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DNA Replication and Transcription Biosynthesis of DNA and RNA

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1 DNA Replication and Transcription Biosynthesis of DNA and RNA
Replication of DNA Action of DNA Polymerases DNA Damage and Repair Synthesis of RNA Post-transcriptional Modifications of RNA Base Sequences in DNA

2 Replication of DNA During replication, each original strand of DNA is used as a template. DNA replication is semiconservative. Each new DNA duplex is composed of an original strand and a new one. It has been demonstrated that this semiconservative process is universal for all cells.

3 DNA replication semiconservative conservative Parent DNA First
generation Second semiconservative conservative

4 Replication of DNA Replication begins at a disecrete point on the
DNA molecule and proceeds bidirectionally.

5 DNA replication

6 Replication process In the late 1950, John Cairns observed structures during the replication of DNA in E. coli cells - replication forks. In circular DNA, replication is observed as occurring at a discrete point and proceeding in both directions. In linear DNA, replication is initiated as several sites which grow in both directions - replication bubbles.

7 Circular DNA replication
forks terminus

8 Linear DNA Replication bubbles Blue original Red daughter

9 Action of DNA polymerases
DNA polymerase I The first enzyme discovered that would catalyze the synthesis of DNA. dNTP + (dNMP)n (dNMP)n+1 + PPi dNTP deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, dTTP) (dNMP) preformed DNA with n or n mononucleotides PPi pyrophosphate Mg2+

10 DNA polymerase I Mg2+ complexes the nucleotide.
Energy is supplied from the release of pyrophosphate. DNA acts as a template. The DNA must have a primer segment with a free 3’-hydroxyl group - for attachment of the new nucleotide. Elongation occurs at the 3’ end and proceeds in the 5’ -> 3’ direction.

11 DNA polymerases II and III
Additional polymerases have been discovered in E. coli. These enzymes have many of the same reaction requirements of DNA polymerase I. It is now believed the DNA polymerase III is the main enzyme used for DNA replication. The other forms most likely serve proofreading or repair functions.

12 DNA polymerases Polymerase Characteristic I II III
Molecular weight , , ,000 Polypeptide subunits Polymerization rate (nucleotides/sec) Activity 3’ -> 5’ exonuclease Yes Yes Yes 5’ -> 3’ exonuclease Yes No No

13 Okazaki fragments All know DNA polymerases catalyze chain elongation in the 5’ 3’ direction. The two template strands are oriented in an antiparallel fashion. Only one strand can be processed in a continuous fashion - 3’ 5’ parent strand. The complementary strand is synthesized in the 5’ 3’ direction as discontinuous fragments - Okazaki fragments.

14 Okazaki fragments The fragments are still added in the 5’ 3’ direction. They are covalently linked in later steps. Leading strand 3’ 5’ 3’ 5’ Lagging strand (Okazaki fragments)

15 DNA Replication Prokaryotic DNA replication - E. coli
This multistep process involves several proteins. Protein Function Helicase Begins unwinding of DNA helix DNA gyrase Assists unwinding SSB proteins Stabilizes single DNA strands Primase Synthesis of RNA primer DNA polymerase III Elongation of chain by DNA synthesis DNA polymerase I Removal of RNA primer and fill in gap with DNA DNA ligase Closes last phosphoester gap

16 Prokaryotic Replication
Step one Helicase recognizes and binds to the origin for replication. It catalyzes the separation of the two DNA strands. DNA gyrase assists in unwinding and the replication fork is formed. DNA gyrase 5’ 3’ Helicase ADP ATP

17 Prokaryotic Replication
Step two Exposed single strands of DNA must then be stabilized and protected from cleavage of the phosphodiester bonds. SSB proteins perform this function. Complementary strands are now available as templates. SSB protein

18 Prokaryotic Replication
Step three Primase initiates synthesis by producing a short strand of RNA (4-10 nucleotides.) This is only required once for the leading strand. Separate initiation is required for all Okazaki fragments. DNA polymerase III can then process the 3’-hydroxyl group. DNA polymerase III primer RNA primase

19 Prokaryotic Replication
Step four After ‘priming’, DNA polymerase III can then process the 3’-hydroxy group. The leading strand continues in the direction of the advancing replication fork. The lagging strand fragments stop when they reach another fragment.

20 Prokaryotic Replication
Step five The RNA primers are removed by the 5’->3’ nuclease action of DNA polymerase I. Remaining gaps are filled by DNA polymerase I. DNA polymerase I

21 Prokaryotic Replication
Step six DNA ligase is used to complete the final phosphoester bond. Termination of replication occurs when the two replication forks meet on the circular DNA. DNA ligase ADP ATP

22 Eukaryotic replication
There is much we do not understand about this more complicated process. Important difference Telomeres - specialized DNA ends that consist of hundreds of repeating hexanucleotide sequences The sequence for humans is AGGGTT

23 Eukaryotic replication
Telomerase The enzyme that catalyzes the synthesis of DNA ends. Unusual enzyme (ribozyme) that contains an RNA molecule that serves as a template. The RNA guides the addition of the correct nucleotides. Varying activity levels of telomerase may serve to regulate cell division and aging.

24 DNA Damage and Repair Mutation
Sudden, random alteration of original DNA code that changes the genotype. It may be as simple as one wrong nucleotide. It can be harmful/deadly or be a positive change. (evolution - rare) Can be caused by chemical or environmental factors - mutagens.

25 DNA Damage and Repair There are ~4000 know human genetic diseases. Many result from mutation of a single gene. Sickle cell anemia. Mutation of gene that makes part of hemoglobin. Male pattern baldness. Characteristic thinning of hair in males. Linked to a pair of genes. Albinism. Lack of ability to produce tyrosinase which catalyzes the conversion of tyrosine to DOPA.

26 Mutations Spontaneous mutations
Changes that occur during normal genetic and metabolic function. Two types Mistakes in the incorporation of deoxyribonucleotides during DNA replication. Base modifications caused by hydrolytic reactions.

27 Spontaneous Mutations
Final mistakes during E. coli replication are very rare. 1 error in every 1010 base pairs. Actual error rate for base incorporation may be much higher ( 1 in every ). Repair mechanisms will correct most mismatched bases.

28 Spontaneous Mutations
Replication errors are of three types: Point mutation - substitution of one base pair for another. Insertion of one or more extra base pairs. Deletion of one or more base pairs. Substitution is the most common type of spontaneous mutation.

29 Spontaneous Mutations
E. coli cells have systems to detect and repair mismatched bases. The general mechanism proceeds in four steps. Endonuclease-catalyzed cleavage of phosphoester bond holding the incorrect base. Removal of mismatched base by an exonuclease. Incorporation of the correct base by DNA polymerase I or III. Closure of the final gap by DNA ligase.

30 Photodimerization UV light Exposure to UV light can cause adjacent
thymines to covalently link. This results in a distortion of the DNA molecule and breaks the hydrogen bonding with the adenine. thymine dimer

31 Thymine dimer repair in E. Coli
To repair the damage, a photoreactivating enzyme binds to thymine dimer. Visible light activates the enzyme which breaks the dimer, restoring original structure. The enzyme is then released from the repaired DNA.

32 Thymine repair in humans
DNA repair is more complex than in E. Coli requiring at least 5 enzymes. Human repair mechanism must: cleave the sugar phosphate backbone remove bad section rebuild a new section Xeroderma Pigmentosum Genetic disorder where repair mechanism does not work. Can result in multiple skin cancers by age 20.

33 Induced mutation A number of environmental factors can induce a mutation - mutagens. Radiation Ionizing - X-rays,  rays, cosmic rays Nonionizing - UV light Intercalating agents Flat, hydrophobic chemicals that are typically aromatic. They can insert between stacked base pairs resulting in insertion or deletion of bases.

34 Intercalating agents intercalating agent

35 Induced mutation Chemical mutagens Reactant with bases - formaldehyde
Base analogs - 2-aminopurine, 5-bromouracil Acridine dyes - proflavin Alkylating agents - mustard gases Others - carcinogens

36 Synthesis of RNA Some basic terms
Template - the strand of DNA used for the synthesis of RNA. It is read in the 3’-> 5’ direction. Coding strand - the ‘other’ DNA strand. Transcript - the RNA molecule. It is synthesized in the 5’ -> 3’ direction. The first base in a gene is numbered +1. Additional bases are numbered sequentially. ‘Upstream’ bases are assigned negative numbers. There is no zero value.

37 DNA-directed RNA synthesis
Prokaryotic cells rely on DNA-directed polymerase (RNA polymerase)to catalyze all steps in the transcription of RNA. The process occurs in three stages: Initiation Elongation Termination

38 RNA synthesis

39 RNA synthesis In the first step, RNA polymerase binds
to a promoter sequence on the DNA chain. This insures that transcription occurs in the correct direction. The initial reaction is to separate the two DNA strands.

40 RNA synthesis ‘Special’ base sequences in the DNA strand
initiation sequence termination sequence ‘Special’ base sequences in the DNA strand indicate where RNA synthesis starts and stops.

41 RNA synthesis The elongation process continues until an entire gene is transcribed. This is catalyzed by RNA polymerase NTP + (NMP)n (NMP)n PPi DNA template NTP ribonucleoside triphosphate, ATP, GTP, CTP, UTP (NMP) preformed RNA with n or n+1 mononucleotides

42 RNA synthesis Once the termination sequence is reached, the
new RNA molecule and the RNA synthase are released. The DNA recoils.

43 RNA synthesis The transcription process differs for eukaryotic cells
Three classes of RNA polymerases for the transcription process (I, II, II) I transcribes large ribosomal RNA genes. II is for protein-encoding genes. III is used during transcription of tRNA and 5S rRNA

44 RNA-directed RNA synthesis
An alternate mode of RNA synthesis is found in RNA viruses. They induce formation of RNA replicase in the host cell and use RNA as a template. Direction of synthesis is 5’ -> 3’. Same basic mechanism. RNA transcript is complementary to the RNA template. There are no editing, proofreading or repair activities.

45 Post-transcriptional modifications of RNA
Primary transcripts Newly synthesized RNA molecules. They are typically inactive. Several types of post-processing may be conducted to produce a mature form of RNA that is active. The processing varies based on the type of RNA.

46 tRNA and rRNA processing
Four types of processes Trimming of the ends by phosphoester bond cleavage Splicing to remove introns Addition of terminal sequences Hetrocyclic base removal Prokaryotic cells do not demonstrate intron removal.

47 tRNA post-processing pre-tRNA tRNA Several steps some catalyzed
- OH 3’ 5’ pre-tRNA tRNA Several steps some catalyzed by ribonuclease P

48 mRNA processing While prokaryotic mRNA requires little or no alteration, eukaryotic mRNA must be modified in the nucleus before use. There are three processes that occur Capping Poly A addition Splicing of coding sequences

49 Capping Modification of the 5’ end.
Hydrolytic removal of a phosphate from the triphosphate functional group. Guanosine triphosphate (GTP) is used to attach a GMP, resulting in a 5’ -> 5’ triphosphate covalent linkage. The end guanine residue is then methylated at N7 Additional capping may include methylation at ribose hydroxyl groups.

50 Poly A addition Modification of the 3’ end.
Most mature mRNA have a 3’ tail of from 20 to 250 nucleotides. Initially an endonuclease catalyzes the removal of a few 3’ base residues. Addition of adenine residues is catalyzed by polyadenyl polymerase. This tail is thought to stabilize mRNA by increasing resistance to cellular nucleases.

51 Intron removal Gene Exon Intron Exon Intron Exon transcription
Primary transcript removal of introns Splicing reseal transcript Mature RNA

52 Base sequences in DNA Efforts are underway to characterize the entire human genome. This requires methods for simple and inexpensive sequencing of DNA. Two methods meet this need. Maxam-Gilbert chemical cleavage Sanger chain-termination sequencing

53 The US human genome project
Methods for DNA sequencing are greatly assisting this project. It is a joint program of the Department of Energy and the National Institutes of Health. This project is a part of a larger international effort to characterize the genomes of humans and several model organisms.

54 X chromosome growth control factor, X-linked Xg blood group
ocular albinism sensorineural deafness anemia, sideroblastic, with spinocerebellar ataxia cleft palate lymphoproliferative syndrome Simpson dysmorphia syndrome coagulation factor IX, hemophilia B blue-monochromatic color blindness coagulation factor VIIIc, hemophilia A homosexuality, male

55 Y chromosome ribosomal protein S4, Y-linked testis determing factor
zinc finger protein, Y-linked acetylserotonin methyltransferase testis-specific protein, Y-linked Xg blood group stature, Y-linked


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