1. DNA replication an overview These slides provide an overview of the basic rules governing DNA replication and introduce key proteins that are typically involved in replication in general Dr. Momna Hejmadi, University of Bath N.B. Some images used in these slides are from the textbooks listed and are not covered under the Creative Commons license as yet DNA basics resources created by Dr. Momna Hejmadi, University of Bath, 2010, is licensed under the Creative Commons Attribution-Non-Commercial-Share Alike 2.0 UK: England & Wales License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/2.0/uk/ or send a letter to Creative Commons, 171 Second Street, Suite 300, San Francisco, California 94105, USA.http://creativecommons.org/licenses/by-nc-sa/2.0/uk/

2. Learning objectives Understand the basic rules governing DNA replication Understand the function of key proteins involved in a generalised replication model References Biochemistry (3e) by D Voet & J Voet Molecular biology of the cell (4th ed) by Alberts et al Essential Cell Biology by Alberts et al Life: The Science of Biology by Sadava et al (8th ed ) Optional Nature (2003) vol 421,pp431-435 http://www.dnai.org/lesson/go/2166/1973

3. `It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material’ Watson & Crick Nature (1953) Original drawing by Francis Crick

4. Four requirements for DNA to be genetic material Must carry information Cracking the genetic code Must replicate DNA replication Must allow for information to change Mutation Must govern the expression of the phenotype Gene function

5. Much of DNA’s sequence-specific information is accessible only when the double helix is unwound Proteins read the DNA sequence of nucleotides as the DNA helix unwinds. Proteins can either bind to a DNA sequence, or initiate the copying of it. DNA stores information in the sequence of its bases Some proteins recognize the base sequence of DNA without unwinding it (e.g. a restriction enzyme). restriction enzyme EcoR V

6. DNA replication occurs with great fidelity Somatic cell DNA stability and reproductive-cell DNA stability are essential. Why? Pan troglodytes 98.77% sequence identity Identity Genetic diseases

7. DNA Replication Process of duplication of the entire genome prior to cell division Biological significance extreme accuracy of DNA replication is necessary in order to preserve the integrity of the genome in successive generations In eukaryotes, replication only occurs during the S phase of the cell cycle. Replication rate in eukaryotes is slower resulting in a higher fidelity/accuracy of replication in eukaryotes

8. The mechanism of DNA replication Arthur Kornberg et al Initiation Proteins bind to DNA and open up double helix Prepare DNA for complementary base pairing Elongation Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA Termination Proteins release the replication complex

9. Flash Animation http://bcs.whfreeman.com/thelifewire8e /content/cat_010/1104001.html

10. Basic rules of replication A.Semi-conservative B.Starts at the ‘origin’ C.Can be uni or bidirectional D.Semi-discontinuous E.Synthesis always in the 5-3’ direction F.RNA primers required

11. DNA replication Of the 3 possible models, replication is… A) Semi- conservative Meselson-Stahl experiments

12. B) Starts at origin Initiator proteins identify specific base sequences on DNA called sites of origin Prokaryotes – single origin site E.g E.coli - oriC Eukaryotes – multiple sites of origin (replicator) E.g. yeast - ARS (autonomously replicating sequences) Prokaryotes Eukaryotes

13. C) Uni or bidirectional Replication forks move in one or opposite directions

14. D) Semi-discontinuous replication Anti parallel strands replicated simultaneously  Leading strand synthesis continuously in 5’– 3’  Lagging strand synthesis in fragments in 5’-3’

15. Semi-discontinuous replication New strand synthesis always in the 5’-3’ direction

16. E) Synthesis is ALWAYS in the 5’-3’ direction Nucleotide recognition Enzyme catalysed polymerisation (DNA polymerase) Complementary base pair copied Substrate used is dNTP

17. Where does energy for addition of nucleotide come from? What happens if a base mismatch occurs? DNA polymerase has 3’  5’ exonuclease activity in order to correct errors From cleavage of high energy phosphate of incoming triphosphate

18. Why does DNA replication only occur in the 5’ to 3’ direction?

19. F) RNA primers required DNA polymerase can only join an incoming nucleotide to one that is base-paired RNA primase provides a base paired 3’ end as a starting point for DNA pol by synthesising ~10 nucleotide primers

20. Basic rules of replication A.Semi-conservative B.Starts at the ‘origin’ C.Can be uni or bidirectional D.Semi-discontinuous E.Synthesis always in the 5-3’ direction F.RNA primers required

21. Topoisomerases Helicases Primase Single strand binding proteins DNA polymerase Tethering protein DNA ligase - Prevents torsion by DNA breaks - separates 2 strands - RNA primer synthesis - prevent reannealing of single strands - synthesis of new strand - stabilises polymerase - seals nick via phosphodiester linkage Core proteins at the replication fork

22. Nature (2003) vol 421,pp431-435Figure in ‘Big’ Alberts too

23. Why has DNA evolved as the genetic material but not RNA? Because DNA is more stable RNA is prone to base-catalysed hydrolysis.

24. Review 1. DNA replication animat tutorial plus revision tutorials and self quizzes http://life9e.sinauer.com/Life9e.html Life: The science of Biology by Sadava et al 2. Use the questions on the following slides

25. What kind of enzyme synthesizes the new DNA strand? 1)RNA polymerase 2)DNA Polymerase 3)Primase 4)Helicase 5)Topoisomerase

26. Eukaryotic chromosomes have multiple origins of replication 1.True 2.False

27. In what direction is the newly synthesized DNA produced? 1.5'-3' 2.3'-5' 3.In the direction of the major groove 4.Both 5'-3' and 3'-5' depending on which strand is being replicated

28. Nucleotides are always added to the growing DNA strand at the 3’ end, at which the DNA has a free ______ on the 3’ carbon of its terminal deoxyribose. 1.Phosphate group 2.Hydroxyl group 3.Nitrogen base 4.Methyl group

29. The E. coli chromosome has 4.7x10 6 bp; a bi-directional replication fork progresses at about 1000 nucleotides/sec. Therefore, the minimum time required to complete replication is 1)12 min. 2)24 min. 3)39 min 4)78 min 5)120 min

30. What is the sequence (1 to 6) in which these proteins function during DNA replication ____ RNA primase ____ DNA ligase ____ DNA polymerase ____ Topoisomerase ____ DNA helicase ____ tethering proteins 1 4 6 2 5 3

31. Why is an RNA primer necessary for DNA replication? A.The RNA primer is necessary for the activity of DNA ligase. B.The RNA primer creates the 5’ and 3’ ends of the strand. C.DNA polymerase can only add nucleotides to RNA molecules. D.DNA polymerase can only add nucleotides to an existing strand