1. Chapter 14 DNA: The Genetic Material

2. 14.1 The Genetic Material 1. Frederick Griffith- 1928- worked with pathogenic bacteria Took a virulent strain of pneumococci (S form) and injected it into mice; the mice died Took a harmless strain of pneumococci (R form) and injected it into mice; the mice lived Heat killed the harmful form of bacteria and injected it into mice and the mice lived. Mixed the heat killed form with live harmless form and injected into mice; the mice died. Transformation-transfer of genetic material from one cell to another and can alter the genetic make up of recipient cell.

3. 14.1 The Genetic Material 2. Avery, MacLeod & McCarthy- hypothesized that the protein coat on bacteria was the transforming factor. Repeated Griffith’s work except he removed almost all of the protein from both strains of bacteria by digesting it with enzymes Transforming activity did not reduce Concluded that nucleic acid of deoxyribose type is the transforming factor. 3. Hershey & Chase- used bacteriophages (virus) to prove it was the nucleic acid that was the transforming factor. Used T2 bacteriophage; radioactively labeled the DNA and protein coat. 35 S was used to label protein coat 32 P was used to label DNA 32 P appeared to be the component transferred to interior of bacteria-hence the hereditary material is DNA.

4. 14.2 DNA Structure 1.P.A. Levene- found DNA contains three main components Phosphate group 5 carbon sugar (deoxyribose or ribose) Nitrogen base (A & G = purines; T, C, U= pyrimidines) These three components make a nucleotide 2. Chargaff’s Rules Proportion of A=T; G=C Equal proportion of purines (A & G) and pyrimidines (C & T)

5. 14.2 The Structure of DNA 3. Rosalind Franklin- carried out X-ray diffraction analysis of DNA Had to use DNA fibers in her work and found that DNA had the shape of a helix with a diameter of 2nm and a complete helical turn every 3.4 nm. 4. Watson & Crick- 1953- using Franklin’s results, built model of nucleotides (held together by phosphodiester bonds) and assembled them into a double helix with bases (A, T, C, G) pointing inward; double helix is stabilized as a duplex; DNA molecule is composed of two anti-parallel strands held together by hydrogen bonds.

6. 14.3 Basic Characteristics of DNA Replication Meselson & Stahl Experiment- confirmed that DNA replicates itself in semi- conservative manner. Semi-conservative- the sequence of the original duplex is conserved after one round of replication. The duplex itself is not.

7. 14.3 Basics Characteristics of DNA Replication The Experiment: Used bacteria grown in 15 N (heavy isotope) and bacteria grown in 14 N (light) The DNA was extracted and suspended in a cesium chloride (salt) solution. DNA will sink in gradient created by cesium, until they reach a place where the density matches. Meselson and Stahl Experiment

8. 14.4 Prokaryotic Replication Replication in E. coli initiates at a specific site, the origin, and ends at a specific site, the terminus. After initiation, replication proceeds bi-directionally from this unique origin to the unique terminus. E. coli possess three different DNA polymerase enzymes DNA poly III- main replication enzyme responsible for the bulk of DNA synthesis DNA poly I- acts on the lagging strand to remove primers and replace them with DNA DNA poly II- does not appear to play a role in replication, but is involved in DNA repair processes.

9. 14.4 DNA Replication The Replication Process- must be fast and accurate 1.Opening of the double helix and separation of the two original strands Stage One- initiator proteins bind to replication origin to start interactions that open the helix. Stage Two- unwinding the helix-enzymes called helicases bind to and move along DNA. Stage Three- stabilizing the single strands- single strand binding (SSB’s) proteins bind to exposed strands to protect them from cleavage and from rewinding. 2.Building a Primer DNA polymerase III requires a 3 ̸ primer to initiate replication. The primer is a short stretch of RNA. Process uses RNA because the starting territory can be marked as “temporary”, making it an error-prone stretch that can be easily excised later. 3.Assembling Complimentary Strands DNA polymerase III bind to strands; catalyzes the formation of complementary sequences on each strand at the same time.

10. 14.4 Leading strand- bases are added continuously in the 5 ̷ to 3 ̷ direction Lagging strand- bases are added in the direction opposite the growing replication fork. Replication of this strand is done in small, separated segments called Okazaki fragments. 4.Removing the Primer DNA polymerase I removes the RNA primer and fills the gap. Will also fill gaps between the Okazaki fragments. (100 to 200 nucleotides long) 5. Joining the Okazaki Fragments DNA ligase joins any remaining fragments on the lagging strand. Essential Biochemistry - DNA Replication

11. 14.5 Eukaryotic Replication Requires multiple origins, much more complex than prokaryotic replication. Linear chromosomes have specialized ends called telomeres that protect the ends of the chromosomes from degradation. The ends are composed of specific DNA sequences, but are not made by the replication complex. Telomerase enzymes use internal RNA as a template instead of DNA to copy the telomeres. A gradual shortening of the ends of chromosomes occurs in the absence of telomerase activity. There is a relationship between cell senescence (aging) and telomere length. Normal cells undergo only a specified number of divisions when grown in culture; this is at least partially based on telomere length. Cancer cells divide indefinitely and show activation of their telomerase; this allows these cells to maintain their telomere length.

12. 14.6 DNA Repair DNA polymerases have 3 / to 5 / exonuclease activity that allows “proofreading” of added bases. This increases the accuracy of replication, but errors still occur. Cells are constantly exposed to mutagens. These include: radiation, UV light, X-rays and chemicals in the environment. There are two types of DNA repair 1.Specific repair systems- target a single kind of lesion in DNA and repair only that damage. Ex: photorepair- specific to damage done by UV light, namely the thymine dimer 1.Nonspecific repair systems- use a single mechanism to repair multiple kinds of lesions in DNA. Ex: excision repair- nonspecific- removes damaged region and is followed by DNA synthesis.