DNA REPLICATION BIT 220 MCCC Chapter 11

Replication Meselson and Stahl

CsCl Equilibrium Density Gradient Centrifugation DNA will position itself in the salt solution where the DNA density equals the salt density If use a heavy isotope in formation of nucleotides, density will increase Protocol: Grow E coli in presence of 15 N. Replace medium with 14 N As multiple rounds of replication... you will see bands of DNA of different densities. Figure 11.3 and Technical sidelight

Requirements of Replication 1. Primer 5’-phosphate of new nucleotide must be added to a free 3’-OH group DNA can not synthesize de novo 2. Template DNA strand which is used to make complementary strand 3. Synthesis is ONLY in 5’ to 3’ direction

Terms Replication Fork: Y-shaped structure Bidirectional Replication Bubble Theta Structures in circular DNA Figure 11.5 Replicon

Enzymes Involved DNA Polymerase Helicase Topoisomerase (Gyrase) Primase Ligase Telomerase Figure 11.26

Origin of Replication unique site in bacteria and viruses for beginning of replication multiple sites in eukaryotes OriC - E. coli 245 base pairs Three tandem repeats of 13mer AT rich allows strand separation Figure 11.6

Separating DNA strands 1. HELICASE Break attachment between strands opening DNA Unwindind DNA Replication Fork (Two = bubble) Y shaped region where 2 new DNA strands elongate 2. Single Stranded DNA Binding Protein (SSBs) hold strands apart prevents DNA strand from folding on itself

DNA polymerase can only work in 5’-3/ direction 1. Catalyzes the formation of phoshodiester between 3’OH of terminal nucleotide with interior phosphate (5’) of incoming nucleotide Figure Proofreading function: excises wrong nucleotide (5-3’ exonuclease OR 3’-5’ exonuclease) Figure Multiple polymerases in both E and P Also see Figures and 11.16

Replication DNA POLYMERASE 1. enzyme which adds new nucleotides complementary bases A-T, G-C new nt attached to 3’-OH of sugar of preceding nt phosphodiester linkage ONLY in 5’-3’ direction 2. Corrects nucleotide mistakes (Proofreading, exonuclease) 3. Removes primer Both sides serve as template to make two additional strands Bidirectional Replication

Continuous vs Discontinuous 1. Leading strand 5’-3’direction - Figure Lagging strand (discontinuously) overall 3’-5’ direction shorter strands ‘Okazaki fragments’ Okazaki fragments made in 5’-3’ LIGASE - Figure enzyme used to seal fragments together catalyzes covalent bond formation between sugar and phosphate to repair ‘nicks’ NICK broken phosphodiester bond

Primase DNA can NOT start new DNA strand without pre-existing nucleic acid Requires a free -OH end Enzyme which adds RNA primer Primer is excised after used and gap is filled with deoxyribonucleotides (DNA polymerase) Figure 11.21

Topoisomerase Fig Problem: unwinding DNA (HELICASE) would cause positive supercoils in circular DNA (in front of rep fork) Solution: Topoisomerase nicks (cuts) DNA to relieve pressure Nicks one or both strands of DNA provides a swivel point or axis of rotation Fig GYRASE topoisomerase II introduce negative supercoils in E coli chromosome when DNA unwinds; chromosome becomes relaxed Figure 11.25

Telomerase Figure Eukaryotes need to add TELOMERES ends of chromosomes/G rich 1. Primer is removed on lagging strand 2. Can not fill gap because no free 3’-OH and can not move in 3’-5’ direction 3. Telomerase adds G rich sequence at 3’ end of chromosome (LEADING) (has RNA template) 4. Complementary strand synthesis

Prokaryotes vs Eukaryotes 1 Origin of Replication (ori) 1000nt/sec Multiple sites of initiation 50 nt/sec Telomerase enzymes which completes ends of chromosomes Nucleosomes FIGURES 11.26, 11.28