2. The work of Messelson & Stahl showed semi-conservative replication. 4. Cairn's experiments showed chromosomes are semi-conservatively replicated.

Transcription

1 BIOLOGY Dr.McDermid Lecture#2/3 DNA Structure & Replication Readings: Griffiths et al, 7 th Edition: Ch. 8 pp (corrected) Problems: Griffiths et al, 7 th Edition: Ch. 8 Tier 1: # 2,3,5,9,13 Tier 2: #1,4,6,7,12 Concepts: How are DNA and chromosomes replicated? 1. The structure of DNA suggests it is replicated semi-conservatively. 2. The work of Messelson & Stahl showed semi-conservative replication. 3. DNA is synthesized by enzymes at a replication fork. 4. Cairn's experiments showed chromosomes are semi-conservatively replicated. 5. Eukaryotes have multiple origins and telomerase to solve the "size" and "ends" problems. Double helix structure provides a chemical explanation for the transmission of genes Double helix structure explains: 1) replication 2) mutations 3) linear sequence of genes Examine one prediction of Watson & Crick Double Helix model - semi conservative replication Meselson & Stahl Page 249. Fig 8-11, -12, and 13 - replication is semi-conservative 1. - grew E. coli in 15 N (heavy) ( 14 N normal) N 14 N for one and two more cell divisions 3. - DNA extracted density gradient Lecture#2 Page 1

2 Conclusion: -DNA is replicated in a semi conservative manner DNA synthesis / chromosome replication Rules for chain elongation by DNA polymerases: 1) Copy already existing chain of DNA 2) Growth of the chain is in one direction only (Figure 8-21-note error) 3) Need a primer to start DNA synthesis Duplex DNA replication (replicate both strands) creates a growing fork structure Lecture#2 Page 2

3 Replication Fork Figure 8-20, 27, -28, -29, -30, & -31 CD has animation (Fig 8-20, 8-21) One parental duplex will produce two daughter duplexes 1)-Leading strand - continuous synthesis on 3' end - initially started with RNA primer 2)-Lagging strand - synthesized in short discontinuous segments - each segment consists of RNA primer and replicated DNA (Okazaki fragments) - polymerase removes RNA primer - ligase seals the discontinuous fragments RESULT: Both chains replicated and continuous Both strands replicated simultaneously Can you see a replication fork? John Cairns' (1963) Experiment E coli can incorporate 3 H-thymidine into DNA Replication is semi conservatively, therefore: 1.- New strand Parental strand - Cairns Experiment: - Replicate cells in 3 H-thymidine medium for 1 cycle - Extracted DNA spread on a microscope slide - Put photographic emulsion over top - expose film - Detect silver grains under light microscope Lecture#2 Page 3

4 After 1 replication cycle in 3 H-thymidine Figure 8-17 During the second replication cycle - Figure ) 1/2 labeled - 1/2 not 2) both labeled - see the replication forks -> branch in the ring of dots (silver grains) - circular DNA molecule theta q structure - density of silver grains reflects - one strand ->1/2 labeled - 1/2 not - other strand -> both labeled Conclusions: 1) Observations are consistant with the semi-conservative model of replication 2) E. coli parental DNA remains circular throughout the DNA replication cycle 3) One origin of replication Where does the Replication Fork begin? - At fixed Origins of Replication Lecture#2 Page 4

5 - proceeds bidirectionally (both directions) E. coli origin of replication - unique - called oric See Fig Initiation Process 1) Proteins bind to origin site, unwind the local duplex, and put it in "open" conformation that is single stranded. 2) Forms a replication fork in both directions -> bidirectional Fig ) Replication proceeds around the chromosome and stops at the terminus Fig > result is two daughter duplexes DNA replication in Eukaryotes - two problems: SIZE and ENDS Size How do eukaryotes replicate their DNA in a short time? -> Lecture#2 Page 5

6 - all ori initiate replication - join up - produce long region of replicated DNA - replication bubbles. Note: 1) not every ori need "fire" at the same time 2) ori not all equi-distant 3) don't need specific termination sequences Ends - Telomeres Eukaryotic chromosome are linear Replication problem - Figure 8-32 Lecture#2 Page 6

7 Problem: Leading strand - Lagging strand - Potential result - Special mechanism for replicating the telomere Enzyme called telomerase adds bases to the end. - telomere tandem arrays of simple DNA sequences. eg. TTAGGGTTAGGGTTAGGGTTAGGG.. Telomerase adds repeat units - Figure extends 3' end by many repeat units - polymerase can then fill in the remaining section - see animation gif at: Lecture notes: Copyright 2002 Heather McDermid and the Department of Biological Sciences, University of Alberta Images are Copyright 2000 by W.H. Freeman & Co. in Griffiths et al, Introduction to Genetic Analysis Lecture#2 Page 7