Chapter 28

1. Chapter 28 DNA Replication, Repair, and Recombination

2. Outline • DNA Replication is Semiconservative • General Features of DNA Replication • DNA Polymerases • The Mechanism of DNA Replication • Eukaryotic DNA Replication • Telomeres and Telomerase • DNA Repair • Reverse Transcriptase

3. DNA Replication Double Helix Facilitates the Accurate Transmission of Hereditary Information • Semiconservative replication:

4. Meselson & Stahl Experiment Experiment of DNA semiconservative replication Density-gradient equilibrium sedimentation • Parent DNA is labeled with 15N by growing E. Coli in 15N containing medium (15NH4Cl) • Transfer E. Coli in 14N containing medium • Look at distribution

5. Significance of semiconservative replication The genetic information is transferred from one generation to the next generation with high fidelity.

6. DNA Replication: Melting of double helix • Replication requires separation of the two strands of double helix • Hydrogen bonds between the base pairs are disrupted • Heat • Acid/alkali • Inside a cell is done with the help of helicases which use ATP • Dissociation of double helix is termed as melting • It occurs abruptly at a certain temperature • Melting temperature (Tm): • Melting is monitored by measuring absorbance at 260 nm

7. DNA Replication: Melting of double helix • The temperature at the midpoint of the transition (tm) is the melting point. It depends on • pH • ionic strength • the size • base composition of the DNA

8. DNA Replication: Melting of double helix Relationship between tm and the G+C content of a DNA

10. Annealing/hybridization • DNA strands with similar sequences will form partial duplexes or hybrid with each other. • Closer evolutionary relationship • between species • Similar DNA sequences • DNA hybridize • This property is used to “fish out” • (clone) a similar gene from different • species, if the gene sequence from • a species is known. Why human DNA hybridizes much more extensively with mouse DNA than with yeast DNA?

11. DNA Replication Replication: polymerization of deoxyribonucleosidetriphosphates along a template What is required?

12. DNA Replication DNA Polymerase • The first DNA Polymerase(short for DNA-pol I) was discovered in 1958 by Arthur Kornberg • received Nobel Prize in physiology or medicine in 1959

13. Structure of DNA polymerase enzymes • First determined DNA polymerase structure • “Klenow fragment” of E. Coli DNA polymerase I

14. DNA Polymerases • 5 structural classes • Finger and thumb domains wrap around DNA and hold it across the enzyme’s active site • Similar overall shape • Similar mechanism

15. What DNA polymerases require for replication? • Template • DNA polymerase is a …………………………..that synthesizes a product with a base sequence complimentary to that of the template • Primer • DNA polymerase requires a primer with a free 3’-hydroxyl group already base-paired to the template.

16. Polymerase reaction • Two bound metal ions participate in the reaction • One metal ion attaches to dNTPand 3’-OH group of the primer • Second metal ion interacts only with dNTP. • Two metal ions bridged by carboxylate groups of two Asp residues.

17. Polymerase reaction

18. How accuracy is maintained during DNA replication? • Binding of dNTP with correct base is favored by formation of a base pair with its partner on the template strand • H-bonds contribute to this formation • Can direct the incorporation • of thymidine • shape complimentarity

19. Why shape complementarity is important? First reason: • Minor groove interactions • DNA polymerases donate 2H bonds to base pairs in minor groove • Hydrogen bond acceptors are present in these 2 positions for all Watson-Crick base pairs

20. Why shape complementarity is important? Second reason: • Shape selectivity: • Binding of dNTP to DNA polymerase induces conformational change • generates a tight pocket • residues lining this pocket ensure the efficiency and fidelity of DNA synthesis

21. Synthesis of RNA primer • Primase: An RNA polymerase • Synthesizes a short stretch of RNA • complimentary to one of the template DNA strands • Later removed by hydrolysis and replaced by DNA

22. How replication proceeds along the parent DNA? • Both strands of parental DNA serve as templates. • Site of DNA synthesis called “replication fork”. Parental DNA

23. How replication proceeds along the parent DNA? • Unwinding of any single DNA replication fork proceeds in one direction • Problem • The two DNA strands are of opposite polarity and DNA polymerases only synthesize DNA 5’ to 3’ • Solution:DNA is made in opposite directions on each template • Leading strand -synthesized 5’ to 3’ in the direction of the replication fork • ………………….. • -requires a single RNA primer • Lagging strand -synthesized 5’ to 3’ in the opposite direction. • -……………………… • -requires many RNA primers • DNA is synthesized in short fragments called Okazaki fragments

24. How are Okazaki fragments joined? • DNA ligase reaction: • DNA ligase catalyzes formation of phosphodiesterbond • In eukaryotes, this is and ATP-driven reaction • In bacteria, this is NAD-driven reaction • DNA ligase seals breaks in dsDNA

26. DNA ligase mechanism

27. DNA ligase mechanism

28. How are DNA strands separated? • Helicases separate DNA strands for replication • Helicases utilizes energy of …………….to do so • Typically oligomers with 6 subunits • Each subunit has P loop NTPasedomain • Neighboring subunits interact closely in the ring structure • Only a single strand of DNA can fit through the center of the ring • DNA strand binds to loops on 2 adjacent subunits

29. Helicase Mechanism • Initially both domains bind ssDNA • Upon ATP binding, • Cleft between domains closes • A1 domain slides along DNA • On ATP hydrolysis • Cleft opens up • Pulls DNA from B1 domain toward A1 • dsDNA separated

30. DNA Unwinding and Supercoiling • As helicase unwinds DNA • the DNA in front becomes overwound • torsionally stressed DNA double helices • fold up on themselves to form tertiary structures

31. Topoisomers • Circular DNA molecules with • same nucleotide sequence • different linking numbers An electron micrograph showing negatively supercoiled and relaxed DNA

32. Linking number • It is equal to the number of times that a strand of DNA winds in the right-handed direction around the helix axis when the axis lies in a plane • The linking number for a relaxed B-DNA molecule: • = the number of base pairs present/ 10.4 • ………….. is the number of base pairs per turn

33. Other Terms • Right-handed vs Left-handed • Important numbers • Linking number (Lk) • Must be integer • Molecules differing only in linking number are topoisomers • Twisting number (Tw): a measure of the helical winding of DNA around each other • Does not have to be integer • Writhing number (Wr): a measure of the coiling of the axis of the double helix. i.e. supercoiling • Does not have to be integer • Lk = Tw + Wr

34. Linking number Unstressed DNA

35. Unwinding the linear duplex by two turns before joining its ends • Two limiting conformations are possible: • The DNA can fold into a structure containing 23 turns of B helix and an unwound loop • The double helix can fold up to cross itself • Such crossings are called ……………..

36. Supercoiling • Why is supercoiling biologically important? • Supercoiled DNA has more compact shape (packaging becomes easy) • Supercoiling affects DNA’s interactions with other molecules

37. Dealing with supercoiling during replication • Negative supercoils must be removed and the DNA relaxed as the double helix unwinds • Topoisomerases introduce or eliminate supercoils • Type I Topoisomerases • Catalyze relaxation of supercoiled DNA • Type II Topoisomerase • Adds negative supercoils to DNA

38. Dealing with supercoiling during replication They alter the linking number of DNA in a 3-step process • Cleave one or both strands • Type I cleaves one strand • Type II cleaves two strands • Passage of a segment of DNA through this break • Reseal DNA break

39. Type I Topoisomerases • Human type I topoisomerase comprises • Four domains around a central cavity • Diameter of 20 Å (diameter of B-DNA) • Includes a tyrosine residue (Tyr 723)

40. Topoisomerase I Mechanism On binding to DNA, TopoI cleaves one strand of the DNA through a Tyr (Y) residue attacking a phosphate. When the strand is cleaved, it rotates in a controlled manner around the other strand. The reaction is completed by religation of the cleaved strand. This relaxes the DNA!

41. Topoisomerase I Mechanism

42. Type II Topoisomerases A more complex mechanism • cuts dsDNA Will not be covered for Chem 361

43. Clinical importance of Types I and II topoisomerases • Human topoisomerase I • Inhibited by Camptothecin, an antitumor agent • Bacterial topoisomerase II (DNA gyrase) • Target of several antibiotics • Novobiocin blocks binding of ATP to gyrase • Nalidixic acid and ciprofloxacin interfere with breakage and rejoining of DNA chains • Used to treat urinary track and other infections • Including Bacillus anthracis (anthrax)

44. Coordination of enzyme activity is required for precise and rapid replication of genome. -Requires highly processive polymerases : Example: DNA Pol III DNA Replication is Highly Coordinated Structure of sliding clamp It allows the polymerase to move with DNA

45. The leading and lagging strands are synthesized in a coordinated fashion DNA polymerase III synthesizes

46. The leading and lagging strands are synthesized in a coordinated fashion • DNA-poly III begins synthesis of the leading strand starting from RNA primer • Helicase unwinds DNA • ss-binding proteins bind to the unwound strands, keeping the strands separated so that both strands can serve as templates • Lagging synthesis more complex • DNA-poly III makes Okazaki fragments • DNA-poly I removes …………………. • DNA ligase connects fragments • DNA synthesis in eukaryotes, more complex

47. The leading and lagging strands are synthesized in a coordinated fashion • DNA-poly III begins synthesis of leading strand using RNA primer • Helicase unwinds DNA • ss-binding proteins keep strands separated so both can be templates. • Lagging strand synthesis more complex Lagging strand

48. The leading and lagging strands are synthesized in a coordinated fashion • The mode of synthesis of the lagging strand is more complex • Lagging strand is synthesized in fragments • such that 5′ → 3′ polymerization leads to overall growth in the 3′ → 5′ direction • Yet the synthesis of the lagging strand is coordinated with the synthesis of the leading strand

49. The leading and lagging strands are synthesized in a coordinated fashion • How is this coordination accomplished? • DNA polymerase III • The holoenzyme includes two copies of the polymerase core enzyme • The core enzymes are linked to a central structure having the subunit composition γτ2δδ′χφ • The entire apparatus interacts with the hexamerichelicaseDnaB

50. The leading and lagging strands are synthesized in a coordinated fashion • Okazaki fragments (RNA polymerase initiates) • Looping the template for the lagging strand places it in position for 5’--->3’ polymerization • DNA poly III lets go off the lagging strand after adding 1000 nucleotides • New loop formed • RNA primer made by primase • Gaps filled by ……………….(it removes primers too)