Part 3. Genetic Information Transfer. The biochemistry and molecular biology department of CMU

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1 Part 3 Genetic Information Transfer The biochemistry and molecular biology department of CMU

2 Cell cycle

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4 Replication: synthesis of daughter DNA from parental DNA Transcription: synthesis of RNA using DNA as the template Post-transcriptional modification: maturation of nascent mrna Translation: protein synthesis using RNA molecules as the template Protein targeting: delivery of matured proteins to their destination

5 Central dogma replication transcription translation DNA RNA protein Reverse transcription

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7 Chapter 10 DNA Replication

8 Section 1 General Concepts of DNA Replication

9 DNA replication a reaction in which daughter DNAs are synthesized using the parental DNAs as the template Transferring the genetic information to the descendant generation with a high fidelity replication parental DNA daughter DNA

10 Phosphodiester bond formation

11 Daughter strand synthesis Chemical formulation: (dnmp) n + dntp (dnmp) n+1 + PPi DNA strand substrate elongated DNA strand The nature of DNA replication is a series of 3-5 phosphodiester bond formation catalyzed by a group of enzymes.

12 DNA replication system Template: Substrate: Primer: Enzyme: Product: double stranded DNA dntp short RNA fragment with a free 3 -OH end DNA-dependent DNA polymerase (DDDP) other enzymes, protein factor dsdna with base pair complementary

13 Characteristics of replication Semi-conservative replication Bidirectional replication Semi-continuous replication High fidelity

14 1.1 Semi-Conservative Replication

15 Clever experiment Grow cells in 15 NH 4 Cl-containing media, isolate DNA material and analyze it using density gradient centrifugation. Grow the 15 N-cells in 14 NH 4 Clcontaining media for 20 min, isolate the first generation of daughter DNA and analyze using density gradient centrifugation. Continue the cell growth in 14 NH 4 Cl media for the second generation of daughter DNA, isolate and analyze it.

16 Data-supported model

17 Experiment of DNA semiconservative replication "Heavy" DNA(15N) grow in 14N medium The first generation grow in 14N medium The second generation

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19 Significance Each of the parental DNA strands serves as the template for making a daughter DNA strand. Thus, each of the two new dsdna consists of a parental strand and a daughter strand. Consequently, the genetic information is ensured to be transferred from one generation to the next generation with a high fidelity.

20 Eukaryotes and prokaryotes DNA replication of prokaryotic systems is relatively simple, and is well studied using E. coli as a model. Eukaryotes are complicated systems. Although sharing some replication similarities with prokaryotic systems, they demonstrate specific and unique features.

21 1.2 Bidirectional Replication Replication starts from unwinding the dsdna at a particular point (called origin), followed by the synthesis on each strand. The parental dsdna and two newly formed dsdna form a Y-shape structure called replication fork.

22 Replication fork 5' 3' 3' 5' 5' 3' direction of replication 5' 3'

23 Bidirectional replication Once the dsdna is opened at the origin, two replication forks are formed spontaneously. These two replication forks move in opposite directions as the syntheses continues.

24 Bidirectional replication

25 Replication bubble

26 Replication of prokaryotes The replication process starts from the origin, and proceeds in two opposite directions. It is named q replication.

27 Replication of eukaryotes Chromosomes of eukaryotes have multiple origins. The space between two adjacent origins is called the replicon, a functional unit of replication. The length of replicons varies from 13 kb 900 kb.

28 origins of DNA replication (every ~150 kb) 5' 3' 3' 5' bidirectional replication fusion of bubbles 5' 3' 5' 3' 3' 5' 3' 5'

29 1.3 Semicontinuous Replication The daughter strands on two parental strands are synthesized differently since the replication process obeys the principle that the DNA synthesis is in the 5 3 direction. The a P atom of the free dntp attaches the 3 -OH group of the existing nucleotide chain to form a phosphoester bond.

30 Leading strand On the parental strand having the 3 end, the daughter strand is synthesized continuously. This strand is referred to as the leading strand. (Primer is required.) 5' 3' 3' direction of unwinding 3' 5' 5'

31 Discontinuous synthesis On the other parental strand having the 5 -end, the synthesis of the daughter strand does not start until there is enough space on the template. A primer is then synthesized, and a DNA synthesis from 5 to3 is followed. As the replication fork moves, many DNA fragments are synthesized sequentially on the DNA template.

32 Discontinuous replication 3' 3' 5' replication direction 5' 3' 5' Okazaki fragment 3' leading strand 5'

33 Okazaki fragments These DNA fragments are called Okazaki fragment. They are nt long for prokaryotes and nt long for eukaryotes. These DNA fragments are linked together later to form a full-length daughter strand. The daughter strand consisting of Okazaki fragments is called the lagging strand.

34 Continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand represent a unique feature of DNA replication. It is referred to as the semi-continuous replication.

35 Semi-continuous replication

36 Section 2 Enzymology of DNA Replication

37 Sequential actions Initiation: recognize the starting point, separate dsdna, begin replication, Elongation: add dntps to the existing strand, form phosphoester bonds, correct the mismatch bases, extending the DNA strand, Termination: stop the replication

38 Teamwork protein M r # function DnaA protein 50,000 1 recognize origin DnaB protein 300,000 6 open dsdna DnaC protein 29,000 1 assist DnaB binding DNA pol Elongate the DNA strands DnaG protein 60,000 1 synthesize RNA primer SSB 75,600 4 single-strand binding DNA topoisomerase 400,000 4 release supercoil constraint

39 2.1 DNA Polymerase The first DNA- dependent DNA polymerase (short for DNA-pol I) was discovered in 1958 by Arthur Kornberg. Nobel Prize in physiology or medicine, 1959 He isolated 0.5g DNA-pol material from 100kg bacterial cells.

40 Later, DNA-pol II and DNA-pol III were identified in experiments using mutated E.coli cell line. ACTIVITY: 1. 5 fi3 polymerizing activity 2. exonuclease activity

41 DNA-pol of E. coli

42 DNA-pol I A single peptide composed of 18 helical segments Elongate nt with low rate Mainly responsible for proofreading and filling the gaps, not the enzyme responsible for the polymerization

43 N end Klenow fragment DNA-pol Ⅰ caroid C end DNA-pol I can be hydrolyzed into two fragments. A-F segments (323 AAs): small fragment, having 5 3 exonuclease activity G-R segments (604 AAs): large fragment called Klenow fragment, having DNA polymerization and 3 5 exonuclease activity

44 DNA-pol II Low template specificity (the mutated strand can be survived) Temporary functional when DNA-pol I and DNA-pol III are not functional Still capable for doing synthesis on the damaged template Participating in DNA repairing

45 DNA-pol III A heterodimer enzyme composed of ten different subunits Having the highest polymerization activity (10 5 nt/min), capable of elongating 0.5 Mb of DNA during one cycle on the leading strand The true enzyme responsible for the elongation process

46 Subunits a e and q make up the core enzyme, having 5 3 polymerizing activity and 3 5 exonuclease activity. Two b subunits encircle the DNA template in a sliding clamp. The rest subunits constitute the g- complex.

47 Structure of DNA-pol III α: has 5 3 polymerizing activity ε:has 3 5 exonuclease activity and plays a key role to ensure the replication fidelity. θ: maintain heterodimer structure

48 DNA-pol of prokaryotes

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50 Important properties Chain elongation: polymerize the DNA chain by forming a serials of phosphoester bonds sequentially---- DNA-pol III Processivity: the capability of adding individual nucleotides to the nascent DNA chain before disassociation Proofreading: identifies the mismatched nucleotides and makes necessary corrections ---- DNA-pol I

51 DNA-pol of eukaryotes DNA-pol a: initiate replication and synthesize primers DNA-pol b: replication with low fidelity DnaG, primase repairing DNA-pol g: polymerization in mitochondria DNA-pol d: elongation DNA-pol e: proofreading and gap filling DNA-pol III DNA-pol I

52 2.2 Primase A polypeptide of 60kD Also called DnaG Primase is able to synthesize primers using free NTPs using the ssdna as the template with the consumption of ATP. Primer is a short RNA fragment of a several decades of nucleotides long, providing a 3 -OH group.

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54 DNA-pol cannot catalyze the phosphoester bond formation using free dntps. (RNA synthesis does not require primers.) Primers provide free 3 -OH groups to react with the a-p atom of dntp to form phosphoester bonds. Primase, DnaB, DnaC and an origin form a primosome complex at the initiation phase.

55 2.3 Helicase Also referred to as DnaB. Open the double strand DNA with consuming ATP Implement the opening process with the assistance of DnaA and DnaC

56 2.4 SSB protein Stand for single strand DNA binding protein Maintain the DNA template in the single strand form in order to prevent the dsdna formation protect the vulnerable ssdna from nucleases

57 SSB protein Homotetramer of 177 AAs Cover 32 nt on ssdna Have a cooperative behavior to facilitate ssdna binding at the downstream region Dynamic participation: constantly associate to and dissociate from the DNA templates

58 2.5 Topoisomerase Opening the dsdna will create supercoil ahead of replication forks. The supercoil constraint needs to be released by topoisomerases. Both prokaryotic and eukaryotic systems have topoisomerases.

59 The interconversion of topoisomers of dsdna is catalyzed by a topoisomerase in a three-step process: Cleavage of one or both strands of DNA Passage of a segment of DNA through this break Resealing of the DNA break

60 Topoisomerase I (topo I) Also called w-protein in prokaryotes It cuts a phosphoester bond on one DNA strand (i.e., a single-strand break) a small distance ahead of the replication fork, rotates the broken DNA freely around the other strand to relax the constraint, and reseals the cut.

61 Topoisomerase II (topo II) It is named gyrase in prokaryotes. It cuts a phosphoester bond on both strands (i.e., a double-strand break) of dsdna, releases the supercoil constraint, and reforms the phosphoester bonds. It can change double-strand DNA into negative supercoil state consuming ATP as the energy source.

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63 2.6 DNA Ligase 3' 5' 5' 3' RNAase 3' 5' 5' OH P 3' dntp DNA polymerase 3' 5' 5' P 3' ATP DNA ligase 3' 5' 5' 3'

64 Connect two adjacent ssdna strands by joining the 3 -OH of one DNA strand to the 5 -P of another DNA strand Only for ssdna hybridized with another ssdna Not working for ssdna and ssrna

65 2.7 Replication Fidelity Replication based on the base pair principle is crucial to the high accuracy of the genetic information transfer. Enzymes use two mechanisms to ensure the replication fidelity. Proofreading and real-time correction Base selection

66 Proofreading and correction DNA-pol I has the function to correct the mismatched nuncleotides. It identifies the mismatched nucleotide, removes it using the 3-5 exonuclease activity, add a correct base, and continues the replication. The exonuclease is an enzyme that hydrolyzes the terminal nucleotides of a nucleotide strand sequentially in a designated direction.

67 Exonuclease functions 5 3 exonuclease activity cut primer or excise mutated segment 3 5 exonuclease activity excise mismatched nuleotides 5' 3' C T T C A G G A G A A G T C C G G C G 3' 5'

68 Base selection It has hypothesized that the phosphoester bond must be formed after the hydrogen bond formation. DNA-pol III without e subunit creates higher frequency of mismatched incorporation. DNA-pol III has the capability of incorporating nucleotides with preference. Mismatched nucleotides appear at different frequencies for the given nucleotides on the template.

69 High fidelity achievement Obey strictly the principle of complementary base pairing Incorporate nucleotides into the elongating chain with selection preference Make instant proofreading and error correction

70 Section 3 DNA Replication Process

71 3.1 Replication of prokaryotes The replication initiation needs to solve three problems. Where does the replication start? How does the dsdna become ssdna to provide the template for synthesis? How is the first free 3 -OH group used for the phosphoester bond formation?

72 3.1 Replication of prokaryotes a. Initiation The replication starts at a particular point called origin. The origin of E. coli, oric, is at the location of 82. The structure of the origin is 248 bp long and AT-rich.

73 Genome of E. coli

74 Structure of oric Three 13 bp consensus sequences Two pairs of anti-consensus repeats

75 Formation of replication fork DnaA recognizes oric. DnaB and DnaC join the DNA-DnaA complex, open the local AT-rich region, and move on the template downstream further to separate enough space. DnaA is replaced gradually. SSB protein binds the complex to stabilize ssdna.

76 Primer synthesis Primase joins and a complex called primosome is formed. Primase starts the synthesis of primers on the ssdna template using NTP as the substrates in the 5-3 direction at the expense of ATP. Primers are short RNA fragments providing a free 3 -OH group for DNA elongation.

77 Releasing supercoil constraint Opening the dsdna causes the downstream supercoiled. Topoisomerase binds to the dsdna region ahead of replication forks to release the supercoil constraint. The negatively supercoiled DNA serves as a better template than the positively supercoiled DNA.

78 Primosome complex Dna A Dna B Dna C primase 3' 5' 3' DNA topomerase 5'

79 b. Elongation dntps are continuously connected to the primer or the nascent DNA chain by DNA-pol III. Two core enzymes (a e and q ) catalyze the synthesis of leading and lagging strands, respectively. The nature of the chain elongation is the series formation of the phosphodiester bonds.

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81 Asymmetric structure Two strands on the replication fork use the same DNA-pol III. The synthesis of the leading strand is ahead of the lagging strand. To do so, the template for the lagging strand needs to fold back in a circular form to associate with DNA-pol III. Replication speed = 2500 bp/sec

82 The synthesis direction of the leading strand is the same as that of the replication fork. The synthesis direction of the latest Okazaki fragment is also the same as that of the replication fork.

83 Lagging strand synthesis Primers on Okazaki fragments are digested by RNase. The gaps are filled by DNA-pol Iin the 5-3 direction. The 5 end of one fragment and the 3 end of the next fragment are sealed by ligase. Okazaki fragments are connected sequentially together to form a complete single strand DNA.

84 3' 5' 5' 3' RNAase 3' 5' 5' OH P 3' dntp DNA polymerase 3' 5' 5' P 3' ATP DNA ligase 3' 5' 5' 3'

85 c. Termination The replication of E. coli is bidirectional from one origin, and the two replication forks must meet at one point called ter at 32. All the primers will be removed, and all the gaps will be filled by DNA-pol I and ligase.

86 3.2 Replication of Eukaryotes Viruses (SV40 ) and simple systems (yeast, Xenopus laevis) are used as the models. DNA replication is closely related with cell cycle. Multiple origins on one chromosome, and replications are activated in a sequential order rather than simultaneously.

87 Cell cycle

88 a. Initiation DNA synthesis occurs in the S phase. The quantity of dntp and the activity of DNA-pol reach the maximal point. Higher replication activities of DNA starts in the early S phase, whereas centromers and telomers are replicated in the later S phase.

89 Initiation The eukaryotic origins are shorter than that of E. coli. Requires DNA-pol a (primase activity) and DNA-pol d (polymerase activity and helicase activity). Needs topoisomerase and replication factors (RF) to assist. Mechanism details are not clear.

90 b. Elongation DNA replication and nucleosome assembling occur simultaneously. Overall replication speed is compatible with that of prokaryotes. Replications in different organs or under different physiological conditions are different. Lots of unanswered questions

91 c. Termination Hydrolyzing the last RNA prime makes the template chromosome become a single stranded that is susceptible to DNase digestion. It seems that the chromosomes will become shorter and shorter after each cell cycle. Chromosomes under the normal physiological condition remain the expected length after replications.

92 3' 5' 3' 5' 3' 5' 3' 5' 5' 3' 5' 3' connection of discontinuous segment 5' 3' 5' 3'

93 Telomere The terminal structure of eukaryotic DNA of chromosomes is called telomere. Telomere is composed of terminal DNA sequence and protein. The sequence of typical telomeres is rich in T and G. The telomere structure is crucial to keep the termini of chromosomes in the cell from becoming entangled and sticking to each other.

94 Telomerase The eukaryotic cells use telomerase to maintain the integrity of DNA telomere. The telomerase is composed of telomerase RNA, telomerase association protein, telomerase reverse transcriptase It is able to synthesize DNA using RNA as the template.

95 Inchworn model

96 Inchworn model

97 Section 4 Other Replication Modes

98 4.1 Reverse Transcription The genetic information carrier of some biological systems is ssrna instead of dsdna (such as ssrna viruses). The information flow is from RNA to DNA, opposite to the normal process. This special replication mode is called reverse transcription.

99 Viral infection of RNA virus

100 Reverse transcription Reverse transcription is a process in which ssrna is used as the template to synthesize dsdna.

101 Reverse transcription Synthesis of ssdna complementary to ssrna, forming a RNA-DNA hybrid Hydrolysis of ssrna in the RNA-DNA hybrid by RNase activity of reverse transcriptase, leaving ssdna. Synthesis of the second ssdna using the left ssdna as the template, forming a DNA-DNA duplex

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103 Reverse transcriptase Reverse transcriptase is the enzyme for the reverse transcription. It has three functions: RNA-dependent DNA polymerase RNase DNA-dependent DNA polymerase

104 Significance of RT An important discovery in life science and molecular biology RNA plays a key role just like DNA in the genetic information transfer and gene expression process. RNA could be the molecule developed earlier than DNA in evolution. RT is the supplementary to the central dogma.

105 Significance of RT This discovery enriches the understanding about the cancercausing theory of viruses. (cancer genes in RT viruses, and HIV having RT function) Reverse transcriptase has become a extremely important tool in molecular biology to select the target genes.

106 4.2 Rolling Circle Replication This replication mode is used in lower biological systems. Virus jx174 is a ssdna virus, and its infective type is ssdna. After infection, the viral ssdna becomes dsdna, the replication type. This dsdnawill be replicated in a special mode.

107 Protein A, having the endonuclease activity, make a cut at the origin on one circular strand. Replication starts from the 3 - end of the open circular single strand using the close circular one as the template. No need for primers. The 5 -end of the open circular strand is separated from the close one, and synthesis of Okazaki fragments stars on the open strand. Finally, protein A cuts the joining point of the new strand and the parental strand, forming two circular DNA.

108 Rolling circle replication 3' 5' 3' 5' 3' 5'

109 4.3 D-loop Replication D-loop replication is the unique replication form for mitochondrial DNA. Mitochondrial DNA is a dsdna having 37 genes, 13 code for proteins, 22 for trna and 2 for rrna. The starting points for the replication are different on the different strands, syntheses on these two strands show a temporal difference.

110 Mitochondrial DNA starts the replication from the 1st origin on one strand. When the elongating DNA chain reaches the 2nd origin on another strand, the 2nd replication starts in an opposite direction. DNA-pol g is the enzyme responsible for the replication in mitochondria. Primers are needed to initiate the replication on both strands.

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112 Section 5 DNA Damage and Repair

113 Checkpoints

114 5.1 Mutation Mutation is a change of nucleic acids in genomic DNA of an organism. The mutation could occur in the replication process as well as in other steps of life process.

115 Consequences of mutation To create a diversity of the biological world; a natural evolution of biological systems To lead to the functional alternation of biomolecules, death of cells or tissues, and some diseases as well Changes of genotype, but no effect on phenotype

116 5.2 Causes of Mutation Physical factors UV radiation DNA damage Chemical modification carcinogens infection spontaneous mutation T viruses G evolution

117 DNA can be damaged either spontaneously or catalyzed by environmental agents. Maintaining the integrity of the genetic information is the most important to the survival of organisms or species.

118 Physical damage O N O N R N O R N O P O N CH 3 UV P CH 3 CH 3 R N O R N O CH 3 O ( T T ) ) N

119 Mutation caused by chemicals Carcinogens can cause mutation. Carcinogens include: Food additives and food preservatives; spoiled food Pollutants: automobile emission; chemical wastes Chemicals: pesticides; alkaline derivatives; -NH 2 OH containing materials

120 5.3 Types of Mutation a. Point mutation Point mutation is referred to as the single nucleotide alternation. Transition: the base alternation from purine to purine, or from pyrimidine to pyrimidine. Transversion: the base alternation between purine and pyrimidine, and vise versa.

121 Hb mutation causing anemia Single base mutation leads to one AA change, causing disease. b chains b mrna HbS CAC GUG HbA CTC GAG AA residue 6 in b chain Val Glu

122 b. Deletion and insertion Deletion: one or more nucleotides are deleted from the DNA sequence. Insertion: one or more nucleotides are inserted into the DNA sequence. Deletion and insertion can cause the reading frame shifted if the number of the deleted or inserted nucleotides is not the even number of three, leading to a mis-expression of the amino acids that translated behind this mutation site.

123 Frame-shift mutation Normal 5 GCAGUA CAU GUC Ala Val His Val Deletion C 5 GAG UAC AUG UC Glu Tyr Met Ser

124 c. Rearrangement It is an exchange of large DNA fragments. It can be either reverse the direction or recombination between chromosomes. 1. Site-specific recombination 2. Homologous genetic recombination 3. DNA transposition

125 5.4 DNA Repairing DNA repairing is a kind response made by cells after DNA damage occurs, which may resume their natural structures and normal biological functions. DNA repairing is a supplementary to the proofreading-correction mechanism in DNA replication.

126 Light repairing O N O N R N O R N O P O N CH 3 UV P CH 3 CH 3 R N O R N O CH 3 N O ) ( T T )

127 Excision repairing One of the most important and effective repairing approach Prokaryotic and eukaryotic cells use different group of enzymes. UvrA and UvrB: recognize and bind the damaged region of DNA. UvrC: excise the damaged segment DNA-pol I: synthesize the DNA segment to fill the gap

128 Xeroderma pigmentosis (XP) XP is an autosomal recessive genetic disease. Patients will be suffered with hyper-sensitivity to UV which results in multiple skin cancers. The cause is due to the low enzymatic activity for the nucleotide excisionrepairing process, particular thymine dimer. 7 genes are involved and 2 of them are responsible for excision.

129 Recombination repair It is used for repairing when a large segment of DNA is damaged. Recombination protein RecA, RecB and RecC participate in this repairing. Rec protein cuts a segment of a healthy strand and fills the gap. The healthy segment can be from parental strand or the replicated one.

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131 SOS repair It is responsible for the situation that DNA is severely damaged and the replication is hard to continue. If workable, the cell could be survived, but may leave many errors. In E. coli, uvr gene and rec gene as well as Lex A protein constitute a regulatory network. This network is of low specificity and low selectivity for nucleotides, and only is activated in the emergence.