DNA Replication DNA → RNA → Protein replication transcription translation DNA → RNA → Protein How DNA controls a cell, part 1: DNA makes an identical copy of itself for each new cell.

Deoxyribonucleic Acid (DNA) Review Made up of nucleotides in a DNA double helix. Nucleotide: 1. Phosphate group 2. 5-carbon sugar (Deoxyribose) 3. Nitrogenous base (A, T, C, G) ~2 nm wide (remember 1 mm = 1,000 μm, 1 μm = 1,000 nm)

DNA Nucleotide O O=P-O N CH2 O C1 C4 C3 C2 Phosphate Group Nitrogenous base (A, G, C, or T) CH2 O C1 C4 C3 C2 5 Sugar (deoxyribose)

DNA Double Helix P O 1 2 3 4 5 P O 1 2 3 4 5 G C T A

BASE-PAIRINGS Base # of Purines Pyrimidines Pairs H-Bonds C G Adenine (A) Thymine (T) A = T 2 Guanine (G) Cytosine (C) C G 3 C G 3 H-bonds

Chargaff’s Rule Adenine must pair with Thymine Guanine must pair with Cytosine Their amounts in a given DNA molecule will be about the same. T A G C

Question: If there is 30% Adenine, how much Cytosine is present? There would be 20% Cytosine. Adenine (30%) = Thymine (30%) = 60% 40% remains, must be C and G equally: Guanine (20%) = Cytosine (20%)

When and where does DNA replication take place？ S phase in interphase of the cell cycle. Nucleus of eukaryotes Cytoplasm of prokaryotes Mitosis -prophase -metaphase -anaphase -telophase G1 G2 S phase interphase DNA replication takes place in the S phase.

DNA Replication Origins of replication 1. Replication Forks: Y-shaped regions of replicating DNA molecules where new strands are growing. Replication Fork Parental DNA Molecule 3’ 5’

DNA Replication Origins of replication 2. Replication Bubbles: a. Hundreds of replicating bubbles (Eukaryotes). b. Single replication fork (bacteria). Bubbles

DNA Replication Strand Separation: 1. Helicase: enzyme which catalyzes the unwinding and separation (breaking H- Bonds) of the parental double helix. 2. Topoisomerase: enzyme which relieves stress on the DNA molecule by allowing free rotation around a single strand. Enzyme DNA Enzyme

DNA Replication Priming: 1. RNA primase: DNA nucleotides can not start a strand; they can only be added to an existing nucleotide strand. Therefore, RNA primase adds several RNA nucleotides (an RNA primer) to allow the addition of DNA nucleotides (DNA Polymerase).

DNA Replication Synthesis of the new DNA Strands: LEADING STRAND 1. DNA Polymerase III: with a RNA primer in place, the DNA Polymerase III enzyme adds DNA nucleotides, leading to the synthesis of a new DNA strand in the 5’ to 3’ direction. RNA Primer DNA Polymerase III Nucleotide 5’ 3’

DNA nucleoside triphosphates are added Nucleotides with three phosphates instead of one Breaking bond between 1st and 2nd phosphate releases energy to bind nucelotides together Similar to ATP use

DNA Replication Synthesis of the new DNA Strands: 3. Lagging Strand: also synthesized in the 5’ to 3’ direction, but discontinuously against overall direction of replication. RNA Primer Leading Strand DNA Polymerase 5’ 3’ Lagging Strand 5’ 3’

DNA Replication Synthesis of the new DNA Strands: Okazaki Fragments: series of short DNA segments on the lagging strand. Lagging Strand RNA Primer DNA Polymerase 3’ 5’ Okazaki Fragment

DNA Replication Synthesis of the new DNA Strands: LAGGING STRAND DNA Polymerase I: removes RNA primers, adds DNA nucleotides beginning with the DNA fragments created by DNA polyermase III. DNA ligase: creates a covalent bond between the edges of Okazaki fragments. Example: joining two Okazaki fragments together. Lagging Strand Okazaki Fragment 2 DNA ligase Okazaki Fragment 1 5’ 3’

DNA Replication Example of Ligase healing Okazaki fragments Proofreading: initial base-pairing errors are usually corrected by DNA polymerase or removed by correcting enzymes (50+ types!) and corrected by DNA polymerase and ligase.

DNA Replication Semiconservative Model: 1. Watson and Crick showed: the two strands of the parental molecule separate, and each functions as a template for synthesis of a new complementary strand. DNA Template New DNA Parental DNA

Some useful resources: Animations: http://bioteach.ubc.ca/TeachingResources/MolecularBiology/DNAReplication.swf http://www.johnkyrk.com/DNAreplication.html