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Genetics 2011 O tli off Ch Outline Chapter t 6 • Ho How investigators in estigators pinpointed DNA as the genetic material • The elegant Watson Watson-Crick Crick model of DNA structure • How DNA structure provides for the storage of genetic information • How DNA structure gives rise to the semiconservative model of molecular replication • How DNA structure promotes the recombination of genetic information Lecture 6 DNA structure, structure replication replication, and recombination ᯘ⩶ె ⪁ᖌ http://lms.ls.ntou.edu.tw/course/136 1 2 Lectured by Han-Jia Lin Genetics 2011 The chemical composition of DNA Chemical characterization localizes DNA in the chromosomes. • 1869 – Friedrich Meischer extracted a weakly y acidic,, p phosphorous p rich material from nuclei of human white blood cells which he named nuclein. nuclein • DNA – deoxyribonucleic acid DNA contains four kinds of nucleotides linked in a long chain • Deoxyribose – a sugar; acidic • Four subunits belonging g g to class of compounds called nucleotides linked together by y phosphodiester p p bonds •Lectured Chromosomes are composed of DNA. by Han-Jia Lin Genetics 2011 3 Phosphodiester bonds – covalent bonds joining adjacent nucleotides Polymer – linked chain of subunits b it Fig. 6.2Lectured by Han-Jia Lin 4 Genetics 2011 Genetics 2011 Are genes composed of DNA or protein? Bacterial transformation implicates DNA as the substance of genes. • DNA • 1928 – Frederick Griffith – experiments with ith smooth th (S), (S) virulent i l t strain t i Streptococcus pneumoniae, and rough (R) nonvirulent (R), i l t strain t i • Only four different subunits make up DNA DNA. • Chromosomes contain less DNA than protein t i b by weight. i ht • Bacterial transformation demonstrates transfer of genetic material. • Protein • 20 different subunits – greater potential variety of combinations • Chromosomes contain more protein than DNA by weight. weight • 1944 – Oswald Avery, y Colin MacLeod, and Maclyn McCarty determined that DNA is the transformation material. 5 Lectured by Han-Jia Lin 6 Lectured by Han-Jia Lin Genetics 2011 Genetics 2011 G iffith experiment Griffith i t G iffith experiment Griffith i t Rough colony Smooth colony Fi 6 Fig. 6.3 3 7 Fig. 6.3a Lectured by Han-Jia Lin Fig. 6.3 b Lectured by Han-Jia Lin 8 Genetics 2011 A Avery, M MacLeod, L d M McCarty C t E Experiment i t Genetics 2011 H Hershey h and d Chase Ch experiments i t • 1952 – Alfred Hershey and Martha Chase provide convincing evidence that genetic material. DNA is g • Waring blender experiment using T2 bacteriophage and bacteria • Radioactive labels 32P for DNA and 35S for protein Fig. 6.4 a 9 Lectured by Han-Jia Lin 10 Lectured by Han-Jia Lin Genetics 2011 Hershey and Chase Waring blender experiment Genetics 2011 Hershey and Chase Waring blender experiment Fi 6.5 Fig. 6 5 a,b b 11 Lectured by Han-Jia Lin Fig. 6.5 c Lectured by Han-Jia Lin 12 Genetics 2011 The Watson Watson-Crick Crick Model: DNA is a double helix. • 1951 – James Watson learns about x-ray diff ti pattern diffraction tt projected j t db by DNA • Knowledge of the chemical structure of nucleotides (deoxyribose sugar, phosphate, and nitrogenous base) • Erwin Chargaff’s experiments demonstrate that ratios of A and T are 1:1,, and G and C are 1:1. • 1953 – James Watson and Francis Crick propose their double helix model of DNA Lectured by Han-Jia Lin structure Genetics 2011 X-ray X ray diffraction patterns produced by DNA fibers – Rosalind Franklin and Maurice Wilkins 13 14 Fig. 6.6Lectured by Han-Jia Lin Genetics 2011 DNA’ chemical DNA’s h i l constituents tit t Deoxyribose Phosphate DNA’ chemical DNA’s h i l constituents tit t Four nitrogenous bases Purines Genetics 2011 Attachment off base to sugar Pyrimidine Nucleoside P i Purine nucleotide Pyrimidine P i idi nucleotide Fig. 6.9b Fig. 6.9a 15 Fig. Lectured 6.7a by Han-Jia Lin Addition of phosphate to nucleoside 16 Fig. Lectured 6.7b by Han-Jia Lin Genetics 2011 A detailed look at DNA DNA䇻䇻s chemical constituents Genetics 2011 Ch Chargaff’s ff’ ratios ti • In all organisms, g , ratios of A to T and G to C are roughly 1:1 Nucleotides linked in a directional chain Phosphodiester bonds always form covalent link between 3' carbon of one nucleoside and 5' 5 carbon of the next nucleoside Note the 5 5'-to-3 to 3' polarity 17 Fig. 6.7c Lectured by Han-Jia Lin 18 Lectured by Han-Jia Lin Genetics 2011 Genetics 2011 Complementary base pairing by f formation ti off h hydrogen d b bonds d explain l i Chargaff’s g ratios. The double helix structure of DNA • DNA is double helix • Strands are antiparallel with a sugar-phosphate backbone on outside and pairs of bases in the middle. • Two T o strands wrap rap around aro nd each other every 30 Angstroms, g , once every y 10 base pairs. • Two chains are held t together th by b h hydrogen d bonds between A-T and G-C base p pairs. • Base pairs consist of h d hydrogen b bonds d ((weak k electrostatic bonds) between a purine and a pyrimidine (G with C, A with T) • Consistent with Chargaff's rules l • Each base p pair has ~ same shape Fig. 6.8 Lectured by Han-Jia Lin 19 20 Fig. Lectured 6.9 by Han-Jia Lin Genetics 2011 Genetics 2011 Double helix may assume alternative forms. • Structurally, purines (A and G) pair best with pyrimadines (T and C). Thus A pairs with T and G pairs • Thus, with C, also explaining Chargaff’s ratios. ratios B form DNA forms rightB-form right handed helix and has a smooth backbone Fig. 6.12 21 Lectured by Han-Jia Lin Prokaryotes Mitochondria Chloroplasts Vi Viruses • Some viruses carry y single-stranded g DNA. • 1. 1 bacteriophages • Some viruses carry RNA. • 1. e.g., AIDS Lectured by Han-Jia Lin Genetics 2011 Four q questions about how DNA structure relates to genetic functions • S Some DNA molecules l l are circular i l instead of linear. 1. 2 2. 3. 4 4. 22 Lectured by Han-Jia Lin Genetics 2011 • • • • Z form DNA forms leftZ-form left handed helix and has an irregular backbone 23 How does the molecule carry information? • Base sequence How is that information is copied for transmission to future generations? • DNA replication li ti What mechanisms allow the genetic information to change? • Recombination • Mutations M t ti ((chapter h t 7) How does DNA-encoded information govern the expression of phenotype? • Gene functions (chapter 8) Lectured by Han-Jia Lin 24 Genetics 2011 Genetics 2011 DNA stores information in the sequence of its bases. Some viruses use RNA as the repository of genetic information. (a) Most genetic information is "read" from unwound DNA e.g. synthesis of DNA or RNA (b) Some S genetic ti iinformation f ti is accessible within doublestranded st a ded DNA e.g. DNA-binding proteins that regulate gene expression Fig. 6.14 25 Lectured by Han-Jia Lin Fig. 6.13 26 Lectured by Han-Jia Lin Genetics 2011 Genetics 2011 DNA replication: Copying genetic information for transmission to the next g generation • Complementary base pairing produces semiconservative replication. • Double helix unwinds • Each strand acts as template • Complementary base pairing ensures that T signals addition of A on new strand, and G signals addition of C C. • Two daughter helices produced after replication li i 27 Lectured by Han-Jia Lin Fig. 6.14 Lectured by Han-Jia Lin 28 Genetics 2011 Experimental proof of semiconservative replication – three possible models Genetics 2011 Meselson Stahl experiments confirm Meselson-Stahl semiconservative replication. • Semiconservative replication – Watson and Crick model • Conservative replication: parental double helix The p remains intact; both strands of the daughter double helix are newly synthesized synthesized. • Dispersive replication: At completion, p , both strands of both double helices contain both original and newly synthesized material material. 29 Lectured by Han-Jia Lin 30 Lectured by Han-Jia Lin Genetics 2011 Genetics 2011 DNA synthesis proceeds in a 5' to 3' direction 3 Th mechanism The h i off DNA replication li ti • Arthur Kornbuerg, a nobel prize winner and other biochemists deduced steps of replication. Template and newly synthesized strands are antiparallel • Initiation I iti ti • Proteins bind to DNA and open up double helix. • Prepare DNA for complementary base pairing • Elongation • Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA DNA. DNA polymerase adds nucleotides to 3'-OH of the new strand 32 31 Lectured by Han-Jia Lin Lectured by Han-Jia Lin Fig. 6.19 Genetics 2011 The mechanism of DNA replication: Initiation Genetics 2011 The mechanism of DNA replication: Initiation (cont) • Preparation of double helix for complementary base pairing • Single-strand binding proteins keep the DNA helix open • Primase synthesizes RNA primer • Primers are complementary and antiparallel to each t template l t strand t d Initiation begins at the origin (Ori) of replication Initiator protein binds to Ori Helicase unwinds the helix Two replication forks are formed Feature Fig. g 6.20a 33 Lectured by Han-Jia Lin 34 Lectured by Han-Jia Lin Genetics 2011 The mechanism of DNA replication: Elongation The correct nucleotide sequence is copied from template strand to newly synthesized strand of DNA DNA polymerase III catalyzes phosphodiester bond formation between adjacent nucleotides (polymerization) Feature Fig. 6.20a (cont) Genetics 2011 The mechanism of DNA replication: Elongation (cont) • Leading strand has continuous synthesis • Lagging strand has discontinuous synthesis • Okazaki fragment – short DNA fragments on lagging strand Feature Fig. 6.20b (cont) 35 Lectured by Han-Jia Lin Feature Fig. 6.20b 36 Lectured by Han-Jia Lin Genetics 2011 The mechanism of DNA replication: Elongation (cont) • DNA polymerase I replaces RNA primer with DNA sequence • DNA ligase g covalently y jjoins successive Okazaki fragments together Genetics 2011 E Enzymes involved i l d in i replication li ti • Pol III – produces new stands of complementary DNA • Pol I – fills in gaps between newly synthesized Okazaki segments • DNA helicase – unwinds double helix • Single-stranded binding proteins – keep helix open • Primase – creates RNA primers to initiate synthesis • Ligase Li – welds ld ttogether th Ok Okazaki ki ffragments t 37 Lectured by Han-Jia Lin Feature Fig. 6.20b (cont) 38 Lectured by Han-Jia Lin Genetics 2011 The bidirectional replication of a circular bacterial chromosome: An overview The bidirectional replication of a circular bacterial chromosome: An overview (cont) DNA topoisomerases relax supercoils by cutting the sugar phosphate backbone bonds strands of DNA • Replication proceeds in two directions from a single i l O Orii • Unwinding of DNA creates supercoiled DNA ahead of replication fork Unwound U db broken k strands t d then sealed by ligase Synthesis continues bidirectionally until replication li i fforks k meet Fig. 6.21a, b 39 Lectured by Han-Jia Lin Genetics 2011 40 Lectured by Han-Jia Lin Fig. 6.21c-f Genetics 2011 Cells must ensure accuracy of genetic information. Genetics 2011 Recombination reshuffles the information content of DNA. • During recombination, DNA molecules b k and break d rejoin. j i g - Experimental p • Meselson and Weigle evidence from viral DNA and radioactive p isotopes • Coinfected E. coli with light and heavy strains of virus after allowing time for recombination • Separated on a CsCl density gradient Three ways to ensure fidelity of DNA information Redundancy •Redundancy • Basis for repair of errors that occur during replication or during storage •Enzymes repair chemical damage to DNA. Errors during replication are rare rare. •Errors 41 Lectured by Han-Jia Lin 42 Lectured by Han-Jia Lin Genetics 2011 Heteroduplexes mark the spot of recombination. DNA molecules break and rejoin during recombination: The experimental evidence • M. Meselson and J. Weigle, co-infected E. coli with radio-labeled phage • Bacteriophage lambda with genetic markers grown on E. coli in media with heavy (13C and 15N) or light (12C and d 14N) isotopes i Separated phage DNA on CsCl density gradient Genetic recombinants had DNA with hybrid densities 43 Lectured by Han-Jia Lin Genetics 2011 • Products of recombination are always in exact register; not a single base pair is lost or gained. • Two strands do not break and rejoin at the same location; often they are hundreds of base pairs apart. apart • Region between break points is called heteroduplex. heteroduplex • One strand is maternal and other ot e iss paternal pate a • Strands can have mismatches Lectured by Han-Jia Lin 44 Mismatches in h t heteroduplexes d l can b be repaired Genetics 2011 Genetics 2011 Experimental observations that led to development of a model of recombination • DNA repair enzymes eliminate mismatches • Tetrad analysis in yeast showed that only two of the four chromatids are recombinant • Either allele can be converted • Recombination occurs only between homologous regions and is highly accurate • Gene conversion – deviations from expected 2:2 segregation e segregation, e.g. g 3:1 or 1:3 • Crossover sites often associated with heteroduplex regions • In yeast, gene conversion occurs 50:50 50 50 with ith and d without ith t crossing over of flanking markers Lectured by Han-Jia Lin Fig. 6.23c 45 • Gene conversion can occur in absence of crossing over • Not all recombination leads to crossovers Genetics 2011 Double stranded break model of meiotic recombination • Homologs physically break break, exchange parts parts, and rejoin. • Breakage g and repair p create reciprocal p p products of recombination. • Recombination events can occur anywhere along l the th DNA molecule. l l • Precision in the exchange prevents mutations from occurring during the process process. • Gene conversion can give rise to unequal yield of two different alleles alleles. 50% of gene conversions are associated with crossing over of adjacent chromosomal regions, and 50% of gene conversions i are nott associated i t d with ith 47 crossing over. Lectured by Han-Jia Lin 46 Lectured by Han-Jia Lin Genetics 2011 Double-strand Doublestrand--break repair model of meiotic recombination • Homologous chromosomes break, exchange DNA, and rejoin j • Breakage and repair creates reciprocal products of recombination • Recombination events can occur anywhere along the DNA • Precision in the exchange g ((no g gain or loss of nucleotide pairs) prevents mutations from occurring • Gene conversion can give rise to an unequal yield of 48 two different alleles Lectured by Han-Jia Lin Genetics 2011 Step 1 in the model of recombination: Double--strand break formation Double Genetics 2011 Step 2 in the model of recombination: Resection Dmc1 D 1 breaks b k phosphodiester h h di t b bonds d off b both th strands t d off one chromatid Spo11 in yeast is homologous to Dmc1 of multicellular eukaryotes • 5' ends of each broken strand are degraded to create 3䇻 3 single single-stranded stranded tails Feature Fig. 6.24 Feature Fig. 6.24 (cont) 49 Lectured by Han-Jia Lin 50 Lectured by Han-Jia Lin Genetics 2011 Step 3 in the model of recombination: First strand invasion Genetics 2011 Step 4 in the model of recombination: Formation of double Holliday junctions • One single-strand tail invades a nonsister chromatid and forms stable heteroduplex • Displacement Di l t lloop (D (D-loop) l ) ffrom invaded chromatid is stabilized by single-strand binding protein • D-loop enlarged by new DNA synthesis at 3'end of invading g strand • New DNA synthesis fills in gap in bottom strand using displaced strand as template Feature Fig. 6.24 (cont) 51 Lectured by Han-Jia Lin 52 Lectured by Han-Jia Lin Feature Fig. 6.24 (cont) Genetics 2011 Step 5 in the model of recombination: Branch migration Genetics 2011 Step 6 in the model of recombination: The Holliday intermediate • Heteroduplex region of both DNA molecules is lengthened Feature Fig. 6.24 (cont) 53 Lectured by Han-Jia Lin 54 Lectured by Han-Jia Lin Feature Fig. 6.24 (cont) Genetics 2011 Step 7 in the model of recombination: Alternative resolutions Genetics 2011 Step 7 in the model of recombination: Alternative resolutions • Cutting of Holliday junctions by endonucleases in either vertical or horizontal plane is equally likely • Cutting of Holliday junctions by endonucleases is q y likelyy in either vertical or horizontal p plane equally Feature Fig. 6.24 (cont) Feature Fig. 6.24 (cont) 55 Lectured by Han-Jia Lin 56 Lectured by Han-Jia Lin Genetics 2011 Genetics 2011 Step 8 in the model of recombination: Probability of crossover occurring E Essential ti l Concepts C t • N Non-crossover occurs when h b both th jjunctions ti are resolved in same plane • Crossover occurs with the two junctions are resolved in different planes Feature Fig. 6.24 (cont) 57 Lectured by Han-Jia Lin • DNA is the nearly universal genetic material. • The Watson-Crick model shows that DNA is a double helix composed of two antiparallel strands of nucleotides: each nucleotide consists of one of four nitrogenous bases ((A,T,C, , , , or G), ), a deoxyribose y sugar, g , and a phosphate. An A pairs with a T and a G pairs with a C. • DNA carries information in the sequence of its bases which may follow one another in any bases, 58 order. Lectured by Han-Jia Lin