1. Chapter 10 DNA Metabolism: Replication, Recombination, and Repair

2. Outline How Is DNA replicated? What are the properties of DNA polymerases? Why are there so many DNA polymerases? How is DNA replicated in eukaryotic cells? How are the ends of chromosomes replicated? How are RNA genomes replicated? How is the genetic information shuffled by genetic recombination? Can DNA be repaired? What is the molecular basis of mutation? Do proteins ever behave as genetic agents? Special Focus: Gene rearrangements and immunology – is it possible to generate protein diversity using genetic recombination?

3. DNA = Deoxyribose nucleic acid Made out of sugars (deoxyribose), phosphates and nitrogen bases. Structure was discovered in 1953 by James Watson and Francis Crick. “Chargoff’s rule”: A = T & C = G

4. Identical base sequences 5’ 3’ Watson/Crick proposed mechanism of DNA replication

5. How is DNA Replicated?  This is a fundamental process occurring in all living organisms to copy their DNA.  Following DNA replication, two identical DNA molecules produced from a single double-stranded DNA molecule.

6. Mode of DNA Replicated? DNA replication: Strand separation followed by the copying of each strand.  DNA replication is bidirectional Bidirectional replication involves two replication forks, which move in opposite directions. The double helix must be unwound - by helicases. Supercoiling must be compensated - by DNA gyrase  DNA replication is semidiscontinuous The leading strand copies continuously. The lagging strand copies in segments (Okazaki fragments) which must be joined. This was discovered by Tuneko and Reiji Okazaki

7. The Semidiscontinuous Model for DNA Replication Newly synthesized DNA is red. (a) Leading and lagging strand synthesis. (b) The action of DNA polymerase.

8. In the late 1950s, three different mechanisms were proposed for the replication of DNA Conservative model: Both parental strands stay together after DNA replication Semiconservative model: The double-stranded DNA contains one parental and one daughter strand following replication Dispersive model: Parental and daughter DNA are interspersed in both strands following replication Proposed Models of DNA Replication

9.  Grow E. coli in the presence of 15 N (a heavy isotope of Nitrogen) for many generations Cells get heavy-labeled DNA  Switch to medium containing only 14 N (a light isotope of Nitrogen)  Collect sample of cells after various times.  Analyze the density of the DNA by centrifugation using a CsCl gradient. 1958: Matthew Meselson & Frank Stahl’s Experiment Semiconservative model of DNA replication

10. Cell division and DNA replication Cells divide  Growth, Repair, Replacement Before cells divide they have to double cell structures, organelles and their genetic information

11. Initiation of replication, major elements: Segments of single-stranded DNA are called template strands. Gyrase (a type of topoisomerase) relaxes the supercoiled DNA. Initiator proteins and DNA helicase binds to the DNA at the replication fork and untwist the DNA using energy derived from ATP (adenosine triphosphate). (Hydrolysis of ATP causes a shape change in DNA helicase) DNA primase next binds to helicase producing a complex called a primosome (primase is required for synthesis), Primase synthesizes a short RNA primer of 10-12 nucleotides, to which DNA polymerase III adds nucleotides. Polymerase III adds nucleotides 5’ to 3’ on both strands beginning at the RNA primer. The RNA primer is removed and replaced with DNA by polymerase I, and the gap is sealed with DNA ligase. Single-stranded DNA-binding (SSB) proteins (>200) stabilize the single-stranded template DNA during the process. Steps of DNA replication: In detail

12. Steps of DNA replication Initiation The origin of replication in E. coli is termed oriC –origin of Chromosomal replication Important DNA sequences in oriC –AT-rich region –DnaA boxes DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequences –causes the region to wrap around the DnaA proteins and separates the AT-rich region

13. Uses energy from ATP to unwind the duplex DNA SSB

14. Each origin of replicaton Initiates the formation of bidirectional replication forks

15. Ahead of the replication fork the DNA becomes supercoiled The supercoiling ahead of the fork needs to be relieved or tension would build up (like coiling as spring) and block fork progression. This supercoiling is relieved by Topoisomerase Supercoiling is relieved by the action of Topoisomerases Type I topoisomerases: Make nicks in one DNA strands Can relieve supercoiling Type II topoisomersases Make nicks in both DNA strands (double strand break) Can relieve supercoiling and untangle linked DNA helices

16. Elongation and termination Direction of fork movement Direction of synthesis Of lagging strand Direction of synthesis of leading strand Protein complexes of the replication fork: DNA polymerase DNA primase DNA Helicase ssDNA binding protein Sliding Clamp Clamp Loader DNA Ligase DNA Topoisomerase

19. Schematic representation of DNA Polymerase III Structure resembles a human right hand Template DNA thread through the palm; Thumb and fingers wrapped around the DNA

20. Concepts and terms to understand: Home work Why are gyrase and helicase required? The difference between a template and a primer? The difference between primase and polymerase? What is a replication fork and how many are there? Why are single-stranded binding (SSB) proteins required? How does synthesis differ on leading strand and lagging strand? Which is continuous and semi-discontinuous? What are Okazaki fragments?

21. 1.Two replication forks result in a theta-like (  ) structure. 2.As strands separate, positive supercoils form elsewhere in the molecule. 3.Topoisomerases relieve tensions in the supercoils, allowing the DNA to continue to separate. Replication of circular DNA in E. coli

22. 1.Common in several bacteriophages including. 2.Begins with a nick at the origin of replication. 3.5’ end of the molecule is displaced and acts as primer for DNA synthesis. 4.Can result in a DNA molecule many multiples of the genome length (and make multiple copies quickly). 5.During viral assembly the DNA is cut into individual viral chromosomes. Rolling circle model of DNA replication

23. The Eukaryotic Cell Cycle The stages of mitosis and cell division define the M phase. G 1 is typically the longest part of the cell cycle; G 1 is characterized by rapid growth and metabolic activity. Cells that are quiescent, that is, not growing and dividing (such as neurons), are said to be in G 0 phase. The S phase is the time of DNA synthesis. S is followed by G 2, a relatively short period of growth when the cell prepares for division.

24. Copying each eukaryotic chromosome during the S phase of the cell cycle presents some challenges: Major checkpoints in the system 1.Cells must be large enough, and the environment favorable. 2.Cell will not enter the mitotic phase unless all the DNA has replicated. 3.Chromosomes also must be attached to the mitotic spindle for mitosis to complete. 4.Checkpoints in the system include proteins call cyclins and enzymes called cyclin-dependent kinases (Cdks). DNA replication in eukaryotes:

25. Each eukaryotic chromosome is one linear DNA double helix Average ~10 8 base pairs long With a replication rate of 2 kb/minute, replicating one human chromosome would require ~35 days. Solution ---> DNA replication initiates at many different sites simultaneously. Rates are cell specific!

26. What about the ends (or telomeres) of linear chromosomes? DNA polymerase/ligase cannot fill gap at end of chromosome after RNA primer is removed. this gap is not filled, chromosomes would become shorter each round of replication! Solution: 1.Eukaryotes have tandemly repeated sequences at the ends of their chromosomes. 2.Telomerase (composed of protein and RNA complementary to the telomere repeat) binds to the terminal telomere repeat and catalyzes the addition of of new repeats. 3.Compensates by lengthening the chromosome. 4.Absence or mutation of telomerase activity results in chromosome shortening and limited cell division.

27. Summary of DNA replication  DNA Replication is the process in which the DNA within a cell makes an exact copy of itself.  DNA replication is the process where an entire double-stranded DNA is copied to produce a second, identical DNA double helix.  Helicase unwinds the double helix starting at a replication bubble.  The two strands separate as the hydrogen bonds between base pairs are broken.  Two replication forks form and the DNA is unwound in opposite directions.  After helicase has completed unwinding the DNA strand, Single strand Binding Proteins (SSB) keep the two strands from re-annealing (coming back together).  Primase is an RNA polymerase that makes the RNA primer.  These primers “tell” the DNA polymerase where to start copying the DNA.  The DNA polymerase starts at the 3’ end of the RNA primer of the leading stand CONTINUOUSLY.  DNA is copied in 5’ to 3’ direction.  DNA polymerase copies the lagging strand DIS- continuously.

28.  The dis-continuous pieces of DNA copied on the lagging strand are known as Okazaki fragments.  Another DNA Polymerase removes the RNA primers and replaces them with DNA.  Finally the gaps in the sugar phosphate backbone are sealed by DNA ligase. There are now 2 identical double helices of DNA.

29. Notice:  No third examination. This means next week no examination.  I will give one topic to each student related to Biochemistry II and you have to submit assignment to me by 31 st May.  I will upload this assignment in this wek on portal. Please see your name and topic.  I suggest you to please share your topic each other to know more.

30. Next class About details of DNA polymerase & DNA Recombination