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Chapter 28 DNA Replication, Repair, and Recombination.

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1 Chapter 28 DNA Replication, Repair, and Recombination

2 Outline DNA Replication is Semiconservative General Features of DNA Replication DNA Polymerases The Mechanism of DNA Replication Eukaryotic DNA Replication Telomeres and Telomerase DNA Repair Reverse Transcriptase

3 Double Helix Facilitates the Accurate Transmission of Hereditary Information Semiconservative replication: DNA Replication

4 Experiment of DNA semiconservative replication Parent DNA is labeled with 15 N by growing E. Coli in 15 N containing medium ( 15 NH 4 Cl) Transfer E. Coli in 14 N containing medium Look at distribution Density-gradient equilibrium sedimentation Meselson & Stahl Experiment

5 Significance of semiconservative replication The genetic information is transferred from one generation to the next generation with high fidelity.

6 Replication requires separation of the two strands of double helix Hydrogen bonds between the base pairs are disrupted Heat Acid/alkali Inside a cell is done with the help of helicases which use ATP Dissociation of double helix is termed as melting It occurs abruptly at a certain temperature Melting temperature (Tm): Melting is monitored by measuring absorbance at 260 nm DNA Replication: Melting of double helix

7 The temperature at the midpoint of the transition (t m ) is the melting point. It depends on pH ionic strength the size base composition of the DNA DNA Replication: Melting of double helix

8 Relationship between t m and the G+C content of a DNA DNA Replication: Melting of double helix

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10 DNA strands with similar sequences will form partial duplexes or hybrid with each other. Closer evolutionary relationship between species Similar DNA sequences DNA hybridize This property is used to fish out (clone) a similar gene from different species, if the gene sequence from a species is known. Why human DNA hybridizes much more extensively with mouse DNA than with yeast DNA? Annealing/hybridization

11 Replication: polymerization of deoxyribonucleoside triphosphates along a template What is required? DNA Replication

12 The first DNA Polymerase (short for DNA-pol I) was discovered in 1958 by Arthur Kornberg – received Nobel Prize in physiology or medicine in 1959 DNA Polymerase DNA Replication

13 First determined DNA polymerase structure Klenow fragment of E. Coli DNA polymerase I Structure of DNA polymerase enzymes

14 DNA Polymerases 5 structural classes – Finger and thumb domains wrap around DNA and hold it across the enzymes active site Similar overall shape Similar mechanism

15 What DNA polymerases require for replication? Template – DNA polymerase is a …………………………..that synthesizes a product with a base sequence complimentary to that of the template Primer – DNA polymerase requires a primer with a free 3-hydroxyl group already base- paired to the template.

16 Polymerase reaction Two bound metal ions participate in the reaction One metal ion attaches to dNTP and 3-OH group of the primer Second metal ion interacts only with dNTP. Two metal ions bridged by carboxylate groups of two Asp residues.

17 Polymerase reaction

18 How accuracy is maintained during DNA replication? Binding of dNTP with correct base is favored by formation of a base pair with its partner on the template strand – H-bonds contribute to this formation Can direct the incorporation of thymidine shape complimentarity

19 Minor groove interactions DNA polymerases donate 2H bonds to base pairs in minor groove Hydrogen bond acceptors are present in these 2 positions for all Watson- Crick base pairs Why shape complementarity is important? First reason:

20 Shape selectivity: Binding of dNTP to DNA polymerase induces conformational change generates a tight pocket residues lining this pocket ensure the efficiency and fidelity of DNA synthesis Second reason: Why shape complementarity is important?

21 Synthesis of RNA primer Primase: An RNA polymerase Synthesizes a short stretch of RNA complimentary to one of the template DNA strands Later removed by hydrolysis and replaced by DNA

22 How replication proceeds along the parent DNA? Both strands of parental DNA serve as templates. Site of DNA synthesis called replication fork. Parental DNA

23 Unwinding of any single DNA replication fork proceeds in one direction Problem The two DNA strands are of opposite polarity and DNA polymerases only synthesize DNA 5 to 3 Solution: DNA is made in opposite directions on each template Leading strand -synthesized 5 to 3 in the direction of the replication fork ………………….. -requires a single RNA primer Lagging strand -synthesized 5 to 3 in the opposite direction. -……………………… -requires many RNA primers DNA is synthesized in short fragments called Okazaki fragments How replication proceeds along the parent DNA?

24 DNA ligase reaction: DNA ligase catalyzes formation of phosphodiester bond In eukaryotes, this is and ATP-driven reaction In bacteria, this is NAD-driven reaction DNA ligase seals breaks in dsDNA How are Okazaki fragments joined?

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26 DNA ligase mechanism

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28 Helicases separate DNA strands for replication Helicases utilizes energy of …………….to do so Typically oligomers with 6 subunits Each subunit has P loop NTPase domain Neighboring subunits interact closely in the ring structure Only a single strand of DNA can fit through the center of the ring DNA strand binds to loops on 2 adjacent subunits How are DNA strands separated?

29 Initially both domains bind ssDNA Upon ATP binding, Cleft between domains closes A1 domain slides along DNA On ATP hydrolysis Cleft opens up Pulls DNA from B1 domain toward A1 dsDNA separated Helicase Mechanism

30 DNA Unwinding and Supercoiling As helicase unwinds DNA – the DNA in front becomes overwound – torsionally stressed DNA double helices fold up on themselves to form tertiary structures

31 Circular DNA molecules with same nucleotide sequence different linking numbers An electron micrograph showing negatively supercoiled and relaxed DNA Topoisomers

32 Linking number It is equal to the number of times that a strand of DNA winds in the right- handed direction around the helix axis when the axis lies in a plane The linking number for a relaxed B-DNA molecule: = the number of base pairs present/ 10.4 ………….. is the number of base pairs per turn

33 Other Terms Right-handed vs Left-handed Important numbers Linking number (Lk) Must be integer Molecules differing only in linking number are topoisomers Twisting number (Tw): a measure of the helical winding of DNA around each other Does not have to be integer Writhing number (Wr): a measure of the coiling of the axis of the double helix. i.e. supercoiling Does not have to be integer Lk = Tw + Wr

34 Unstressed DNA Linking number

35 Unwinding the linear duplex by two turns before joining its ends Two limiting conformations are possible: The DNA can fold into a structure containing 23 turns of B helix and an unwound loop The double helix can fold up to cross itself Such crossings are called ……………..

36 Supercoiling Why is supercoiling biologically important? Supercoiled DNA has more compact shape (packaging becomes easy) Supercoiling affects DNAs interactions with other molecules

37 Dealing with supercoiling during replication Negative supercoils must be removed and the DNA relaxed as the double helix unwinds Topoisomerases introduce or eliminate supercoils Type I Topoisomerases – Catalyze relaxation of supercoiled DNA Type II Topoisomerase – Adds negative supercoils to DNA

38 Dealing with supercoiling during replication They alter the linking number of DNA in a 3-step process 1.Cleave one or both strands – Type I cleaves one strand – Type II cleaves two strands 2.Passage of a segment of DNA through this break 3.Reseal DNA break

39 Type I Topoisomerases Human type I topoisomerase comprises – Four domains around a central cavity Diameter of 20 Å (diameter of B-DNA) Includes a tyrosine residue (Tyr 723)

40 Topoisomerase I Mechanism On binding to DNA, TopoI cleaves one strand of the DNA through a Tyr (Y) residue attacking a phosphate. When the strand is cleaved, it rotates in a controlled manner around the other strand. The reaction is completed by religation of the cleaved strand. This relaxes the DNA!

41 Topoisomerase I Mechanism

42 Type II Topoisomerases A more complex mechanism cuts dsDNA Will not be covered for Chem 361

43 Clinical importance of Types I and II topoisomerases Human topoisomerase I – Inhibited by Camptothecin, an antitumor agent Bacterial topoisomerase II (DNA gyrase) – Target of several antibiotics Novobiocin blocks binding of ATP to gyrase Nalidixic acid and ciprofloxacin interfere with breakage and rejoining of DNA chains – Used to treat urinary track and other infections » Including Bacillus anthracis (anthrax)

44 Coordination of enzyme activity is required for precise and rapid replication of genome. -Requires highly processive polymerases : Example: DNA Pol III DNA Replication is Highly Coordinated Structure of sliding clamp It allows the polymerase to move with DNA

45 DNA polymerase III synthesizes The leading and lagging strands are synthesized in a coordinated fashion

46 DNA-poly III begins synthesis of the leading strand starting from RNA primer Helicase unwinds DNA ss-binding proteins bind to the unwound strands, keeping the strands separated so that both strands can serve as templates Lagging synthesis more complex – DNA-poly III makes Okazaki fragments – DNA-poly I removes …………………. – DNA ligase connects fragments – DNA synthesis in eukaryotes, more complex

47 DNA-poly III begins synthesis of leading strand using RNA primer Helicase unwinds DNA ss-binding proteins keep strands separated so both can be templates. Lagging strand synthesis more complex Lagging strand The leading and lagging strands are synthesized in a coordinated fashion

48 The mode of synthesis of the lagging strand is more complex Lagging strand is synthesized in fragments such that 5 3 polymerization leads to overall growth in the 3 5 direction Yet the synthesis of the lagging strand is coordinated with the synthesis of the leading strand The leading and lagging strands are synthesized in a coordinated fashion

49 How is this coordination accomplished? DNA polymerase III The holoenzyme includes two copies of the polymerase core enzyme The core enzymes are linked to a central structure having the subunit composition γτ 2 δδχφ The entire apparatus interacts with the hexameric helicase DnaB The leading and lagging strands are synthesized in a coordinated fashion

50 Okazaki fragments (RNA polymerase initiates) Looping the template for the lagging strand places it in position for 5--->3 polymerization DNA poly III lets go off the lagging strand after adding 1000 nucleotides New loop formed RNA primer made by primase Gaps filled by ……………….(it removes primers too)

51 In E. coli: a unique site origin of replication is called oriC locus Prokaryotes: Replication Origin of Replication

52 The DnaA proteins bind to the five high-affinity sites in oriC DnaA molecules form an oligomer a cyclic hexamer The DNA is wrapped around the outside of the DnaA hexamer The binding of DnaA molecules to one another signals the start of the preparatory phase Prokaryotes: Replication

53 DnaA oriC: Preparation for replication DnaB (hexameric helicase) + DnaC (helicase loader) SSB Prepriming complex DnaG (primase) inserts the RNA primer Single DNA strands are exposed in the prepriming complex makes single-stranded DNA accessible to other proteins Prokaryotes: Replication

54 DNA pol III holoenzyme + Prepriming complex ATP hydrolysis within DnaA Breakup of DnaA (prevents additional round of replication!) The polymerase holoenzyme assembles Prokaryotes: Replication

55 Eukaryote oriC is more complex E. Coli Replicates 4.6 million bp Genetic information contained in 1 chromosome Circular chromosome Human diploid cell Replicates 6 billion bp 23 pairs of chromosomes must be replicated Linear chromosome Eukaryotes: Replication

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57 Greatest replication problem with linear chromosomes Complete replication of DNA ends is difficult – polymerases act only in the 5 3 direction – the lagging strand would have an incomplete 5 end after the removal of the RNA primer – each round of replication would further shorten the chromosome

58 Telomers (from Greek: telos = end) Ends of chromosomes are different Hundreds of tandem repeats of six-nucleotide sequence One of the strands is G rich at the 3 end, and it is slightly longer than the other strand Proposed model Single-stranded region invades duplex to form large duplex loop

59 Telomeres are replicated by telomerase, a specialized polymerase that carries its own RNA template Telomerase contains an RNA molecule that serves as the template for the elongation of the G-rich strand carries the information necessary to generate the telomere sequences

60 DNA Damage DNA does become damaged in the course of replication through other processes Damage to DNA can be simple as the misincorporation of a single base complex chemical modification of bases chemical cross-links between the two strands of the double helix breaks in one or both of the phosphodiester backbones Results cell death or cell transformation changes in the DNA sequence that can be inherited by future generations blockage of the DNA replication process itself

61 Sources of Damage: Bases can be damaged by oxidizing agents, alkylating agents, and light Oxidation: Reactive oxygen species – hydroxyl radical reacts with guanine to form 8-oxoguanine – 8-Oxoguanine is mutagenetic Deamination: potentially deleterious process – adenine can be deaminated to form hypoxanthine – mutagenic hypoxanthine pairs with cytosine rather than thymine OxidationDeamination

62 Sources of Damage: Bases can be damaged by oxidizing agents, alkylating agents, and light Alkylation: – Electrophilic centers can be attacked by nucleophiles N-7 of guanine and adenine form alkylated adducts Aflatoxin B 1 – produced by molds that grow on peanuts and other foods – converted into a highly reactive epoxide by a cytochrome P450 enzyme – reacts with the N-7 atom of guanosine to form a mutagenic adduct that frequently leads to a G–C-to-T–A transversion

63 Sources of Damage: Bases can be damaged by oxidizing agents, alkylating agents, and light Ultraviolet light: ubiquitous DNA-damaging agent – covalently links adjacent pyrimidine residues along a DNA strand – pyrimidine dimer cannot fit into a double helix blocks replication and gene expression – A thymine dimer is an example of an intrastrand cross-link -Cross-links between bases on opposite strands also can be introduced by various agents

64 Sources of Damage: Bases can be damaged by oxidizing agents, alkylating agents, and light High-energy electromagnetic radiation: X-rays produces high concentrations of reactive species in solution induces several types of DNA damage single double-stranded breaks in DNA

65 DNA repair pathways: Mismatch repair: correction in place Nucleotide excision repair: a stretch of DNA is removed Base excision repair: damaged base is removed and replaced DNA damage can be detected and repaired by a variety of systems

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67 DNA polymerases – able to correct many DNA mismatches produced in the course of replication – the ε subunit of E. coli DNA polymerase III functions as a 3-to-5 exonuclease Error correction by the 35 exonuclease activity of DNA polymerase

68 DNA polymerases As a new strand of DNA is synthesized, it is proofread – incorrect base slows down DNA synthesis difficulty of threading a non-Watson–Crick base pair into the polymerase – mismatched base is weakly bound able to fluctuate in position – slowdown allows time for these fluctuations to take the newly synthesized strand out of the polymerase active site and into the exonuclease active site – the DNA is degraded, one nucleotide at a time, until it moves back into the polymerase active site and synthesis continues Error correction by the 35 exonuclease activity of DNA polymerase

69 Exonuclease activity ahead of the polymerase activity A mismatched base (here, a C–A mismatch) impedes translocation of DNA polymerase I to the next site. Sliding backward, the enzyme corrects the mistake with its 35 exonuclease activity, then resumes its polymerase activity in the 53 direction. Contributes to remarkable fidelity of DNA replication with an error rate of less than ………………………. Error correction by the 35 exonuclease activity of DNA polymerase

70 Mismatch-repair systems consist of at least two proteins MutS for detecting the mismatch MutL for recruiting an endonuclease (MutH) cleaves the newly synthesized DNA strand close to the lesion to facilitate repair Mismatch-repair systems

71 Direct Repair Example: photochemical cleavage of pyrimidine dimers Nearly all cells contain a photoreactivating enzyme called DNA photolyase – The enzyme binds to the distorted region of DNA – Uses light energy the absorption of a photon by the N 5,N 10 -methenyltetrahydrofolate coenzyme forms an excited state – cleaves the dimer into its component bases

72 DNA repair enzyme:AlkA Base-Excision Repair

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74 T-dimer is repaired by three enzymes: 1. Excinuclease detects the distortion and then cuts the damaged DNA strand at two sites 8 nucleotides away from the damaged site on the 5 side 4 nucleotides away on the 3 side. 2. DNA polymerase I For repair synthesis 3. DNA ligase Nucleotide Excision repair:

75 U formed by the deamination of C excised and replaced by C!! Uracil Repair

76 Why is T instead of U in DNA? T or U pairs with A – Only difference: a methyl group in T C in DNA spontaneously deaminates forming U (100 events per day!, deamination of A and G much slower) Potentially mutagenic because U-A occurs rather than C-G Prevented by DNA glycosidase – This enzyme cuts U but does not attack T the -CH3 in T is a tag that distinguishes T from deaminated C

77 Defective repair of DNA--->Cancer Xeroderma pigmentosum (AR) Extreme sensitivity to UV Skin is dry Keratosis Skin cancer Death before the age of 30! Defect: Excinuclease part of repair system

78 Mutagen Detection Many human cancers caused by chemicals! Chemicals usually cause mutations How do we identify them? Bruce Ames developed a simple, sensitive test called Ames for detecting chemical mutagens. – 0.5 microg of 2-aminoantharacene gives 11,000 colonies; only 30 colonies in its absence!

79 Ames test Thin layer of agar with salmonella bacteria cannot synthesize His Addition of chemical mutagen to center results in many mutations (one making bacteria synthesize His again) Revertants make many colonies What if P-450 is involved in mutagenesis?

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83 Double stranded DNA molecules with similar sequences sometimes recombine DNA replication copies genetic messages as faithfully as possible Several biochemical processes require recombination of genetic material between two DNA molecules Recombination plays important role in Making molecular diversity for Ab Manipulating genes Generation of gene knockout mice

84 RECOMBINATION: two DNA molecules can recombine to form new DNA molecules with segments from both parental molecules

85 DNA Replication

86 The genes of some viruses are made of RNA Genes in all pro and eukaryotes made of DNA In viruses, genes made of DNA or RNA RNA is like DNA but: Sugar is ribose U instead T – RNA can be single or double stranded Reverse Transcription

87 Genetic info of RNA virus is contained in its RNA Example: Tobacco mosaic virus which infects tobacco plants It consists of a single strand of RNA surrounded by a protein coat An RNA-directed RNA polymerase copies the viral RNA Infected cells die as the virus instructs the cell to commit suicide and results in discoloration of tobacco leaf Example: Retrovirus Known as retroviruses because information flows BACKWORD (RNA--- DNA )not from DNA--- RNA. Includes HIV-1 as well as a number of RNA viruses that produce tumors The genes of some viruses are made of RNA Reverse Transcription

88 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. Reverse Transcription

89 Viral infection of RNA virus Viral DNA gets incorporated into the chromosomal DNA of the host and is replicated along with the normal DNA Later the integrated viral genome is expressed to form viral RNA and viral proteins Reverse Transcription


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