The focus of this study is the interaction between DNA and I-Msol, an endonuclease. An endonuclease is a protein that cuts DNA at a certain sequence with a certain length; I-Msol cuts DNA at approximately 20-24 base pairs and is highly specific in what sequence it is designed to cut.
The interaction between DNA and an endonuclease like I-Msol is difficult to determine just by looking at the amino acid sequence of the protein. The difficulty arises because of the many molecular interactions such as hydrogen bonding and electrostatic interactions are affected by how the protein specifically interacts with the DNA, 3-dimensionally. The best method of understanding these complexities is to create model that considers all these dynamics of the DNA/protein interaction. Thus, a I-Msol/DNA model was created in silico to study the areas of contact between the two macromolecules as well as what happened when changes were made to the base pairs of the DNA.
In summary:
* It is impossible to predict DNA and protein interaction by looking at the amino acid sequence
* hydrogen-bonding and electrostatic interactions alter amino acid sequence
* I-Msol is a endonuclease, which cuts DNA around 20-24 base pairs with high specificity
* Preliminary testing resulted in determining many of areas of contact between I-Msol and the DNA molecule
* The researches made base pair substitutions that affected the interaction of the protein with the DNA
Results
After the protein-DNA contacts were redesigned, then, in this complex, the amino acids that were likely to cause disruption were changed. These amino acids were categorized on affinity for the designed protein for the new site and the decrease in affinity for the original wild type I-Msol enzyme As from the table, the largest predicted change came from the -6C*G, + 6 C*G, because this automatically prevents direct hydrogen bonding. Different amino acids had different disruption predictions.
Using a formula, the binding energies were calculated by using the value of the wild type and then subtracting the facilitate comparison. A difference map represented certain values such as electron density for the new amino acids. A gel electrophoretic shift was done to observe the binding specificity.
As the experiment indicates the experimental changes in binding affinities differed from the predicted ones. The results indicate that the future looks very bright for medicine and biochemistry as the redesign of the of DNA complexes is predicted to allow us to make mass breakthroughs in the future.
Upon completion of the experiment we were able to examine the DNA. First, the electrophorese
Miller, Kenneth R. and Joseph S. Levine. “Chapter 12: DNA and RNA.” Biology. Upper Saddle River: Pearson Education, Inc., 2002. Print.
The molecule consisted of a double helix with phosphates, deoxyribose sugar molecules, and nitrogenous bases. If the spirals were split, the DNA could replicate, which explained why genes were transferred from parents to their children. Additionally, the order of compounds on the DNA indicated that there was a unique ‘code’ on each strand. Watson and Crick believed that this ‘code’ was translated into specific proteins. , ,
Also in a PRC reaction, DNA Polymerase is made of many complicated proteins with the function of duplicating DNA before division occurs (2).
3. Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS. Eric Wickstrom, Ed. Wiley-Liss, Inc., NY, 1991. pp 25-33, 35-51, 125-141.
...l electrophoresis (SCGE) also known as comet assay has become one of the standard methods for assessing DNA damage, with applications ranging from testing genotoxicity, human bio-monitoring and molecular epidemiology to its use in fundamental research in DNA damage and repair (Collins, 2004). The comet assay is a simple method for detecting DNA strand breaks within cells in eukaryotes. The procedure of comet assay includes Embedding the cells in agarose in a microscope slide, followed by lysing of cells with detergent and high salts to form nucleotides containing supercoiled loops of DNA linked to the nuclear matrix, and then undergoing Electrophoresis at high pH, which results in formation of structures resembling as comets, observed by fluorescence microscopy. The intensity of the comet tail relative to the head reflects the number of DNA breaks (Collins, 2004).
... starts relaxing the supercoils and altering of DNA and interacts with DNA helicase SGS1 and plays a role in DNA recombination, also cellular aging and maintenance of genome stability. Alternate splicing results in multiple transcript variants. Additional spliced variants of the gene have been described, but their complete length is unknown.
Due to the nature of amino acids, a titration curve can be employed to identify
Tsou, J. A., Hagen, J. A., Carpenter, C. L., & Laird-Offringa, I. A. (2002, August 05). DNA
Modern techniques , rather than the gene map , maps the map of the DNA within the gene itself : the positions of short sequences " marker " are used as markers signaling over the cromosssomas . Once a gene is discovered, it is necessary to unravel its base sequence prior to its function being studied . The sequencing has become easier with the development of methods for cloning the DNA - producing large amounts of identical fragments. In the method most widely used DNA sequencing , the chain is denatured into single strands . These are then used as templates for DNA synthesis , but such that replication to as the double helix reaches a certain growth in the mold base . In addition to provide DNA polymerase and the four bases, A - G -C- T, also using small amounts of these dideoxynucleotide bases. This is incorporated , as the normal bases, the double helix growth but prevent the continuation of the chain. The fragments are then separated by gel electrophoresis and the base seq...
n.d. - n.d. Peptides and Proteins. Proteins. Retrieved July 25, 2008, from http://www.cd http://www.cem.msu.edu/reusch/VirtualText/protein2.htm Ophardt, C. E. (2003).
A polypeptide chain is a series of amino acids that are joined by the peptide bonds. Each amino acid in a polypeptide chain is called a residue. It also has polarity because its ends are different. The backbone or main chain is the part of the polypeptide chain that is made up of a regularly repeating part and is rich with the potential for hydrogen-bonding. There is also a variable part, which comprises the distinct side chain. Each residue of the chain has a carbonyl group, which is good hydrogen-bond acceptor, and an NH group, which is a good hydrogen-bond donor. The groups interact with the functional groups of the side chains and each other to stabilize structures. Proteins are polypeptide chains that have 500 to 2,000 amino acid residues. Oligopeptides, or peptides, are made up of small numbers of amino acids. Each protein has a precisely defined, unique amino acid sequence, referred to as its primary structure. The amino acid sequences of proteins are determined by the nucleotide sequences of genes because nucleotides in DNA specify a complimentary sequence in RNA, which specifies the amino acid sequence. Amino acid sequences determine the 3D structures of proteins. An alteration in the amino acid sequence can produce disease and abnormal function. All of the different ways
Record, C.~F. Anderson and T.~M. Lohman, ‘Thermodynamic Analysis of Ion Effects on the Binding and Conformational Equilibria of Proteins and Nucleic Acids: The Roles of Ion Association or Release, Screening, and Ion Effects on Water Activity’, Q.~Rev.~Biophys., 11.June 1978 (1978), 103–78 .
The Use of Recombinant DNA Technology Recombinant DNA technology is the technology of preparing recombinant DNA in vitro by cutting up DNA molecules and splicing together fragments from more than one organism.(1) This is the process of using recombinant DNA technology to enable the rapid production of human protein from a single gene of insulin. Firstly the single gene required must be isolated. This can be done three ways: Either by working backwards from the protein- Finding the amino acid sequence for the protein needed, the order of bases can be established using known genetic code. New DNA can be made from this sequence of bases resulting in artificial gene made from complementary DNA.
The scientific and medical progress of DNA as been emense, from involving the identification of our genes that trigger major diseases or the creation and manufacture of drugs to treat these diseases. DNA has many significant uses to society, health and culture of today. One important area of DNA research is that used for genetic and medical research. Our abi...