DNA Structure and Replication: A Detailed Overview

DNA Structure and Replication Genetic Material Francis Crick - co-discoverer of the 3 dimensional structure of DNA in 1953 DNA - "A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way" Genetic Material Friedrich Miesch...

DNA Structure and Replication Genetic Material Francis Crick - co-discoverer of the 3 dimensional structure of DNA in 1953 DNA - "A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way" Genetic Material Friedrich Miescher - a Swiss physician and biochemist who described the DNA in the mid-19th century. o He isolated nuclei from white blood cells in pus on soiled bandages, and he found out that there is an unusual acidic substance containing nitrogen and phosphorus in the nuclei. Genetic Material o Nuclein - was the term called by Miescher in an 1871 paper since the material was discovered in cell nuclei o It was then called nucleic acid Genetic Material Archibald Garrod - an English Physician who was the first one to link inherited disease and protein in 1902. o he noted that people born with certain errors of metabolism lacks certain enzymes. Genetic Material Frederick Griffith - an English microbiologist who took the first step in identifying DNA as the genetic material. o Through studying pneumonia during the years' post-1918 flu pandemic, he noticed that mice with a certain form of pneumonia harbored one of two types of Streptococcus pneumoniae bacteria. Genetic Material Type R bacteria - rough in texture Type S bacteria - smooth since they were enclosed in a polysaccharide capsule. Genetic Material Findings: o Mice injected with type R bacteria did not develop pneumonia. o when injected with the latter, the mice did. It is because type R did not have a protective coat to shield them from the organism's immune system. This is how the type S bacteria cause severe or virulent infection--because of the capsule's presence. o Heating type S bacteria killed them, which no longer causes pneumonia in mice. o The mixture of type R bacteria with heat-killed type S bacteria caused death to mice because of pneumonia. Genetic Material Griffith termed the phenomenon as transformation, he suggested that the transforming principle might be some part of the polysaccharide capsule or some compound required for capsule synthesis. Genetic Material Oswald Avery, Colin MacLeod, and Maclyn McCarty were U.S. physicians who hypothesized that a nucleic acid might be Griffith’s "transforming principle." Genetic Material By adding enzymes that either destroy proteins (protease) or DNA (deoxyribonuclease or DNase) to bacteria that were broken apart to release their contents, they demonstrated that DNA transforms bacteria-and that protein does not. Genetic Material Their observation: o treating broken-open type S bacteria with a protease did not prevent the transformation of a nonvirulent strain, but treating such bacteria with deoxyribonuclease or DNase did disrupt transformation. ▪ Protease - an enzyme that dismantles the protein. ▪ DNase - an enzyme that dismantles the DNA only. o 1944 - they confirmed that DNA transformed the bacteria. Genetic Material They concluded: DNA passed from type S bacteria into type R, enabling the type R to manufacture the smooth coat necessary for infection. Once type R bacteria encase themselves in smooth coats, they are no longer type R. Protein is NOT the Hereditary Molecule Alfred Hershey and Martha Chase - U.S. microbiologists who used Escherichia coli bacteria infected with a virus that consisted largely of a protein "head" surrounding DNA in 1953. Protein is NOT the Hereditary Molecule Viruses infect bacterial cells by injecting their DNA or RNA into them. Then, the infected bacteria produce more viruses. The viral protein coats remain outside the bacterial cells. Culture medium - a way that researchers can analyze viruses in which the medium. It contains a radioactive chemical that the viruses take up. Protein is NOT the Hereditary Molecule Their experiment: o "Labeled" viral nucleic acid - emits radiations o The two microbiologists observed if protein contains sulfur but not phosphorus, and that nucleic acids contain phosphorus but not sulfur. o Viruses grew in the presence of radioactive sulfur ▪ Viral protein coats took up and emitted radioactivity. ▪ had their protein marked, but not their DNA (because protein incorporates sulfur, but DNA does not). Protein is NOT the Hereditary Molecule o Viruses grew in the presence of radioactive phosphorus ▪ the viral DNA emitted radioactivity. ▪ had their DNA marked, but not their protein, because this element is found in DNA but not protein. o If protein is the genetic material, then the infected bacteria would have radioactive sulfur. But if DNA is the genetic material, then the bacteria would have radioactive phosphorus. Protein is NOT the Hereditary Molecule ▪ In the end, viruses labeled with sulfur, the virus coats were radioactive, but the virus-infected bacteria containing viral DNA were not. ▪ In the other tube, where the virus had incorporated radioactive phosphorus, the virus coats carried no radioactive label, but the infected bacteria did. Protein is NOT the Hereditary Molecule Conclusion: Part of the virus could enter the bacteria and direct them to mass-produce more virus was the part that had incorporated phosphorus--the DNA. Therefore, the genetic material is DNA. Discovery of the Structure of DNA Phoebus Levene - Russian-American biochemist who identified the 5-carbon sugar ribose as part of some nucleic acids in 1909. He discovered deoxyribose in other nucleic acids in 1929. ▪ The major chemical distinction between DNA and RNA: RNA has ribose, while DNA has deoxyribose. Discovery of the Structure of DNA o He also discovered the three parts of a nucleic acid ▪ Sugar ▪ nitrogen-containing base ▪ Phosphorus-containing component Discovery of the Structure of DNA In the early 1950s, Erwin Chargaff showed that DNA in several species contains equal amounts of the bases adenine (A) and thymine (T) and equal amounts of the bases guanine (G) and cytosine (C). Discovery of the Structure of DNA Maurice Wilkins (English physicist) and Rosalind Franklin (English chemist) - bombarded DNA with X-rays using a technique called X-ray diffraction and then deduced the overall structure of the molecule from the patterns in which the X rays were deflected. Discovery of the Structure of DNA o Franklin distinguished two forms of DNA-a dry, crystalline "A" form and the wetter type seen in cells, the "B" form. It took her 100 hours to obtain "photo 51" of the B form. o Wilkins showed photo 51 to Watson at the end of January 1953 made the men realize that the symmetry of the molecule fits the shape of a regular helix. Discovery of the Structure of DNA Linus Pauling - a famed biochemist who suggested a triple helix structure for DNA, but it was incorrect Discovery of the Structure of DNA Watson and Crick found the answer using cardboard cut outs of the DNA components when Watson was playing with the cutouts while waiting for a meeting with Crick. Discovery of the Structure of DNA o When he assembled A next to T and G next to C, he noted the similar shapes, and suddenly all the pieces fit. o Hydrogen bonds are the chemical attractions between the DNA bases that create the "steps" of the double helix. DNA Structure Gene - a section of a DNA molecule whose sequence of building blocks specifies the sequence of amino acids in a particular protein. Inherited traits are diverse because proteins have diverse functions. Malfunctioning or inactive proteins can devastate health. DNA Structure Nucleotide - a single building block of DNA that consists of one deoxyribose sugar, one phosphate group (a phosphorus atom bonded to four oxygen atoms), and one nitrogenous base. Purines have a two-ring structure. These are adenine (A) and guanine (G). Pyrimidines have a single-ring structure. These are cytosine (C) and thymine (T). Nitrogenous bases - information-containing parts of DNA because they form sequences. DNA Structure Polynucleotide chains - combinations of multiple nucleotides attached by strong attachments called phosphodiester bonds between the deoxyribose sugars and the phosphates. This creates a continuous sugar- phosphate backbone Antiparallelism - the opposing orientation of the two nucleotide chains in a DNA molecule. o The carbons of deoxyribose are numbered from 1 to 5, starting with the carbon found by moving clockwise from the oxygen. o One strand of the double helix runs in a 5′ to 3′ direction and the other strand runs in a 3′ to 5′ direction DNA Structure Complementary base pairs - specific purine- pyrimidine couples o A is paired with T, and G is paired with C. Hydrogen bonds - chemical attractions that hold the DNA base pairs together. o A hydrogen atom on one molecule is attracted to an oxygen atom or nitrogen atom on another molecule. o They are weak individually, but over the many bases of a DNA molecule, they impart great strength. ▪ 2 hydrogen bonds join each A and T. ▪ 3 hydrogen bonds join each G and C. DNA Configuration Several types of proteins compress DNA without damaging or tangling it. Scaffold proteins form frameworks that guide DNA strands. Histones - proteins; DNA coils around them. o Together, they form structures that resemble beads on a string. DNA Configuration Nucleosome - DNA "bead" DNA wraps at several levels until it is compacted into a chromatid. o Chromatid - a chromosome consisting of one double helix, in the unreplicated form. The fifth type of histone protein anchors nucleosomes to short "linker" regions of DNA, which tighten the nucleosomes into fibers. At any given time, only small sections of the DNA double helix are exposed. DNA Configuration Chromatin - "colored material;" it is a chromosome substance. o it is about 30 percent histone proteins, 30 percent scaffold proteins and other proteins that bind DNA, 30 percent DNA, and 10 percent RNA. DNA Configuration When chromatin is loose (not condensed into chromosomes that are visible upon staining), it forms loops at about 10,000 places in the genome. o An "anchor" protein called CTCF brings together parts of the DNA sequence within the same long DNA molecule to form the overall "loop-ome" structure. o Loop formation - is not done by in random manner o Chromatin loops - rarely overlap and affect swaths of the DNA sequence that is smaller than 2 million base pairs. Progeria, the genetic disorder that resembles rapid aging, disrupts chromatin binding to the nuclear envelope. DNA Replication DNA must be copied, or replicated, so that the information it holds can be maintained and passed to future cell generations, even as that information is accessed to guide the manufacture of proteins. DNA Replication When two strands of the DNA double helix unwind and separates, the exposed unpaired bases would attract their complements from free, unattached nucleotides available in the cell from nutrients. The two identical double helices would form from one original, the parental double helix. o Semiconservative - each new DNA double helix conserves half of the original. DNA Replication Matthew Meselson and Franklin Stahl - they demonstrated the semiconservative mechanism of DNA replication with a series of "density shift" experiments in 1957. They labeled replicating DNA from bacteria with a dense, heavy form of nitrogen and traced the pattern of distribution of the nitrogen. The higher- density nitrogen was incorporated into one strand of each daughter's double helix. DNA Replication The semiconservative model ruled out mechanisms such as: o Conservative mechanism ▪ replicating a daughter DNA double helix built of entirely "heavy" labeled nucleotides o Dispersive mechanism ▪ a daughter double helix in which both strands were composed of joined pieces of "light" and "heavy" nucleotides. DNA Replication DNA amplification - a biotechnology that researchers use when replicating DNA conducted outside cells Polymerase chain reaction (PCR) - first and best-known DNA amplification technique. It uses DNA polymerase to rapidly replicate a specific DNA sequence in a test tube. DNA Replication PCR requires the following: ✓ Knowing a target DNA sequence from the suspected pathogen ✓ Two types of lab-made, single-stranded short pieces of DNA called primers these are complementary in sequence to opposite ends of the target sequence ✓ Many copies of the four types of DNA nucleotides DNA Replication ✓ Taq1, which is a DNA polymerase from a bacterium that lives in hot springs. The enzyme eases PCR because it withstands heat, which is necessary to separate the DNA strands. ✓ In the first step of PCR, the temperature is raised. ✓ The temperature is then lowered. ▪ This separates the two strands of the target DNA DNA Replication ✓ Taq1, which is a DNA polymerase from a bacterium that lives in hot springs. The enzyme eases PCR because it withstands heat, which is necessary to separate the DNA strands. ✓ In the first step of PCR, the temperature is raised. ✓ The temperature is then lowered. ▪ This separates the two strands of the target DNA DNA Replication ✓ Many copies of the two short DNA primers and Taq1 DNA polymerase are added. ▪ Primers bind by complementary base pairing to the separated target strands. ▪ the polymerase then fills in the bases opposite their complements, creating two daughter strands from each separated target double helix DNA Replication ✓ The four double helices resulting from the first round of amplification then serve as the target sequences for the next round. ✓ The process continues by again raising the temperature. Pieces of identical DNA accumulate exponentially. The number of amplified pieces of DNA equals 2n, where n is the number of temperature cycles. After 30 cycles, PCR yields more than 10 billion copies of the target DNA sequence. DNA Replication Real-time PCR In this type, the DNA amplification is detected in real-time with the help of a fluorescent reporter. Nested PCR This was designed to improve sensitivity and specificity. Multiplex PCR This is used for the amplification of multiple targets in a single PCR experiment DNA Replication Quantitative PCR It uses the DNA amplification linearity to detect, characterize and quantify a known sequence in a sample. Arbitrary Primed PCR It is a DNA fingerprinting technique based on PCR. It uses primers the DNA sequence of which is chosen arbitrarily. DNA Sequencing Frederick Sanger - a British biochemist and two-time Nobel Prize winner who invented a way to determine the base sequence of a small piece of DNA in 1977. DNA Sequencing Sanger Sequencing o It generates a series of DNA fragments of an identical sequence that is complementary to the DNA sequence of interest, which serves as a template strand. o It deduces a DNA sequence by aligning pieces of different sizes that differ from each other at the end base. Variations on this theme label, cut, and/or immobilize the DNA pieces in different ways, greatly speeding the process. oThe fragments differ in length from each other by one end base DNA Sequencing Next-generation sequencing uses a massively parallel approach, and different types of materials on which to immobilize DNA pieces, to read and overlap millions of pieces at once. It greatly speeds DNA sequencing.