Chapter 16 The Molecular Basis of Inheritance

Fig In 1953, James Watson and Francis Crick introduced an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA

Fig Living S cells (control) Pathogenic Living R cells (control) Heat-killed S cells (control) Mixture of heat-killed S cells and living R cells Mouse dies Mouse healthy Living S cells RESULTS EXPERIMENT Frederick Griffith in 1928 Transformation, now defined as a change in genotype and phenotype due to assimilation of foreign DNA

In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNA

Hershey and Chase 1952, studying T2 virus infecting Escherichia coli –Bacteriophage or phage Phage coat made entirely of protein DNA found inside capsid

Fig EXPERIMENT Phage DNA Bacterial cell Radioactive protein Radioactive DNA Batch 1: radioactive sulfur ( 35 S) Batch 2: radioactive phosphorus ( 32 P)

Fig EXPERIMENT Phage DNA Bacterial cell Radioactive protein Radioactive DNA Batch 1: radioactive sulfur ( 35 S) Batch 2: radioactive phosphorus ( 32 P) Empty protein shell Phage DNA

Fig EXPERIMENT Phage DNA Bacterial cell Radioactive protein Radioactive DNA Batch 1: radioactive sulfur ( 35 S) Batch 2: radioactive phosphorus ( 32 P) Empty protein shell Phage DNA Centrifuge Pellet Pellet (bacterial cells and contents) Radioactivity (phage protein) in liquid Radioactivity (phage DNA) in pellet

Chargaff ’ s rules It was known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group In 1950, Erwin Chargaff reported that DNA composition varies from one species to the next Chargaff ’ s rules state that in any species there is an equal number of A and T bases, and an equal number of G and C bases

Fig Sugar–phosphate backbone 5 end Nitrogenous bases Thymine (T) Adenine (A) Cytosine (C) Guanine (G) DNA nucleotide Sugar (deoxyribose) 3 end Phosphate

Building a Structural Model of DNA: Scientific Inquiry After most biologists became convinced that DNA was the genetic material, the challenge was to determine how its structure accounts for its role Maurice Wilkins and Rosalind Franklin were using a technique called X-ray crystallography to study molecular structure Franklin produced a picture of the DNA molecule using this technique

Fig (a) Rosalind Franklin (b) Franklin’s X-ray diffraction photograph of DNA

Fig (c) Space-filling model Hydrogen bond 3 end 5 end 3.4 nm 0.34 nm 3 end 5 end (b) Partial chemical structure(a) Key features of DNA structure 1 nm

DNA is –Double stranded –Helical –Sugar-phosphate backbone –Bases on the inside –Stabilized by hydrogen bonding –Base pairs with specific pairing

AT/GC or Chargoff ’ s rule –A pairs with T –G pairs with C Keeps with consistent 10 base pairs per turn 2 DNA strands are complementary –5 ’ – GCGGATTT – 3 ’ –3 ’ – CGCCTAAA – 5 ’ 2 strands are antiparallel –One strand 5 ’ to 3 ’ –Other stand 3 ’ to 5 ’ two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior

Space-filling model shows grooves –Major groove Where proteins bind –Minor groove Fig. 16-7b

Replication 3 different models for DNA replication proposed in late 1950s –Semiconservative –Conservative –Dispersive Newly made strands are daughter strands Original strands are parental strands

Fig Parent cell First replication Second replication (a) Conservative model (b) Semiconserva- tive model (c) Dispersive model

Fig EXPERIMENT RESULTS CONCLUSION Conservative model Semiconservative model Dispersive model Bacteria cultured in medium containing 15 N Bacteria transferred to medium containing 14 N DNA sample centrifuged after 20 min (after first application) DNA sample centrifuged after 40 min (after second replication) More dense Less dense Second replicationFirst replication

Fig a EXPERIMENT RESULTS Bacteria cultured in medium containing 15 N Bacteria transferred to medium containing 14 N DNA sample centrifuged after 20 min (after first application) DNA sample centrifuged after 20 min (after second replication) Less dense More dense a rare heavy form (15N) a common light form (14N)

Fig b CONCLUSION First replicationSecond replication Conservative model Semiconservative model Dispersive model

DNA Replication: A Closer Look The copying of DNA is remarkable in its speed and accuracy More than a dozen enzymes and other proteins participate in DNA replication Origin of replication –Site of start point for replication Bidirectional replication –Replication proceeds outward in opposite directions Bacteria have a single origin Eukaryotes require multiple origins

Fig Origin of replication Parental (template) strand Daughter (new) strand Replication fork Replication bubble Two daughter DNA molecules (a) Origins of replication in E. coli Origin of replicationDouble-stranded DNA molecule Parental (template) strand Daughter (new) strand Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes 0.5 µm 0.25 µm Double- stranded DNA molecule

Fig a Origin of replication Parental (template) strand Daughter (new) strand Replication fork Replication bubble Double- stranded DNA molecule Two daughter DNA molecules (a) Origins of replication in E. coli 0.5 µm

Fig b 0.25 µm Origin of replicationDouble-stranded DNA molecule Parental (template) strand Daughter (new) strand Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes

Fig Topoisomerase Helicase Primase Single-strand binding proteins RNA primer DNA helicase Binds to DNA and travels 5’ to 3’ using ATP to separate strand and move fork forward DNA topoisomerase:Relives additional coiling ahead of replication fork Single-strand binding proteins:Keep parental strands open to act as templates DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end Primerase:The initial nucleotide strand is a short RNA primer The primer is short (5– 10 nucleotides long), and the 3 end serves as the starting point for the new DNA strand

At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating Helicases are enzymes that untwist the double helix at the replication forks Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template Topoisomerase corrects “ overwinding ” ahead of replication forks by breaking, swiveling, and rejoining DNA strands

Fig A C T G G G GC CC C C A A A T T T New strand 5 end Template strand 3 end 5 end 3 end 5 end 3 end Base Sugar Phosphate Nucleoside triphosphate Pyrophosphate DNA polymerase

DNA polymerases Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork Most DNA polymerases require a primer and a DNA template strand The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells

Fig Leading strand Overview Origin of replication Lagging strand Leading strandLagging strand Primer Overall directions of replication Origin of replication RNA primer “Sliding clamp” DNA poll III Parental DNA

Fig a Overview Leading strand Lagging strand Origin of replication Primer Overall directions of replication

Fig b Origin of replication RNA primer “Sliding clamp” DNA pol III Parental DNA

Fig Overview Origin of replication Leading strand Lagging strand Overall directions of replication Template strand RNA primer Okazaki fragment Overall direction of replication

Fig a Overview Origin of replication Leading strand Lagging strand Overall directions of replication 1 2

Fig b1 Template strand

Fig b2 Template strand RNA primer

Fig b3 Template strand RNA primer Okazaki fragment

Fig b4 Template strand RNA primer Okazaki fragment

Fig b5 Template strand RNA primer Okazaki fragment

Fig b6 Template strand RNA primer Okazaki fragment Overall direction of replication

Fig Overview Origin of replication Leading strand Lagging strand Overall directions of replication Leading strand Lagging strand Helicase Parental DNA DNA pol III PrimerPrimase DNA ligase DNA pol III DNA pol I Single-strand binding protein

Table 16-1

Proofreading and Repairing DNA DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides In mismatch repair of DNA, repair enzymes correct errors in base pairing DNA can be damaged by chemicals, radioactive emissions, X-rays, UV light, and certain molecules (in cigarette smoke for example) In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA

Fig Nuclease DNA polymerase DNA ligase

Replicating the Ends of DNA Molecules Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes The usual replication machinery provides no way to complete the 5 ends, so repeated rounds of replication produce shorter DNA molecules

Fig Ends of parental DNA strands Leading strand Lagging strand Last fragment Previous fragment Parental strand RNA primer Removal of primers and replacement with DNA where a 3 end is available Second round of replication New leading strand New lagging strand Further rounds of replication Shorter and shorter daughter molecules

Telomeres and aging Body cells have a predetermined life span Skin sample grown in a dish will double a finite number of times –Infants, about 80 times –Older person, 10 to 20 times Senescent cells have lost the capacity to divide

Progressive shortening of telomeres correlated with cellular senescence Telomerase present in germ-line cells and in rapidly dividing somatic cells Telomerase function reduces with age Inserting a highly active telomerase gene into cells in the lab causes them to continue to divide

Telomeres and cancer When cells become cancerous they divide uncontrollably In 90% of all types of human cancers, telomerase is found at high levels Prevents telomere shortening and may play a role in continued growth of cancer cells Mechanism unknown

Fig µm

Fig a DNA double helix (2 nm in diameter) Nucleosome (10 nm in diameter) Histones Histone tail H1 DNA, the double helixHistones Nucleosomes, or “beads on a string” (10-nm fiber) Concept 16.3 A chromosome consists of a DNA molecule packed together with proteins

Fig b 30-nm fiber Chromatid (700 nm) LoopsScaffold 300-nm fiber Replicated chromosome (1,400 nm) 30-nm fiber Looped domains (300-nm fiber) Metaphase chromosome

Chromatin is organized into fibers 10-nm fiber –DNA winds around histones to form nucleosome “ beads ” –Nucleosomes are strung together like beads on a string by linker DNA 30-nm fiber –Interactions between nucleosomes cause the thin fiber to coil or fold into this thicker fiber Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

300-nm fiber –The 30-nm fiber forms looped domains that attach to proteins Metaphase chromosome –The looped domains coil further –The width of a chromatid is 700 nm Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Most chromatin is loosely packed in the nucleus during interphase and condenses prior to mitosis Loosely packed chromatin is called euchromatin During interphase a few regions of chromatin (centromeres and telomeres) are highly condensed into heterochromatin Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Histones can undergo chemical modifications that result in changes in chromatin organization –For example, phosphorylation of a specific amino acid on a histone tail affects chromosomal behavior during meiosis Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Fig RESULTS Condensin and DNA (yellow) Outline of nucleus Condensin (green) DNA (red at periphery) Normal cell nucleus Mutant cell nucleus

You should now be able to: 1.Describe the contributions of the following people: Griffith; Avery, McCary, and MacLeod; Hershey and Chase; Chargaff; Watson and Crick; Franklin; Meselson and Stahl 2.Describe the structure of DNA 3.Describe the process of DNA replication; include the following terms: antiparallel structure, DNA polymerase, leading strand, lagging strand, Okazaki fragments, DNA ligase, primer, primase, helicase, topoisomerase, single-strand binding proteins 4.Describe the function of telomeres 5.Compare a bacterial chromosome and a eukaryotic chromosome

Fig. 16-UN5