or Medicine was awarded to Arthur Kornberg and Severo Ochoa “for their discovery of the mechanisms in the biological synthesis of ribonucleic acid and deoxyribonucleic acid.”

General Structure
Pol I mainly functions in the repair of damaged DNA. It contains both of the secondary structures, alpha helices and beta sheets. Therefore, Pol I is part of the alpha/beta protein superfamily protein class. The alpha/beta protein class consists of alpha and beta segments that are scattered throughout any given protein. The proteolytic fragment of E. Coli DNA Pol I consists of four domains with two separate enzymatic activities. The fourth domain consists of an exonuclease that proofreads the product of DNA Pol 1 and is able to remove any mistakes committed Pol I. The other three in the fragment domains work together to sustain DNA polymerase activity.

E. Coli bacteria contains 5 different DNA polymerases. Eukaryotic cells contain 5 different DNA polymerases: α, β, γ, δ, and ε. A total of 15 human DNA polymerases have been identified.

Structural and Functional Similarity to Other Polymerases
All DNA polymerases extend DNA only in the 5’ to 3’ direction. Thus, only the leading strand is continuously extended in the direction of replication fork movement; whereas, the lagging stranding runs discontinuously in the opposite direction as Okazaki fragments. DNA polymerases also cannot initiate DNA chains, hence, they must be initiated by short RNA or DNA segments known as primers. In order for DNA polymerization to take place, two requirements must be met. First of all, all DNA polymerases must have both a template strand and a primer strand. Second, DNA polymerases can only add nucleotides to preexisting strand. It cannot synthesize DNA from a template strand, like RNA can, because it requires a primer to do so.

The x-ray structures of the polymerase domain of all DNA polymerases have been said to resemble that of human’s right hand. All DNA polymerases contain three domains. The first domain, which is known as the 'fingers domain’ interacts with the the dNTP and the paired template base. Known as the “'palm domain,” the second domain catalyses the reaction of the transfer of the phosphoryl group. Lastly, the third domain, which is known as the 'thumb domain,” interacts with double stranded DNA.

Function
Pol I possesses four enzymatic activities:
 * 1) A 5'→3' (forward) DNA-Dependent DNA polymerase activity, requiring a 3' primer site and a template strand
 * 2) A 3'→5' (reverse) exonuclease activity that mediates proofreading
 * 3) A 5'→3' (forward) exonuclease activity mediating nick translation during DNA repair.
 * 4) A 5'→3' (forward) RNA-Dependent DNA polymerase activity. Pol I operates on RNA templates with considerably lower efficiency (0.1–0.4%) than it does DNA templates, and this activity is probably of only limited biological significance.[2]

In order to determine whether Pol I was primarily used for DNA replication or in the repair of DNA damage, an experiment was conducted with a deficient Pol I mutant strain of E. Coli. The mutant strain that lacked Pol I was isolated and treated with a mutagen. The mutant strain developed bacterial colonies that continued to grow normally and that also, lacked Pol I. This confirmed that Pol I was not required for DNA replication. However, the mutant strain also displayed characteristics which involved extreme sensitivity to certain factors that damaged DNA, like UV light. Thus, this reaffirmed that Pol I was more likely to be involved in repairing DNA damage rather than DNA replication.

Mechanism
In the replication process, RNase H removes the RNA primer (created by Primase) from the lagging strand and then Polymerase I fills in the necessary nucleotides between the Okazaki fragments (see DNA replication) in a 5'→3' direction, proofreading for mistakes as it goes. It is a template-dependent enzyme—it only adds nucleotides that correctly base pair with an existing DNA strand acting as a template. DNA Ligase then joins the various fragments together into a continuous strand of DNA.

Despite its early characterisation, it quickly became apparent that Polymerase I was not the enzyme responsible for most DNA synthesis—DNA replication in E. coli proceeds at approximately 1,000 nucleotides/second, while the rate of base pair synthesis by Polymerase I averages only between 10 and 20 nucleotides/second. Moreover, its cellular abundance of approximately 400 molecules per cell did not correlate with the fact that there are typically only two replication forks in E. coli. Additionally, it is insufficiently processive to copy an entire genome, as it falls off after incorporating only 25-50 nucleotides. Its role in replication was proven when, in 1969, John Cairns isolated a viable Polymerase I mutant that lacked the polymerase activity.[3] Cairns' lab assistant, Paula De Lucia, created thousands of cell free extracts from E.coli colonies and assayed them for DNA-polymerase activity. The 3,478th clone contained the polA mutant, which was named by Cairns to credit "Paula" [De Lucia].[4] It was not until the discovery of DNA polymerase III that the main replicative DNA polymerase was finally identified.

Studies of Pol I have confirmed that different dNTPs bind to same active site on Pol I. Thus, Pol I can distinguish between the dNTPs only after it undergoes a conformational change. Once this change has occurred, Pol I checks for proper geometry and proper alignment of the base pair, formed between bound dNTP and a matching base on the template strand. The correct geometry of A=T and G ≡ C base pairs are the only ones that fit in the active site. However, it is important to know that one in every 10^4 t to 10^5 nucleotides is added incorrectly. Nevertheless, Pol I will be there to fix this error in DNA replication.

Research Applications
DNA polymerase I obtained from E. coli is used extensively for molecular biology research. However, the 5'→3' exonuclease activity makes it unsuitable for many applications. Fortunately this undesirable enzymatic activity can be simply removed from the holoenzyme to leave a useful molecule called the Klenow fragment, widely used in molecular biology. Exposure of DNA polymerase I to the protease subtilisin cleaves the molecule into a smaller fragment, which retains only the DNA polymerase and proofreading activities. 2313541.20.