Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Evidence that DNA can transform bacteria Frederick Griffith (1928) – Streptococcus pneumoniae bacteria – transformation Mouse Experiment Experiment proved that transformation can happen Avery and colleagues (1944) – announced transformation agent was DNA

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure 16.2 Can the genetic trait of pathogenicity be transferred between bacteria? Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below: Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells. EXPERIMENT RESULTS CONCLUSION Living S (control) cells Living R (control) cells Heat-killed (control) S cells Mixture of heat-killed S cells and living R cells Mouse diesMouse healthy Mouse dies Living S cells are found in blood sample.

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Evidence that viral DNA can program cells Alfred Hershey and Martha Chase (1952) – bacteriophages or phages (viruses that infect bacteria) – discovered DNA is the genetic material NOT protein Blender experiment

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure 16.3 Viruses infecting a bacterial cell Phage head Tail Tail fiber DNA Bacterial cell 100 nm

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Additional evidence that DNA is the genetic material Erwin Chargaff (1947) – Chargaff’s rules – The equivalences for any given species between the number of A and T and G and C bases are equal. Analyzed DNA from different organisms – Humans 30.3% of bases were A’s – E. Coli 26% of bases were A’s

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Rosalind Franklin – (1950’s) – X-ray diffraction photo of DNA – helped Watson and Crick develop their model of DNA structure

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure 16.6 Rosalind Franklin and her X-ray diffraction photo of DNA (a) Rosalind Franklin Franklin’s X-ray diffraction Photograph of DNA (b)

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Structure of DNA Watson & Crick – (1953) – 1 page paper in the British journal Nature “Molecular Structure of Nucleic Acids: A Structure for Deoxynucleic acids”

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure 16.1 Watson and Crick with their DNA model

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure 16.5 The structure of a DNA strand

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure 16.7 The double helix

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure 16.8 Base pairing in DNA H N H O CH 3 N N O N N N NH Sugar Adenine (A) Thymine (T) N N N N Sugar O H N H N H N O H H N Guanine (G) Cytosine (C)

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Unnumbered Figure p. 298 Purine + Purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width Consistent with X-ray data

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings DNA Replication Section 16.2 Semi-conservative model – each of the two daughter molecules will have one old strand, derived from the parent molecules, and one newly made strand

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure 16.9 A model for DNA replication: the basic concept (layer 1) (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. A C T A G T G A T C

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure 16.9 A model for DNA replication: the basic concept (layer 2) (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (b) The first step in replication is separation of the two DNA strands. A C T A G A C T A G T G A T C T G A T C

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure 16.9 A model for DNA replication: the basic concept (layer 3) (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (b) The first step in replication is separation of the two DNA strands. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. A C T A G A C T A G A C T A G T G A T C T G A T C A C T A G T G A T C T G A T C

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure 16.9 A model for DNA replication: the basic concept (layer 4) (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (b) The first step in replication is separation of the two DNA strands. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. (d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand. A C T A G A C T A G A C T A G A C T A G T G A T C T G A T C A C T A G A C T A G T G A T C T G A T C T G A T C T G A T C

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure Three alternative models of DNA replication Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. (a) Semiconserva- tive model. The two strands of the parental molecule separate, and each functions as a template for synthesis of a new, comple- mentary strand. (b) Dispersive model. Each strand of both daughter mol- ecules contains a mixture of old and newly synthesized DNA. (c) Parent cell First replication Second replication

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure Incorporation of a nucleotide into a DNA strand New strandTemplate strand 5’ end 3’ end Sugar A T Base C G G C A C T P P P OH P P 5’ end 3’ end 5’ end A T C G G C A C T 3’ end Nucleoside triphosphate Pyrophosphate 2 P OH Phosphate

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure Origins of replication in eukaryotes Replication begins at specific sites where the two parental strands separate and form replication bubbles. The bubbles expand laterally, as DNA replication proceeds in both directions. Eventually, the replication bubbles fuse, and synthesis of the daughter strands is complete Origin of replication Bubble Parental (template) strand Daughter (new) strand Replication fork Two daughter DNA molecules In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome. In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). (b) (a) 0.25 µm

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Parental DNA DNA pol Ill elongates DNA strands only in the 5 3 direction Okazaki fragments DNA pol III Template strand Leading strand Lagging strand Template strand DNA ligase Overall direction of replication One new strand, the leading strand, can elongate continuously 5 3 as the replication fork progresses. The other new strand, the lagging strand must grow in an overall 3 5 direction by addition of short segments, Okazaki fragments, that grow 5 3 (numbered here in the order they were made). DNA ligase joins Okazaki fragments by forming a bond between their free ends. This results in a continuous strand Figure Synthesis of leading and lagging strands during DNA replication

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure Synthesis of the lagging strand Overall direction of replication DNA ligase forms a bond between the newest DNA and the adjacent DNA of fragment 1. 6 The lagging strand in this region is now complete. 7 DNA pol 1 replaces the RNA with DNA, adding to the 3 end of fragment 2. 5 After the second fragment is primed. DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 4 After reaching the next RNA primer (not shown), DNA pol III falls off. 3 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. 2 Primase joins RNA nucleotides into a primer. 1 Template strand RNA primer Okazaki fragment

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Table 16.1 Bacterial DNA replication proteins and their functions

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Overall direction of replication Helicase unwinds the parental double helix. Molecules of single- strand binding protein stabilize the unwound template strands. The leading strand is synthesized continuously in the 5  3 direction by DNA pol III. Leading strand Origin of replication Lagging strand Lagging strand Leading strand OVERVIEW Leading strand Replication fork DNA pol III Primase Primer DNA pol III Lagging strand DNA pol I DNA ligase Primase begins synthesis of RNA primer for fifth Okazaki fragment. 4 DNA pol III is completing synthesis of the fourth fragment, when it reaches the RNA primer on the third fragment, it will dissociate, move to the replication fork, and add DNA nucleotides to the 3 end of the fifth fragment primer. 5 DNA pol I removes the primer from the 5 end of the second fragment, replacing it with DNA nucleotides that it adds one by one to the 3’ end of the third fragment. The replacement of the last RNA nucleotide with DNA leaves the sugar- phosphate backbone with a free 3 end. 6 DNA ligase bonds the 3 end of the second fragment to the 5 end of the first fragment. 7 Parental DNA Figure A summary of bacterial DNA replication

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Proofreading and repairing DNA Errors do occur – 1 in 10 billion nucleotides on entire DNA – 1 in 100,000 for incoming nucleotides Proofreading is done by DNA pol III as it attaches new nucleotides Mismatch repair cells use special enzymes to fix mismatched nucleotides – A-C for example

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure Nucleotide excision repair of DNA damage Nuclease DNA polymerase DNA ligase A thymine dimer distorts the DNA molecule. 1 Repair synthesis by a DNA polymerase fills in the missing nucleotides. 3 DNA ligase seals the Free end of the new DNA To the old DNA, making the strand complete. 4 A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. 2

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Telomeres Repeated units of bases – TTAGGG in humans Do NOT contain genes They protect the genes from being eroded (getting shorter and shorter) through DNA replication rounds

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure Shortening of the ends of linear DNA molecules End of parental DNA strands Leading strand Lagging strand Last fragmentPrevious fragment RNA primer Lagging strand Removal of primers and replacement with DNA where a 3 end is available Primer removed but cannot be replaced with DNA because no 3 end available for DNA polymerase Second round of replication New leading strand New lagging strand 5 Further rounds of replication Shorter and shorter daughter molecules

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure Telomeres 1 µm