The Cell Cycle, DNA Replication, and Mitosis

1. The Cell Cycle, DNA Replication, and Mitosis Chapter 19

2. The Cell Cycle, DNA Replication, and Mitosis Cell growth is generally accompanied by cell division, whereby one cell gives rise to two new daughter cells All the genetic information in the nucleus must be accurately duplicated and carefully distributed to the daughter cells In doing this a cell passes through a series of stages known as the cell cycle

3. Overview of the Cell Cycle The cell cycle begins when two new cells are formed by division of a parent cell and ends when one of these cells divides again M phase is when the cells actually divide; the nucleus first, followed by the cytoplasm Nuclear division is mitosis and division of the cytoplasm is cytokinesis

4. Chromosomes in mitosis At the beginning of mitosis, chromatin folds and condenses to produce visible chromosomes DNA has replicated, so each chromosome is composed of two sister chromatids The microtubules of the mitoticspindle will distribute the chromatids to opposite ends of the cell

5. Figure 19-1A

6. Mitosis is a relatively short part of the cell cycle Cells spend very little time in M phase Most of the time is spent in interphase, which is composed of G1 phase, S phase (when DNA is replicated), and G2 phase The overall length of the cell cycle is called the generation time; in cultured mammalian cells this is about 18–24 hours

7. Figure 19-1B

8. The G phases G1 is quite variable depending on cell type; G2 is shorter and less variable During G1 a major decision is made, whether a cell will divide again; cells that arrest in G1, waiting for a signal to divide, are said to be in G0 Cells that exit the cell cycle are said to undergo terminal differentiation

9. DNA Replication DNA replication is a central event in the cell cycle The underlying mechanism depends on the double-helical structure of DNA One strand of every new DNA molecule is derived from the parent molecule and the other is new: semiconservative replication

10. Figure 19-2

11. DNA Replication Is Usually Bidirectional DNA replication is especially well understood in Escherichia coli Saccharomyces cerevisiae and the virus SV40 are used in studies of eukaryotic replication Replication is very similar in prokaryotes and eukaryotes

12. Early experiments in replication Cairns studied replication in E. coli; he grew cells in a medium containing 3H-thymidine He visualized the circular chromosomes by autoradiography; he observed replication forks These are formed where replication begins and then proceeds in bidirectionalfashion away from the origin

13. Figure 19-4A

14. Bacterial replication Replication forks move away from the origin, unwind the DNA, and copy both strands as they proceed This is called theta () replication and is observed in replication of circular DNA molecules The two copies of the replicating chromosome bind to the plasma membrane at their origins; when replication is complete the cell divides by binary fission

15. Figure 19-4B

16. Figure 19-4C

17. Eukaryotic DNA Replication Involves Multiple Replicons In eukaryotes replication of linear chromosomes is initiated at multiple sites, creating replication units called replicons At the center of each replicon is a DNA sequence called an origin of replication, where synthesis is initiated by several groups of initiator proteins First, a multisubunit protein complex called the origin recognition complex (ORC) binds the replication origin

18. Figure 19-5A-E

19. Figure 19-5A

20. Figure 19-5B

21. Figure 19-5C

22. Figure 19-5D

23. Figure 19-5E

24. Eukaryotic replication (continued) Next, the minichromosome maintenance (MCM) proteins bind the origin The MCM proteins include several DNA helicases that unwind the double helix; a set of proteins called helicase loaders recruit the MCM proteins At this point all the DNA-bound proteins make up the pre-replication complex and the DNA is “licensed” for replication

25. Origins of replication DNA sequences that act as replication origins are greatly varied in eukaryotes Sequences that confer ability to replicate when introduced into DNA molecules are called autonomously replicating sequences or ARS After replication begins, two replication forks synthesize DNA in opposite directions, forming a replication bubble that grows as replication proceeds

26. Rates of replication S phase is very rapid in embryonic cells, where cell divisions occur in quick succession, but slower in adult cells, in which fewer and more widely spaced replicons are used During S phase in eukaryotes, not all replicons are activated at the same time; some are replicated early and others later Genes that are transcriptionally active are replicated earlier than inactive genes

27. Replication Licensing Ensures That DNA Molecules Are Duplicated Only Once Prior to Each Cell Division Licensing is provided by binding of MCM proteins to the origin, which requires both ORC and helicase loaders It ensures that after DNA is replicated at each origin, the DNA cannot be licensed for replication again until after mitosis After replication begins, the MCM proteins are removed from the origins and cannot bind again

28. Role of Cdk Cyclin-dependent kinase, cdk, has many roles in the cell cycle One form produced early in S phase activates DNA synthesis at licensed origins and prevents origins from being licensed again It catalyzes phosphorylation of ORC proteins and helicase loaders

29. Geminin Multicellular eukaryotes contain an additional inhibitor of relicensing called geminin It is made during S phase that blocks the binding of MCM proteins to DNA When the cell completes mitosis, geminin is degraded and Cdk activity falls so that licensing can occur for the next cycle

30. Figure 19-6

31. DNA Polymerases Catalyze the Elongation of DNA Chains DNA polymerase is an enzyme that can copy DNA molecules Incoming nucleotides are added to the 3 hydroxyl end of the growing DNA chain, so elongation occurs in the 5 to 3 direction Several other forms of DNA polymerase have been identified; the original is now called DNA polymerase I

32. Figure 19-7

33. Table 19-1

34. Temperature-sensitive mutations It is difficult to grow and study mutant strains that lack important functions such as DNA replication One approach involves using temperature-sensitive mutants, which produce proteins that function properly at 37oC but lose their function when the temperature is raised to 42oC

35. Observations on temperature- sensitive bacteria Bacterial strains have been identified in which DNA polymerase III functions normally at 37oC but loses its ability to replicate when the temperature is raised to 42oC The observations indicate that DNA polymerase III is central to bacterial DNA replication However, a variety of other proteins is needed for replication

36. Eukaryote DNA polymerases Eukaryotic cells contain several types of DNA polymerase; more than a dozen Of these, DNA polymerase , , and  are involved in nuclear DNA replication DNA polymerase  is used in mitochondrial DNA replication Those types remaining are involved in DNA repair or replication across regions of DNA damage

37. Biotechnology functions of DNA polymerases DNA polymerases have practical applications in biotechnology The polymerase chain reaction is a technique in which a DNA polymerase is used to amplify tiny samples of DNA

38. DNA Is Synthesized as Discontinuous Segments That Are Joined Together by DNA Ligase DNA is synthesized in the 5 to 3 direction, but the two strands of the double helix are oriented in opposite directions One strand (the lagging strand) is synthesized in discontinuous fragments called Okazaki fragments The other (the leading strand) is synthesized in a continuous chain

39. Okazaki’s experiments Okazaki isolated DNA from bacteria that were briefly exposed to a radioactive substrate incorporated into newly made DNA Much of the radioactivity was located in small fragments about 1000 nucleotides long With longer labeling the radioactivity became associated with longer molecules; this conversion did not take place in bacteria lacking DNA ligase

40. Figure 19-8

41. Figure 19-8A

42. Figure 19-8B

43. Okazaki’s observations illustrate how lagging strand synthesis occurs DNA synthesis from the lagging strand is synthesized in Okazaki fragments These are then joined by DNA ligase to form a continuous new 3 to 5 DNA strand Okazaki fragments are 1000–2000 nucleotides long in bacteria and viruses, but about one-tenth this length in eukaryotic cells

44. Figure 19-9

45. Proofreading Is Performed by the 3→ 5 Exonuclease Activity of DNA Polymerase About 1 of every 100,000 nucleotides incorporated during DNA replication is incorrect Such mistakes are usually fixed by a proofreading mechanism Almost all DNA polymerases have a 3 → 5 exonuclease activity

46. Proofreading Exonucleases degrade nucleic acids from the ends of the molecules Endonucleases make internal cuts in nucleic acid molecules The exonuclease activity of DNA polymerase allows it to remove incorrectly base-paired nucleotides and incorporate the correct base

47. Figure 19-10-1

48. Figure 19-10-2

49. RNA Primers Initiate DNA Replication DNA polymerase can only add nucleotides to the 3 end of an existing nucleotide chain Researchers implicated RNA in the initiation process based on several observations 1. Okazaki fragments usually have short stretches of RNA at their 5 ends

50. Observations about RNA involvement in DNA replication (continued) 2. DNA polymerase is able to add nucleotides to RNA chains as well as DNA chains 3. Cells contain an enzyme called primase that synthesizes short (~10 nt) chains of RNA using DNA as a template 4. Primase is able to initiate RNA strands without a pre-existing chain to add to