Molecular Biology

1. Molecular Biology Miruka Conrad Ondieki Department of Biochemistry-KIU(Western Campus)

2. Organisation of the Genome • Prokaryotic cell’s genome • Eukaryotic cell’s genome

3. Prokaryotic cell’s genome • The DNA of a bacterial cell, such as Escherichia coli, is a circular double-stranded molecule often referred to as the bacterial chromosome • In E. coli this DNA molecule contains 4.6 million base pairs • The circular DNA is packaged into a region of the cell called the nucleoid • In the nucleoid, the DNA is organized into 50 or so loops or domains that are bound to a central protein scaffold, attached to the cell membrane • In the loops, DNA is negatively supercoiled (twisted upon itself) • It is also complexed with several DNA-binding proteins, the most common of which are proteins HU, HLP-1 and H-NS

4. Prokaryotic cell’s genome…

5. Eukaryotic cell’s genome • The genomic DNA of a eukaryotic cell is contained within a specialized organelle, the nucleus • A typical human cell contains 1000 times more DNA than the bacterium E. coli • This very large amount of eukaryotic nuclear DNA is tightly packaged in chromosomes • With the exception of the sex chromosomes, diploid eukaryotic organisms such as humans have two copies of each chromosome, one inherited from the father and one from the mother • Chromosomes contain both DNA and protein • Most of the protein on a weight basis is histones but there are also many thousands of other proteins found in far less abundance

6. Eukaryotic cell’s genome… • The nuclear DNA–protein complex is callechromatin • The mitochondria and chloroplasts of eukaryotic cells also contain DNA but, unlike the nuclear DNA, this consists of double-stranded circular molecules resembling bacterial chromosomes • In the nucleus, each chromosome contains a single linear double-stranded DNA molecule • The extensive packaging of DNA in chromosomes results from three levels of folding involving: • Nucleosomes • 30 nm filaments • Radial loops

7. Eukaryotic cell’s genome…

8. Cell cycle • Although cell division occurs in all organisms, it takes place very differently in prokaryotes and eukaryotes • There are two distinct types of eukaryotic cell division: • Mitosis leads to production of cells that are genetically identical to their parent • Meiosis leads to production of cells with half the genetic content of the parent • Mitosis serves as the basis for producing new cells, meiosis as the basis for producing new sexually reproducing organisms.

9. Cell cycle • In a population of dividing cells, whether inside the body or in a culture dish, each cell passes through a series of defined stages, which constitutes the cell cycle • The cell cycle can be divided into two major phases; • M phase • Interphase

10. M phase • M phase includes • the process of mitosis, during which duplicated chromosomes are separated into two nuclei • cytokinesis, during which the entire cell divides into two daughter cells • Whereas M phase usually lasts only an hour or so in mammalian cells, interphase may extend for days, weeks, or longer, depending on the cell type and the conditions

11. Interphase • Interphase, which is the period between cell divisions, is a time when the cell grows and engages in diverse metabolic activities • Interphase lasts for days, weeks, or longer, depending on the cell type and the conditions • Numerous preparations for an upcoming mitosis occur during interphase, including replication of the cell’s DNA • The period of time between the end of DNA synthesis and the beginning of M phase is termed as G2 (for second gap)

12. S phase • DNA replication occurs during a period of the cell cycle termed S phase • S phase is also the period when the cell synthesizes the additional histones that will be needed as the cell doubles the number of nucleosomes in its chromosomes

16. Control of the Cell Cycle • The Role of Protein Kinases • Checkpoints • Kinase Inhibitors • Cellular Responses

17. DNA Replication Semiconservative Replication • According to this model of replication, each of the daughter duplexes should consist of one complete strand inherited from the parental duplex and one complete strand that has been newly synthesized • Replication of this type is said to be semiconservative because each daughter duplex contains one strand from the parent structure

19. Conservative replication • In conservative replication, the two original strands would remain together (after serving as templates), as would the two newly synthesized strands • As a result, one of the daughter duplexes would contain only parental DNA, while the other daughter duplex would contain only newly synthesized DNA.

21. Dispersive replication • In dispersive replication the parental strands are broken into fragments, and new strands are be synthesized in short segments • Then the old fragments and new segments are joined together to form a complete strand • As a result, the daughter duplexes contain strands that are composites of old and new DNA

23. DNA replication in bacteria • Replication begins at a specific site on the bacterial chromosome called the origin • The origin of replication on the E. coli chromosomeis a specific sequence called oriCwhere a number of proteins bind to initiate the process of replication • Once initiated, replication proceeds outward from the origin in both directions, that is, bidirectionally • The sites where the pair of replicated segments come together and join the nonreplicated DNA are termed replication forks

24. DNA replication in bacteria contd.. • Each replication fork corresponds to a site where • The parental double helix is undergoing strand separation • Nucleotides are being incorporated into the newly synthesized complementary strands • The two replication forks move in opposite directions until they meet at a point across the circle from the origin, where replication is terminated • The two newly replicated duplexes detach from one another and are ultimately directed into two different cells

26. Unwinding the Duplex and Separating the Strands • Separation of the strands of a circular, helical DNA duplex poses major topological problems • Cells contain enzymes, called topoisomerases, that can change the state of supercoiling in a DNA molecul • One enzyme of this type, called DNA gyrase, a type II topoisomerase, relieves the mechanical strain that builds up during replication in E. coli • DNA gyrase molecules travel along the DNA ahead of the replication fork, removing positive supercoils

28. Mechanism of action of DNA gyrase • DNA gyrase removes positive supercoils by cleaving both strands of the DNA duplex, passing a segment of DNA through the double-stranded break to the other side, and then sealing the cuts • This process is driven by the energy released during ATP hydrolysis • Eukaryotic cells possess similar enzymes that carry out this required function.

29. Properties of DNA Polymerases • DNA polymerases are the enzymes that synthesize new DNA strands • For the reaction to proceed, the enzyme requires the presence of DNA and all four deoxyribonucleoside triphosphates (dTTP, dATP, dCTP, and dGTP) • Original DNA strands serve as templates for the polymerization reaction • All DNA polymerases—both prokaryotic and eukaryotic—have the same two basic requirements • A template DNA strand to copy • A primer strand to which nucleotides can be added

30. Semidiscontinuous Replication • Both newly synthesized strands are assembled in a 5' → 3' direction • During the polymerization reaction, the —OH group at the 3' end of the primer carries out a nucleophilic attack on the 5' α-phosphate of the incoming nucleoside triphosphate • The polymerase molecules responsible for construction of the two new strands of DNA both move in a 3'-to-5' direction along the template, and both construct a chain that grows from its 5'-P terminus

31. Semidiscontinuous Replication contd.. • Consequently, one of the newly synthesized strands grows toward the replication fork, where the parental DNA strands are being separated, while the other strand grows away from the fork • The strand that grows away from the replication fork is synthesized discontinuously, as fragments • Before the synthesis of a fragment can be initiated, a suitable stretch of template must be exposed by movement of the replication fork • Once initiated, each fragment grows away from the replication fork toward the 5 end of a previously synthesized fragment to which it is subsequently linked.

32. Semidiscontinuous Replication contd.. • The strand that is synthesized continuously is called the leading strand because its synthesis continues as the replication fork advances • The strand that is synthesized discontinuously is called the lagging strand becauseinitiation of each fragment must wait for the parental strands to separate and expose additional template • Because one strand is synthesized continuously and the other discontinuously, replication is said to be semidiscontinuous

34. Okazaki fragments • The discovery that one strand was synthesized as small fragments was made by Reiji Okazaki of Nagoya University, Japan • Okazaki found that if bacteria were incubated in [3H]thymidine for a few seconds and immediately killed, most of the radioactivity could be found as part of small DNA fragments 1000 to 2000 nucleotides in length • In contrast, if cells were incubated in the labeled DNA precursor for a minute or two, most of the incorporated radioactivity became part of much larger DNA molecules

35. Okazaki fragments • These results indicated that a portion of the DNA was constructed in small segments (later called Okazaki fragments) that were rapidly linked to longer pieces that had been synthesized previously • The enzyme that joins the Okazaki fragments into a continuous strand is called DNA ligase

36. Role of primase enzyme • How does the synthesis of each of the Okazaki fragments begin, when none of the DNA polymerases are capable of strand initiation? • Further research studies revealed that initiation is not accomplished by a DNA polymerase but, rather, by a distinct type of RNA polymerase, called primase, that constructs a short primer composed of RNA, not DNA • The leading strand, whose synthesis begins at the origin of replication, is also initiated by a primase molecule

37. Role of primase enzyme contd.. • The short RNAs synthesized by the primase at the 5' end of the leading strand and the 5' end of each Okazaki fragment serve as the required primer for the synthesis of DNA by a DNA polymerase • The RNA primers are subsequently removed, and the resulting gaps in the strand are filled with DNA and then sealed by DNA ligase

39. Sequence of events at the replication fork • Replication involves more than incorporating nucleotides • Unwinding the duplex and separating the strands require the aid of two types of proteins that bind to the DNA • A helicase(or DNA unwinding enzyme) • Single-stranded DNA binding (SSB) proteins

40. Role of DNA helicases • DNA helicases unwind a DNA duplex in a reaction that uses energy released by ATP hydrolysis to move along one of the DNA strands, breaking the hydrogen bonds that hold the two strands together and exposing the single-stranded DNA templates • E. coli has at least 12 different helicases for use in various aspects of DNA (and RNA) metabolism • In bacteria, the primase enzyme and the helicase associate transiently to form what is called a “primosome”

41. Role of SSB proteins • DNA unwinding by the helicase is aided by the attachment of SSB proteins to the separated DNA strands • These proteins bind selectively to single-stranded DNA, keeping it in an extended state and preventing it from becoming rewound or damaged

44. Replication in Eukaryotic Cells General features: • Eukaryotic cells replicate their genome in small portions, termed replicons • Each replicon has its own origin from which replication forks proceed outward in both directions • In a human cell, replication begins at about 10,000 to 100,000 different replication origins • Approximately 10 to 15 percent of replicons are actively engaged in replication at any given time during the S phase of the cell cycle

45. General features contd… • Replicons located close together in a given chromosome tend to undergo replication simultaneously • The most highly compacted, least acetylated regions of the chromosome are packaged into heterochromatin, and they are the last regions to be replicated • The inactive, heterochromatic X chromosome in the cells of female mammals is replicated late in S phase, whereas the active, euchromatic X chromosome is replicated at an earlier stage

46. The Eukaryotic Replication Fork

47. Synthesis of the strands • The DNA of eukaryotic cells is synthesized in a semidiscontinuous manner • However, the Okazaki fragments of the lagging strand are considerably smaller than in bacteria, averaging about 150 nucleotides in length • Like DNA polymerase III of E. coli, the eukaryotic replicative DNA polymerase is present as a dimer • This suggests that the leading and lagging strands are synthesized in a coordinate manner by a single replicative complex, or replisome

48. Eukaryotic DNA polymerases • Five “classic” DNA polymerases have been isolated from eukaryotic cells, and they are designated α, β, γ, δ and ε • Of these enzymes, polymerase γ replicates mitochondrial DNA, and polymerase β functions in DNA repair • The other three polymerases have replicative functions • Polymerase α is tightly associated with the primase, and together, they initiate the synthesis of each Okazaki fragment • Primase initiates synthesis by assembly of a short RNA primer, which is then extended by the addition of about 20 deoxyribonucleotides by polymerase δ

49. Eukaryotic DNA polymerases contd… • Polymerase δ is thought to be the primary DNA-synthesizing enzyme during replication of the lagging strand • Polymerase ε is thought to be the primary DNA-synthesizing enzyme during replication of the leading strand • After synthesizing an RNA-DNA primer, polymerase α is replaced at each template–primer junction by the PCNA–polymerase δ complex, which completes synthesis of the Okazaki fragment

50. Eukaryotic DNA polymerases contd… • When polymerase δ reaches the 5' end of the previously synthesized Okazaki fragment, the polymerase continues along the lagging-strand template, displacing the primer • The displaced primer is cut from the newly synthesized DNA strand by an endonuclease (FEN-1) and the resulting nick in the DNA is sealed by a DNA ligase • Like bacterial polymerases, all of the eukaryotic polymerases elongate DNA strands in the 5'→3' direction by the addition of nucleotides to a 3' hydroxyl group • None of them is able to initiate the synthesis of a DNA chain without a primer.