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Lecture#2 - DNA Structure and Replication Readings: Griffiths et al (2004) 8th Edition: pp 237-249 Problems: Chap. 7: 1-25, 26, 27

Concepts: How are DNA and chromosomes replicated? 1. The structure of DNA suggests it is replicated semi-conservatively. 2. The work of Meselson & 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 - (transmission genetics) Double helix structure explains: 1) - mechanism for the exact replication of genes - one generation to the next 2) - in chemical terms, the nature of Mutations - cell & organism 3) - the nature of the linear sequence of genes, genes along a chromosome Examine one prediction of Watson & Crick Double Helix model - that of semi conservative replication - (not conservative or dispersive) - each daughter duplex contains one parental and one newly synthesized strand

Messelson & Stahl -1958 8th – Fig 7-12, 7-13 1. - grew E. coli many generations in medium with 15N (heavy) not 14N (normal) - into nitrogen atoms of the bases of DNA 2. - removed from 15N -> 14N and permitted one and two more cell divisions 3. - DNA was extracted from cells and centrifuged at high speed in a CsCI density gradient to separate DNA with light 14N vs heavy 15N DNA Messelson & Stahl Observed: 1st generation - single band between 15N + 14N controls 2nd generation - two bands 1 - between 15N + 14N controls 2 - at 14N control Compare to predictions of various models Conservative: 1st generation - 2 bands therefore wrong Dispersive: 2nd generation - 1 band therefore wrong Semi Conservative : (correct) - 1st generation - 1 band - 2nd generation - 2 bands 1-> 14N 1-> 15N14N mix

Conclusion: -> evidence that DNA is replicated in a semi conservative manner -> supports Watson and Crick Double helix model

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Lecture 2; 2007

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Figure 7-13: The Meselson/Stahl experiment with labeled cells.

DNA synthesis / chromosome replication - well described in E. coli - less is known about eukaryotic systems

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Lecture 2; 2007

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Rules for chain elongation of DNA by DNA polymerases: 1) Copy already existing chain of DNA - template 2) Growth of the chain is in one direction only 5' -> 3’ 3) Need a primer to start DNA synthesis - chain elongation - DNA pol cannot initiate chain synthesis de novo 4) DNA replication (replicate both strands) requires a special growing fork structure

Replication Fork Molecules & Events 8th – Fig 7-16, 7-17, 7-18 You will need to know the details of the replication fork and the events occurring on the leading and lagging strands. The Replication Fork Figure 7-18(8th) 8-20(7th) Replication Complex (multiple enzymes) Sites of Replication in the nucleus (factory) One parental duplex will produce two daughter duplexes At the Replication Fork: 1)-Leading strand - DNA pol -> continuous synthesis on 3' end - initially started with RNA primer 2)-Lagging strand - must be synthesized in short discontinuous segments - each segment consists of RNA primer and replicated DNA (Okazaki fragments) - repeated cycles permit lagging strand to be synthesized - polymerase removes RNA primer - ligase seals the discontinuous fragments into a single strand RESULT: Both chains replicated and continuous Both strands are replicated simultaneously:

Chromosome replication - Prokaryote - Circular DNA John Cairns' (1963) Experiment Tested the semi-conservative model using E coli and allowing the cells to replicate and incorporate (radioactive isotope) 3H-thymidine into DNA)

Hypothesis: If each newly synthesized daughter molecule is semi conservatively replicated then each should contain: 1.- One strand (newly made) that is radioactive - has 3H-thymidine incorporated - "hot" 2.- Other strand (parental) - contains no 3H-thymidine - "cold" Cairns Experiment: - Replicate cells in 3H-thymidine medium for 1 cycle - Extracted DNA -> spread on a microscope slide - Put photographic emulsion over top - expose film-> get grain of silver (Autoradiography) - Detect location of 3H radiation under light microscope Cairns Observed After 1 replication cycle in 3H-thymidine - single rings of dots (silver grains). See Fig 7-14 (8th) 8-17(7th) 3

Lecture 2; 2007

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Figure 7-14 Cairns interpreted - these rings as one labeled strand and one unlabeled strand - evidence that E. coli-bacterial chromosome is circular (confirms previous genetic analysis) - no replication forks are seen, or expected to be seen During the second replication cycle - labeled DNA can be "seen" 1) 1/2 labeled - 1/2 not 2) both labeled - branch in the ring of dots (silver grains) -> replication forks - for a circular DNA molecule - see a theta ⎝ structure Density of silver grains is consistent with - one strand ->1/2 labeled - 1/2 not - other strand -> both labeled (2 X grains) - observation of many molecules correspond with the progressive movement of the replication fork around the ring Conclusions: 1) Observations are consistent with the semi-conservative model of replication - applies to whole circular DNA molecules - Replication forks exist 2) E. coli parental DNA remains circular throughout the DNA replication cycle 3) Replication - bidirectional? -> not proved here 4) One origin of replication - next section

Where does the Replication Fork begin? - Origins of Replication E. coli origin of replication - See Fig. 7-21(8th) 8-23(7th) - unique (only one in the genome of 4.2 Mb) - called oriC - unique in circular E. coli chromosome - has substructure 13 mer + binding sites for protein

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Lecture 2; 2007

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Starts at fixed Origins of Replication and proceeds bidirectionally (both directions) until replication is finished. 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

3) Replication proceeds around the chromosome and stops at the terminus -> result is two daughter duplexes E. coli - chromosome - bi-directional replication - replicated in about 40 minutes ~ 4000 kb in size Therefore each fork replicates 2000 kb in 40 min. or ~ 50 kb/min or ~1000 bases/sec Prokaryotes have a single replication unit (replicon)

DNA replication in Eukaryotes Two problems: size & ends Size How do eukaryotes replicate their DNA in a short time? ->multiple replicons (origins) per chromosome. Comparison of Replicons -> Multiple origins on the same DNA molecule: - both ori initiate replication; are bidirectional (2 forks) - join up - produce long region of replicated DNA - intermediates sometimes called replication bubbles. Note: 1) that not every ori need "fire" at the same time - some early - some late 2) ori not all equi-distant - DNA replicated early (near ori ) while some DNA is replicated late (distant from ori) 3) don't need specific termination sequences. 4) Experiments like Cairns are consistent with bidirectional from many ori sites. Fig 7-22 (8th) 8-25(7th) Ends - Eukaryotic chromosome are linear chromosomes - Telomeres Replication problem at the end of chromosome! Fig 7-24, 7-25(8th) 8-32, -33(7th) Leading strand - complete replication (no problem) 5

Lecture 2; 2007

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Lagging strand - no way to get last section replicated Result -> shortened chromosome each cell generation Enzyme called telomerase adds bases to the end. - consists of tandem arrays of simple DNA sequences. eg. TTGGGG ->->->->->->

Figure 7-24. The replication problems at chromosome ends. Telomerase adds repeat units - extends 3' end many repeat units - polymerase fills in the remaining section - length is genetically regulated

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