DNA and Replication AP Biology Mr. Beaty 2007

The Great Debate Which chemical is used to store and transmit genetic information? Protein or DNA Most Scientists of the day agreed that the substance must be protein.

Evidence for DNA as genetic material Griffith, In his work with Streptococcus pneumoniae, Griffith realized that some “transforming” agent was exchanged between bacteria which enabled to acquire traits from one another. The use of heat to inactivate cells suggested that the agent was not protein. This phenomenon is now called transformation - a change in phenotype by taking genetic material from the environment.

Griffith Experiment

Avery’s Experiment Avery, et al., isolated various chemicals from bacteria and used them to try transform bacteria. Only DNA worked.

Viruses are made of nucleic acid and protein

Hershey Chase Experiment (1952)

Chargaff (1947) Adenine pairs Thymine; Cytosine pairs Guanine If a mixture made from cells contained 20% Adenine, then what is the percentage of Guanine?

Structure of DNA Wilkins and Franklin used X- ray diffraction to attempt to find the structure of DNA.

The Structure of DNA was discovered Watson and Crick (1953) Double Helix Sides: phosphate and sugar Rungs: nitrogenous bases held together by hydrogen bonds

DNA Nucleotide O O=P-O OPhosphate Group Group N Nitrogenous base (A, G, C, or T) (A, G, C, or T) CH2 O C1C1 C4C4 C3C3 C2C2 5 Sugar Sugar(deoxyribose)

Nitrogenous Bases Double ring PURINES Double ring PURINES Adenine (A) Guanine (G) Single ring PYRIMIDINES Single ring PYRIMIDINES Thymine (T) Cytosine (C) T or C A or G

DNA Strands are Anti-parallel P P P O O O P P P O O O G C TA

Base-Pairings Purines only pair with Pyrimidines Three hydrogen bonds required to bond Guanine & Cytosine CG 3 H-bonds

T A Two hydrogen bonds are required to bond Adenine & ThymineTwo hydrogen bonds are required to bond Adenine & Thymine

Question: Adenine Cytosine If there is 30% Adenine, how much Cytosine is present?

Answer: Cytosine There would be 20% Cytosine Adenine (30%) = Thymine (30%) Adenine (30%) = Thymine (30%) Guanine (20%) = Cytosine (20%) Guanine (20%) = Cytosine (20%) Therefore, 60% A-T and 40% C-G Therefore, 60% A-T and 40% C-G

Structure of DNA

DNA Replication (Semiconservative Model)

Semiconservative Model

DNA Replication Video DNA Replication Video

Origin of Replication Origin of replication (“bubbles”): beginning of replication Replication fork: ‘Y’-shaped region where new strands of DNA are elongating Helicase:catalyzes the untwisting of the DNA at the replication fork DNA polymerase:catalyzes the elongation of new DNA

DNA Replication, II Antiparallel nature: sugar/phosphate backbone runs in opposite directions (Crick); one strand runs 5’ to 3’, while the other runs 3’ to 5’; DNA polymerase only adds nucleotides at the free 3’ end, forming new DNA strands in the 5’ to 3’ direction only

DNA Replication, III Leading strand: synthesis toward the replication fork (only in a 5’ to 3’ direction from the 3’ to 5’ master strand) Lagging strand: synthesis away from the replication fork (Okazaki fragments); joined by DNA ligase (must wait for 3’ end to open; again in a 5’ to 3’ direction) Initiation: Primer (short RNA sequence~w/primase enzyme), begins the replication process

DNA Replication: the leaning strand DNA Replication: the leaning strand

DNA Replication: the lagging strand

DNA Repair Mismatch repair: DNA polymerase Excision repair: Nuclease Telomere ends: telomerase

Elongating a new strand After the strands are separated, DNA polymerase “reads” the exposed bases on the template strand and attaches new bases by complementary base pairing. Note that this process is decreasing entropy greatly so it must require energy.

Similar to ATP!! The energy to add new nucleotides comes from the substrates themselves which are nucleoside triphosphates. The loss of two phosphates from the substrate provides the energy to drive the reaction.

Elongating a new strand DNA polymerase can only attach the 5' phosphate (P) of one nucleotide to the 3' hydroxyl (OH) of another nucleotide that is already part of a strand. The enzyme can only work by building a new strand in the 5' ➝ 3' direction.

The two strands of DNA are antiparallel.

Problem of antiparallel strands The C5 phosphate of one nucleotide is attached to the C3 hydroxyl of an adjacent nucleotide. Therefore, the strand has a free 3' OH at one end and a free 5' P at the other. Remember, the molecule is arranged with the strands going in opposite directions so the 3' end of one strand is aligned with the 5' end of the other.

Problem of antiparallel strands DNA polymerase adds nucleotides only to the 3' end but can only do this on one strand, the leading strand. The other strand has a 5' P at the end rather than the 3' OH DNA polymerase needs. This strand, the lagging strand, must be made in an overall 3' ➝ 5' direction. To do this, the new strand is made in short fragments, called Okazaki fragments, going in the opposite direction from the leading strand. Another enzyme, DNA ligase, then fills in the gaps to join the fragments together.

Synthesis of Leading and Lagging strand during DNA replication

Priming DNA Synthesis Remember that DNA can only attach the 5' phosphate (P) of one nucleotide to the 3' hydroxyl (OH) of another nucleotide that is already part of a strand. A primer is a short piece of RNA that is constructed on the template to serve as a starting point for DNA polymerase. The enzyme primase builds the primers, which are about 10 nucleotides long. Later, the primers are replaced by DNA.

DNA Polymerase cannot initiate a polynucleotide strand; it can only add to the 3’ end of an already- started strand. The primer is a short segment of RNA synthesized by the enzyme primase. Each primer is eventually replaced by DNA

Summary of DNA Replication 1. Helicase unwinds the paretental double helix. 2. Single-strand binding proteins stabilize the unwound parental DNA. 3. The leading strand is synthesized continuously in the 5  3 direction by DNA polymerase. 4. The lagging strand is synthesized discontinuously. Primase synthesizes a short RNA primer, whichis extended by DNA polymerase to form an Okazaki fragment. 5. Another DNA polymerase replaces the RNA primer with DNA. 6. DNA Ligase joins the Okazaki framents to the growing strand.

DNA Replication Summary

Error rate Complementary base pairing allows an error rate of 1 in 10,000 bp. DNA polymerase checks for these errors by checking the width of the helix and reduces the rate to 1/10,000. DNA is constantly exposed to chemicals, viruses, and radiation which cause damage. This damage is repaired by>50 known enzymes that constantly check DNA for errors. These combined efforts reduce the error rate to 1 in a billion.

DNA Repair A team of enzymes detects and repairs damaged DNA. Example of repairing a thymine dimer, caused from UV radiation.

The End Replication Problem When a linear DNA molecule replicates, a gap is left at the 5’end of each new strand because DNA polymerase can only add nuceotids to a three end. As a aresult with each round of replication, the DNA molecules get slightly shorter.

Telomeres and Telomerase Eukaryotes deal with the end- replication issue by having expendable, noncoding sequences called telomeres at the ends of their DNA and the enzyme telomerase in some of their cells