1. Chapter 10. DNA Replication  DNA replication requires: Deoxyribonucleoside triphosphates: the four dNTPs (dATP, dGTP, dCTP, dTTP). DNA template RNA primer Enzymes, accessory proteins, and other factors such as Mg 2+.

2.  DNA replication in prokaryotes is different from that in eukaryotes: 1)The templates: prokaryotic DNA is a double- stranded, closed circle, while eukaryotic DNA is a linear, double stranded structure. 2)Location: prokaryotic DNA replication is located in cytosol while that of eukaryotes occurs in the nucleus.

3. 3)Starting origin: prokaryotic DNA replication starts at a single origin while that of eukaryotes starts at multiple origins. 4)Polymerase: E. coli contains three DNA polymerases, named polymerase I, II, and III; animal cells have at least five different DNA polymerases, designated , , , , and .

4. 1.Important terms of DNA replication Semiconservative replication: after DNA replication, one strand of a parent duplex is associated with a newly synthesized strand in the daughter DNA duplex. Origin: the initiation site on a DNA strand for replication.

5. Replication fork: a Y-shaped structure formed by the newly synthesized DNA duplexes and the parent DNA during DNA replication. RNA primer: a short RNA sequence synthesized by primase, from which DNA polymerases initiate DNA biosynthesis. The RNA primer is later degraded and replaced with DNA.

6. 2. Enzymes of replication 1)DNA polymerases: using parent DNA single strand as a template, catalyze the reactions that extend the polydeoxyribo- nucleotide strands. DNA polymerase (DNA) n residues + dNTP (DNA) n+1 residues + PPi

7. DNA polymerase catalyzed reaction

8. DNA polymerases of E. coli Functions Polymerases Pol I Pol II Pol III 5’  3’ polymerization+++ 3’  5’ proofreading exonuclease+++ 5’  3’ repair exonuclease+--

9. Human DNA polymerases  LocationNNMNN Replication +-++- Repair -+--+ Associated functions 5’  3’ polymerase+++++ 3’  5’ exonuclease--+++ 5’  3’ exonuclease ----- Primase+---- N: nucleus M: mitochondrion +: yes -: no

10. 2) DNA ligase: catalyzes the formation of a phosphodiester bond between a free 3’- OH and 5’-PO 3 2- in DNA. OH O - O-P-O O - O O P O O - 5’ 3’ Ligase DNA

11. 3) DNA topoisomerases: enzymes that catalyze the interconversion of topological isomers of DNA (Topology refers to the degree and nature of supercoiling). There two classes of topoisomerase, named I and II. A) Topoisomerase I: makes a nick in only one strand and allows the intact strand to pass through the nick, which is then closed.

12. B) Topoisomerase II: makes a transient break in two strands and allows a duplex segment of DNA to pass through the “gate”, which is then closed by the enzyme. Nalidixic acid inhibits bacterial topoisomerase II (gyrase). This drug is usually used to treat urinary tract infections.

13. 3. DNA replication in E. coli 1)The bacterial DNA replication starts at a single origin. Both DNA strands serve as template and the replication proceeds bi- directionally.

14. 2) The process of DNA replication in E.coli A)Fork formation: helicase binds at the origin and opens the helix with ATP hydrolysis, forming a replication fork. This is followed by binding of single- strand binding (SSB) proteins to the single strands, stabilizing the single strand state. helicase SSB DNA helix

15. B) Synthesis of primer: primase catalyzes formation of a short RNA sequence at the origin, using the parent DNA as template. 3’ 5’ 3’ 5’ 3’ Primer

16. C) Synthesis of the leading strand and Okazaki fragments by polymerase III 3’ 5’ 3’ New DNA sequence 3’ 5’ 3’ Leading strand Okazaki fragments Polymerase III dNTP

17. D) Removal of RNA primers and formation of intact new DNA strands  Leading strand New DNAParent DNA RNA primer Removal of RNA primer Pol I fill up the gap, the ends joined by ligase

18.  Okazaki fragments New DNAParent DNA RNA primer Removal of RNA primer Pol I fill up the gap, fragments joined by ligase Okazaki fragment

19. E) Synthesis of both RNA primers and DNA sequence (leading strand and Okazaki fragments) is in a 5’  3’ direction. During DNA synthesis, the replication complex (polymerase III, SSB, etc.) moves in both directions. The topoisomerase I ahead of the replication fork makes a break to allow the DNA to rotate so that the DNA strands are ready for replication.

20. F) Topoisomerase II separates the interlocked daughter DNA molecules by causing a transient double strand break. Topoisomerase II Double-stranded DNA

21. 4. DNA replication in humans 1)Cell cycle: the cell cycle of eukaryotes consists of G 1, S, G 2, and M phases. S(Synthesis, 7h) G 2 (prepare for Mitosis, 4 h) G 1 (growth, 11h) M(Mitosis, 1-2h) G 0 (quiescent)

22.  In human cells in culture, G 1, S, and G 2 make up interphase, which lasts for about 23 hours. The mitosis occurs in 1-2 hours. DNA replication occurs only in the S phase. 2) The replication process: similar to that in E. coli, it also needs topoisomerases, helicase, SSB, DNA polymerases, and ligase. A) Initiation: DNA polymerase  and  are responsible for replication of chromosomal DNA. Polymerase  has the primase activity and initiates the replication by synthesizing the RNA primers.

23.  A yeast chromosome contains about 400 initiation sites for DNA replication. These origins are called ARS for “autonomous replication sequence”, insertion of which into a bacterial plasmid may cause autonomous replication of DNA.  ARS is recognized by “origin recognition complex (8 proteins)” which initiates replication.

24. B) Extension: DNA polymerase  is responsible for the lagging strand biosynthesis and  for the leading strand biosynthesis. DNA polymerase  is also responsible for proof- reading.  Leading strand: in DNA replication the extending strand that can be continuously synthesized.  Legging strand: refers to the strand that is discontinuously synthesized via Okazaki fragments.

25. C) Termination: DNA fragments synthesized during replication are linked via phosphodiester bonds catalyzed by ligase to form an intact DNA strand. 3’ 5’ 3’ Lagging strand Leading strand

26. Replication of eukaryotic DNA +

27.  The synthesis of the chromosomal ends: the enzyme that replicates the chromosomal ends (telomeres) is called “telomerase”, which carries a short RNA sequence serving as a template for the end replication.  The human telomerase consists of three components: human telomerase RNA (hTR), human telomerase associated protein 1 (hTP1), and human telomerase reverse transcriptase (hTRT).

28. Replication of telomeric DNA GGGTTGGGGTTG-3’ 5’ GGGTTGGGGTTG 5’ GGGTTGGGGTTGGGGTTG 5’ GGGTTGGGGTTGGGGTTGGGGTTG-3’ 5’ CCCAAC-5’ Telomerase hTR

29. 5. Reverse transcription: a way of DNA biosynthesis using RNA as template. Reverse transcriptase is responsible for this type of DNA synthesis, and it is essential for reproduction of RNA viruses, which are called “retroviruses”, such as human immunodeficiency virus (HIV) and RNA tumor viruses. RNA tumor virus DNA provirus RNA tumor virus

30. Mechanism of reverse transcription: R | U5 | B | | U3 | R R | U5 B | | U3 | R R | U5 tRNA DNA synthesis RNA degradation

31. B | | U3 | R R | U5 B | | U3 | R B | | U3 | R | U5 U3 | R | U5 | B First jump DNA extension RNA degradation 2 nd DNA synthesis

32. B | |U3 | R | U5 U3 | R | U5 | B B | |U3 | R | U5 U3 | R | U5 | B U3 | R | U5 | B | |U3 | R | U5 RNA degradation Second jump 2 nd DNA extension Double-stranded proviral DNA

33.  Significance of reverse transcription: the synthesized proviral DNA is integrated into the host cell DNA, from which the viral RNAs can be synthesized in the host cell. + Host DNA Retroviral DNA Integrated proviral DNA Integration

34. 6. Mutations and lesions in DNA A)Mutation: A heritable change in DNA due to an alteration in the base sequence. Types of mutation: substitution, deletion, and insertion. (1) Substitution of one base pair for another is the most common type of mutation. This type includes: transition and transversion.

35.  Transition: replacement of one purine by the other.  Transversion: replacement of a purine by a pyrimidine, or a pyrimidine by a purine. A  T T  A A  T T  A G  C C  G C  G G  C Transitions transversions

36. (2) Deletion or insertion of one or more base pairs in DNA may alter the reading frame in translation, unless an integral multiple of three base pairs is inserted or deleted. This reading-frame alteration is called “frame- shift mutation”. e.g. ACT GGC AGT TCA AGC ACT AGG CAG TTC AAG C

37. B) Lesions and repairs: DNA is damaged by many chemical and physical agents, and cells possess mechanisms for repair.  Ionizing radiation, ultraviolet light, and some chemicals may cause base alteration or deletion, breakdown of phosphodiester bonds, covalently cross-link of DNA strands, etc. Many of these DNA damages can be repaired by the cell, some damages may cause mutation, and some may cause death of the cell.

38.  Pyrimidine dimer formation: adjacent pyrimidine residues on a DNA strand can become covalently linked when the DNA is exposed to ultraviolet light. A C C G C A T—T C A G T G T G G C G T A A G T C A C A C C G C A T T C A G T G T G G C G T A A G T C A C 5’ 3’ 5’ 3’ 5’ UV

39. Repair Formation and recovery of T-T dimer

40.  Once the pyrimidine dimer is formed, it can not fit into the DNA double helix and therefore, the DNA replication and gene expression are blocked until the lesion is removed.  Repair of DNA damage: includes light repair, excision repair, and SOS repair, etc.

41. (1)Light repair (photolyase repair): a mechanism for removal of thymine dimers. Nearly all cells contain a photoreactivating enzyme called DNA photolyase, which cleaves the dimer into its normal bases when the enzyme is excited by photons. (2)Excision repair: a mechanism involving hydrolytic removal of the damaged DNA fragment, synthesis of a new one to repair the gap.

42. Mechanism of excision repair Endonuclease cleavage Exonuclease removal Polymerase synthesis Ligase linking Damaged segment

43. (3) SOS repair (SOS response): when DNA molecules are severely damaged, single- stranded DNA (ssDNA) binds to recA protein. The ssDNA-recA complex activates the SOS genes that encode mRNAs for repair proteins. The repair proteins increase by hundred folds to mediate the repair of damaged DNA molecules.

44. ssDNA-recA  DNA damages activated lexA  SOS genes lexA SOS genes autoproteolysis Activated genes Repair proteins  Inactive genes Degraded lexA fragments Note: lexA is a repressor of SOS genes