Prokaryotic cells divide by pinching in two. Fig. 10-CO, p.240

Transcription

1 Prokaryotic cells divide by pinching in two Fig. 10-CO, p.240

2 Learning Objectives 1. What Is the Flow of Genetic Information in the Cell? 2. What Are the General Considerations in the Replication of DNA? 3. How Does the DNA Polymerase Reaction Take Place? 4. Which Proteins Are Required for DNA Replication? 5. How Is DNA Replicated in Eukaryotes?

3 The Central Dogma The flow of genetic information in biological systems. Fig. 10-1, p.241

4 It states that in all living organisms, the genetic information is stored in chromosomal DNA. The flow of genetic information occurs in one direction from DNA RNA Protein

5 The eukaryotic cell cycle Fig , p.256

6 DNA Replication In this process the two polynucleotide chains of DNA are separated and each is copied in a complementary manner to produce daughter polynucleotide chains. Each daughter DNA molecule will contain one polynucleotide comes from the parental DNA and the other chain is newly formed ( semiconservative replication)

7 Experimental evidence for semiconservative replication. Meselon-Stahl Experiment 1958 ( In Prokaryotes ) Fig. 10-3, p.243

8 Fig. 10-2, p.242

9 DNA replication must occur in order to faithfully transmit genetic material to the progeny of any cell or organism. DNA replication takes place during S phase of the cell cycle. Transcription is the process by which the information contained in a section of DNA is transferred to a newly assembled piece of messenger RNA (mrna). Translation a process in which proteins can be synthesized using the information in mrna as a template.

10 In eukaryotic genome there is no similar linear relationship between genetic information carried in DNA and proteins expression, which was observed in prokaryotic system Occasionally, genetic information flows from RNA to DNA (in reverse of normal transcription). This is known to occur in the case of retroviruses, such as HIV.

11 DNA replication In prokaryotes like E. coli, the replication starts at fixed point called the origin and proceeds bidirectional along the chromosomal DNA (both directions) until the whole circular DNA is completely replicated. By then the replication process is terminated at certain point on DNA.

12 Fig. 10-4a, p.244

13 Fig. 10-5a, p.245

14 Fig. 10-5b, p.245

15 Mechanism of DNA replication in Prokaryotes Relaxation of complex super structure of chromosomal DNA Inside bacteria the circular double helix DNA is present in super helical form in which the DNA is further twisted in more circles. The relaxed double helix is called a secondary structure and the complex super helix called tertiary structure. The super helix is needed to pack the DNA in small space inside the cell but it is not suitable form for starting DNA replication. So, the first step in starting DNA replication is the relaxation of super coiled DNA by making small cut (nicking) with the enzyme Topoisomerase (Gyrase).

16

17 DNA replication involves 3 stages Initiation, elongation and termination. 1.Initiation: Initiation of DNA replication starts by the binding of the initiator factor protein DnaA at the initiation point in DNA, which recognizes a repeat 9 A-T base pair rich region. The binding of this protein helps in local unwinding double helix DNA at the initiation point by the enzyme helicase. After local unwinding the two single strands DNA are kept separated and not folded back again by single strand binding proteins.

18

19

20 Formation of bubble origin point The separation of the two DNA strands expand the DNA region at the origin point to form a bubble shape area that help in the incorporation of deoxynucleotides to synthesis new DNA strands.

21

22 II. Elongation of new DNA At the bubble area, local unwinding and replication will grow in two opposite directions so that both polynucleotides are copied simultaneously.this polynucleotides growth form a Y-shaped replication fork at each direction site of DNA replication (2 replication forks). Since, the DNA synthesis occurs in 5 to 3 direction, one strand called the leading strand, can be synthesized continuously while the other called the lagging strand, must be synthesized backward discontinuously in short fragments (Okazaki fragments) which are later joined to make one long piece.

23 Properties of prokaryotic DNA polymerase enzymes There are 3 different types of prokaryotic DNA polymerases,called pol I,pol II, pol III.Only pol I and III are involved in DNA replication while pol II function is limited to DNA repair of damaged DNA.The pol I enzyme has slower activity than pol III enzyme. Each DNA polymerase enzyme has two types of activities, polymerization and nuclease activities.

24 Table 10-1, p.246

25 The dimer of β-subunits of DNA polymerase III bound to DNA t Fig. 10-7, p.246

26 1) Polymerization activity Add deoxynucleotids ( as monophosphate) from triphosphates deoxyncleotides, using energy liberated from the hydrolysis of pyrophosphates (the process is called polymerization).the monophosphate deoxynuleotides are connected by phosphodiester linkage in 5 3 direction.

27 The building blocks for this process are 5'-ribonucleoside triphosphates, and pyrophosphate released as each phosphodiester bonds made.

28

29 RNA primer DNA polymerases cannot initiate synthesis of a complementary strand of DNA on a totally singlestranded template. Rather, they require an RNA primer that is, a short, double-stranded region consisting of RNA base-paired to the DNA template, with a free OH-group on the 3'-end of the RNA strand.this OH group serves as the first acceptor of a nucleotide by action of DNA polymerase. In de novo DNA synthesis, that free 3'-hydroxys provided by the short stretch of RNA, rather than DNA.

30 Primase A specific RNA polymerase, called primase synthesizes the short stretches of RNA (approximately ten nucleotides long) that are complementary and antiparallel to the DNA template. In the resulting hybrid duplex, the U in RNA pairs with A in DNA. These short RNA sequences are constantly being synthesized at the replication fork on the lagging strand, but only one RNA sequence at the origin of replication required on the leading strand.

31 Primosome Prior to the beginning of RNA primer synthesis on the lagging strand, a prepriming complex of several proteins assembled and binds to the single strand of DNA, displacing some of the single-stranded DNA-binding proteins. This protein complex, plus primase is called the primosome. It initiates Okazaki fragment formation by moving along the template for the lagging strand in the 5 3 direction, periodically recognizing specific sequences of nucleotides that direct it to create an RNA primer thats synthesized in the 5 3 direction (antiparallel to the DNA template chain).

32 2) Exonuclease activity: Repeated cutting of single nucleotide from the terminal end of a polynucleotide 3-Exonuclease activity: Removal of polynucleotide sequence in 3 5 direction from the terminal end associated with pol III activity. 5- Exonuclease activity: Removal of polynucleotide sequence in 5 3 direction from the terminal end, associated with pol I activity.

33 DNA polymerase I proofreading removes nucleotides from the 3 end of the growing DNA chain. Fig , p.250

34 The 5 3 exonuclease activity of DNA polymerase I can remove up to 10 nucleotides in the 5 direction downstream from a 3 -OH single strand nick Fig a, p.252

35 Mechanism of DNA synthesis at each Replication Fork 1)-Leading strand RNA primase enzyme creates a short primer RNA with free 3' end ( 10 RNA nucleotide sequence). DNA polymerase III enzyme - uses a single parent strand of DNA as a template to add new nucleotides to the 3' OH end of initially incorporated RNA primer.

36 The addition is continuous according to the base pairing rule. If a mismatch is accidentally incorporated, the polymerase is inhibited from further extension. Proofreading removes the mismatched nucleotide and extension continues. The mismatched nucleotides are remove by the exonuclease activities of DNA polymerase III. Later, DNA polymerase I removes the RNA primers and replaces them by DNA pieces leaving a small gap of free 3' OH and 5 OH ends to be sealed by DNA ligase.

37 2)-Lagging strand DNA polymerase is unable to work directly on the lagging strand because it lacks a free 3- OH end on the existing DNA strand. The new strand is synthesized in short discontinuous segments, each segment consists of RNA primer formed by primase and replicated DNA piece (Okazaki fragments) about DNA nucleotides in length formed by DNA polymerase III similarly to the leading strand except that the addition takes place in backward direction Later the polymerase I removes the RNA primers and replace them by DNA fragments leaving gaps. Ligase enzyme seals the gaps as described before..

38 Reiji Okazaki ( ) was a pioneer Japanese molecular biologist, known for his research on DNA replication and especially for describing the role of Okazaki fragments which he discovered working with his wife Tsuneko in Okazaki was born in Hiroshima, Japan. He graduated in 1953 from Nagoya University, and worked as a professor there after He died of leukemia (due to Atomic bombings of Hiroshima) in 1975 at the age of 44; he had been heavily irradiated in Hiroshima when the first atomic bomb was dropped. His wife, Tsuneko, won the L'Oréal- UNESCO Awards for Women in Science in 2000 for her work.

39

40

41

42 Final results of replication at the fork level

43 III.Termination: Termination requires that the progress of the DNA replication fork must stop at a specific locus on DNA. This process involves the interaction between two components: (1) A termination site sequence in the DNA (2) A protein which binds to this sequence to physically stop DNA replication. In bacteria this protein is named the DNA replication terminus site-binding protein. Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome. In E. coli the chromosomal DNA replication takes about 40 minutes to replicate the 4000 kb size of DNA. Therefore each fork replicates 2000 kb in 40 min. or ~ 50 kb/min or ~1000 bases/sec.

44 Why Does DNA Contain Thymine & Not Uracil? p.251a

45 The incorporation of thymine instead of uracil helps ensure that the DNA is replicated faithfully. p.251b

46

47 General features of a replication fork Fig , p.249

48 Table 10-3, p.250

49 END Part I

50 Eukaryotic DNA replication

51 Eukaryotic genome is more complex than prokaryotic genome. For example human genome is composed of 30,000-40,000 genes, and each gene is segmented into two types of DNA pieces, exons and introns,each gene has on average between 5 and 8 exons, 8000 base-pairs.

52 Exon: DNA segment which after transcription to RNA codes directly to peptide units of a polypeptide, i.e which `is expressed in protein' (200 base-pairs on average, in human genome) Intron: DNA segment which is not directly expressed for protein, involved in regulation, splicing and other unknown functions each 2000 bp's on average, in human genome.

53 Cell cycle in eukaryotes Actively dividing eukaryote cells pass through a series of stages known collectively as the cell cycle: involving interphase stage between each two mitosis. The interphase stage itself includes 3 phases represented by two gap phases (G1 and G2); and S (for synthesis) phase, in which the genetic material is duplicated. The two gaps are preparative periods for cell division and an opportunity for the cell to make decision whether to go in to division or not. In the M phase a nuclear division takes place before it is followed by cell division that occurs in cytokinesis

54

55 In the S phase. DNA synthesis replicates the genetic material and each chromosome becomes having two sister chromatids. Therefore, only in this period of the interphase that DNA replication occurs which is a necessary step before the cell decides to go in to division(without DNA replication there is no cell division).in human the S period is carried out for about 8 hours in an average total cell cycle period of around 20 hours.

56 Eukaryotic DNA synthesis is similar to synthesis in prokaryotes, except for some complexity. In eukaryotic cells: there is more DNA than prokaryotic cells the chromosomes are linear the DNA complexes with proteins

57 Eukaryotic replication initiated at many points Because the eukaryotic genome is so large (about 100 times the size of bacterial DNA), it would take days to replicate the whole length of eukaryotic chromosome using the same single initiation point as in prokaryotes. Therefore, many initiation points ( about 10,000 in human) are found in each eukaryotic chromosome instead of one, with replication forks moving in opposite directions away from each initiation point until they meet in the middle between each two initiation points. The initiation point is called a replicons which do not need specific termination sequences.

58 Fig. 10-4b, p.244

59 Not all replicons are activated simultaneously. Rather, clusters of adjacent replicons are activated throughout S phase until the whole chromosome is completely replicated.

60 The rate of eukaryotic DNA replication is much slower than E. coli, with only nucleotides bases/sec are replicated in eukaryotic Okazaki fragments. However, the majority of replication forks results in the whole genome being replicated in only about 8 hours. Histones for packaging the DNA are synthesized simultaneously with DNA replication to bind the new DNA.

61 Enzymes involved in eukaryotic DNA replication 1.DNA Polymerase α Initiation the synthesis of RNA primer (about ribonucleotides) then adds DNA to the RNA primers It has low processivity (efficiency) of DNA synthesis and has no 3 5 exonuclease activity. 2.DNA Polymerase δ The principal DNA polymerase in eukaryotic DNA replication which has 3 5 exonuclease activity. When it complexes with PCNA (Proliferating Cell Nuclear Antigen) it becomes highly processive.

62 Additional Proteins Involved in Eukaryotic DNA Synthesis DNA helicase: the enzyme which carries out partial unwinding of double helix DNA at the initiation point before the starting of DNA replication PCNA (Proliferating Cell Nuclear Antigen) Provides high processivity to DNA Polymerase δ RPA (Replication Protein A) ssdna-binding protein that facilitates the unwinding of the helix to create two replication forks. RFC (Replication Factor C) binds PCNA at the end of the primer FEN1/RTH1 (flap endonuclease 1/RAD two homologue 1) exonuclease complex

63 Leading strand synthesis 1) Starts with the primase activity of DNA Pol α to put down RNA primer in 5 3 -direction 2) The same enzyme adds a piece of DNA to the primer 3) RFC binds PCNA at the end of the primer 4) PCNA displaces DNA Pol α. 5) DNA polymerase δ binds to PCNA at the 3 ends of the growing strand to carry out polymerase switching to highly processive DNA synthesis activity. The RFC mediates the polymerase switching by helping in the a) Assembly of PCNA b) Removal of DNA Pol α c) Addition of DNA Pol δ

64 Lagging strand synthesis ) Starts off the same way as leading strand synthesis ) RNA primers synthesized by DNA polymerase α every 50 nucleotides and consist of nucleotides RNA ) DNA polymerase δ switching as before to extend the RNA primers and generating Okazaki fragments ) When the DNA Pol δ polymerizes the RNA primer of the downstream Okazaki fragment, RNase H1 removes all but the last RNA nucleotide of the RNA primer ) The FEN1/RTH1 exonuclease complex removes the last RNA nucleotide ) DNA Pol δ fills in the gap as the RNA primer is being removed ) DNA ligase joins the Okazaki fragment to the growing strand

65 Telomeres problem during human DNA replication Telomeres present at the ends of linear chromosomal DNA and consist of long area of short repeating sequences TTGGGG ->->->->- to protect the integrity and stability of human chromosomes. During DNA synthesis these chromosome ends cannot be replicated with DNA polymerase.

66 This sequence of TTAGGG is repeated approximately 2,500 times in humans. In humans, average telomere length declines from about 11 kilobases at birth to less than 4 kilobases in old age, with average rate of decline being greater in men than in women.

67 Telomeres are found at the termini of chromosomes. The end of a telomere inserts back into the main body of the telomere to form a T-loop

68 Since DNA can only be synthesized at the 3'- end of a pre-exiting DNA or RNA chain, there is no available mechanism for achieving DNA synthesis all of the ways to the end of the lagging strand. Once the primer in the last Okasaki fragment is removed by a 5' to 3' exonuclease it is not possible to replace it with DNA. This is because the 5 - ends of the lagging strands does not have enough space to put a new primer with free 3'-hydroxyl group and therefore it is not copied completely.

69 After maturation of Okazaki fragments, there is a primer gap

70 This problem does not occur with the leading strand which can undergo complete replication round. If this phenomenon is repeated over many rounds of replication, some of the chromosomes will gradually develop major shortening in their ends.

71

72

73 Correction of the chromosome ends by the enzyme Telomerase Telomerase enzyme is a ribonucleo protein complex containing RNA-dependent DNA polymerase activity and 450-nucleotide RNA. It can act as a reverse transcriptase enzyme by using its own repetitive RNA sequence (AAAACCCC ) as a template to add a repeat complementary sequence of TTAGGG to the 3- OH end of leading strand in telomeres of human DNA. This addition step by telomerase is repeated several times until an extend 3- end of the DNA is formed. In this case the role of telomerase enzyme is ended leaving gap in the 5- phosphate end of the opposite lagging DNA strand.this gap will be filled later by primase (adding short RNA primer ) and combined actions of DNA polymerase and ligase activities..

74

75

76 Some somatic cells lack telomerase activity and therefore, their telomeres get shorter with each cell division (About 50 bases are lost from each telomere every time a cell divides) which may end up with cell death. To the contrary cancer cells may have high activity of telomerase enzyme that increases their survivals,

77 Telomerase: Terminates the process of DNA replication only at the telomere ends of chromosomal DNA by adding many repeat units that can not be recognized by the replication complex.

78 p.259a

79 p.259b

80 p.258a

81 Telomere replication (asterisks indicate sequences at the 3 end that cannot be copied by conventional DNA replication p.258b

82 Model for initiation of the DNA replication cycle in eukaryotes ORC is present at the replicators throughout the cell cycle. The pre-replication complex (pre-rc) is assembled through sequential addition of the RAP (replication activator protein) & RLFs (replication licensing factors ) during a window of opportunity defined by the state of cyclin-cdks. Phosphorylation of the RAP,ORC,and RLFs triggers replication. After initiation, a post-rc state is established, and the RAP & RLFs are degraded.

83 Model for initiation of the DNA replication cycle in eukaryotes Fig , p.257

84 The basics of the eukaryotic replication fork Fig , p.261

85 Table 10-4, p.257

86 Table 10-5, p.260

87 END Part II