Chapter 16. DNA The Genetic Material Replication 2005-2006

Scientific History The march to understanding that DNA is the genetic material T.H. Morgan (1908) Frederick Griffith (1928) Avery, McCarty & MacLeod (1944) Hershey & Chase (1952) Watson & Crick (1953) Meselson & Stahl (1958) 2005-2006

Genes are on chromosomes 1908 | 1933 T.H. Morgan working with Drosophila (fruit flies) genes are on chromosomes but is it the protein or the DNA of the chromosomes that are the genes? through 1940 proteins were thought to be genetic material… Why? 2005-2006

The “Transforming Factor” mix heat-killed pathogenic & non-pathogenic bacteria live pathogenic strain of bacteria live non-pathogenic strain of bacteria heat-killed pathogenic bacteria A. B. C. D. mice die mice live mice live mice die Transformation? something in heat-killed bacteria could still transmit disease-causing properties 2005-2006

The “Transforming Factor” 1928 Frederick Griffith Streptococcus pneumonia bacteria was working to find cure for pneumonia harmless live bacteria mixed with heat-killed infectious bacteria causes disease in mice substance passed from dead bacteria to live bacteria = “Transforming Factor” Fred Griffith, English microbiologist, dies in the Blitz in London in 1941 2005-2006

DNA is the “Transforming Factor” 1944 Avery, McCarty & MacLeod purified both DNA & proteins from Streptococcus pneumonia bacteria which will transform non-pathogenic bacteria? injected protein into bacteria no effect injected DNA into bacteria transformed harmless bacteria into virulent bacteria 2005-2006

Avery, McCarty & MacLeod Oswald Avery Colin MacLeod Maclyn McCarty 2005-2006

1952 | 1969 Confirmation of DNA Hershey & Chase classic “blender” experiment worked with bacteriophage viruses that infect bacteria grew phage viruses in 2 media, radioactively labeled with either 35S in their proteins 32P in their DNA infected bacteria with labeled phages 2005-2006

Hershey & Chase 2005-2006 Martha Chase Alfred Hershey

Hershey & Chase Which radioactive marker is found inside the cell? Protein coat labeled with 35S DNA labeled with 32P Hershey & Chase T2 bacteriophages are labeled with radioactive isotopes S vs. P bacteriophages infect bacterial cells bacterial cells are agitated to remove viral protein coats Which radioactive marker is found inside the cell? Which molecule carries viral genetic info? 35S radioactivity found in the medium 32P radioactivity found in the bacterial cells 2005-2006

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Blender experiment Radioactive phage & bacteria in blender 35S phage radioactive proteins stayed in supernatant therefore protein did NOT enter bacteria 32P phage radioactive DNA stayed in pellet therefore DNA did enter bacteria Confirmed DNA is “transforming factor” 2005-2006

1947 Chargaff DNA composition: “Chargaff’s rules” varies from species to species all 4 bases not in equal quantity bases present in characteristic ratio humans: A = 30.9% T = 29.4% G = 19.9% C = 19.8% 2005-2006

1953 | 1962 Structure of DNA Rosalind Franklin Maurice Wilkins Watson & Crick developed double helix model of DNA other scientists working on question: Rosalind Franklin Maurice Wilkins Linus Pauling Watson & Crick’s model was inspired by 3 recent discoveries: Chargaff’s rules Pauling’s alpha helical structure of a protein X-ray crystallography data from Franklin & Wilkins 2005-2006 Franklin Wilkins Pauling

1953 article in Nature Watson and Crick 2005-2006

Rosalind Franklin (1920-1958) 2005-2006 A chemist by training, Franklin had made original and essential contributions to the understanding of the structure of graphite and other carbon compounds even before her appointment to King's College. Unfortunately, her reputation did not precede her. James Watson's unflattering portrayal of Franklin in his account of the discovery of DNA's structure, entitled "The Double Helix," depicts Franklin as an underling of Maurice Wilkins, when in fact Wilkins and Franklin were peers in the Randall laboratory. And it was Franklin alone whom Randall had given the task of elucidating DNA's structure. The technique with which Rosalind Franklin set out to do this is called X-ray crystallography. With this technique, the locations of atoms in any crystal can be precisely mapped by looking at the image of the crystal under an X-ray beam. By the early 1950s, scientists were just learning how to use this technique to study biological molecules. Rosalind Franklin applied her chemist's expertise to the unwieldy DNA molecule. After complicated analysis, she discovered (and was the first to state) that the sugar-phosphate backbone of DNA lies on the outside of the molecule. She also elucidated the basic helical structure of the molecule. After Randall presented Franklin's data and her unpublished conclusions at a routine seminar, her work was provided - without Randall's knowledge - to her competitors at Cambridge University, Watson and Crick. The scientists used her data and that of other scientists to build their ultimately correct and detailed description of DNA's structure in 1953. Franklin was not bitter, but pleased, and set out to publish a corroborating report of the Watson-Crick model. Her career was eventually cut short by illness. It is a tremendous shame that Franklin did not receive due credit for her essential role in this discovery, either during her lifetime or after her untimely death at age 37 due to cancer. 2005-2006

Double helix structure of DNA the structure of DNA suggested a mechanism for how DNA is copied by the cell 2005-2006

Directionality of DNA nucleotide PO4 N base 5 CH2 O 4 1 ribose 3 You need to number the carbons! it matters! nucleotide PO4 N base 5 CH2 O 4 1 ribose 3 2 OH 2005-2006

The DNA backbone 5 PO4 base CH2 O C O –O P O O base CH2 O OH 3 Putting the DNA backbone together refer to the 3 and 5 ends of the DNA the last trailing carbon base CH2 O C O –O P O O base CH2 O OH 3 2005-2006

Anti-parallel strands Phosphate to sugar bond involves carbons in 3 & 5 positions DNA molecule has “direction” complementary strand runs in opposite direction “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick 2005-2006

Bonding in DNA 5’ 3’ 3’ 5’ hydrogen bonds phosphodiester bonds ….strong or weak bonds? How do the bonds fit the mechanism for copying DNA? 2005-2006

Base pairing in DNA Purines Pyrimidines Pairing adenine (A) guanine (G) Pyrimidines thymine (T) cytosine (C) Pairing A : T C : G 2005-2006

Copying DNA Replication of DNA base pairing allows each strand to serve as a pattern for a new strand 2005-2006

Models of DNA Replication Alternative models so how is DNA copied? 2005-2006

Semi-conservative replication 1958 Meselson & Stahl label nucleotides of “parent” DNA strands with heavy nitrogen = 15N label new nucleotides with lighter isotope = 14N “The Most Beautiful Experiment in Biology” parent replication 2005-2006

Semi-conservative replication 1958 Make predictions… 15N strands replicated in 14N medium 1st round of replication? 2nd round? 2005-2006

DNA Replication Large team of enzymes coordinates replication more than a dozen enzymes & other proteins participate in DNA replication 2005-2006

single-stranded binding proteins Replication: 1st step Unwind DNA helicase enzyme unwinds part of DNA helix stabilized by single-stranded binding proteins 2005-2006 single-stranded binding proteins

Replication: 2nd step Bring in new nucleotides to match up to template strands 2005-2006

Incorporation of a Nucleotide into a DNA Strand 2005-2006

Energy of Replication ATP GTP TTP CTP The nucleotides arrive as nucleosides (a sugar and a base) DNA bases arrive with their own energy source for bonding bonded by DNA polymerase III ATP GTP TTP CTP 2005-2006

Replication DNA P III leading strand Adding bases 5' 3' 3' 5' energy can only add nucleotides to 3 end of a growing DNA strand strand grows 5'3’ energy energy The energy rules the process. energy 3' 5' 2005-2006 leading strand

Leading & Lagging strands Leading strand - continuous synthesis Okazaki Lagging strand - Okazaki fragments - joined by ligase 2005-2006

Okazaki fragments 2005-2006

Priming DNA synthesis DNA polymerase III can only extend an existing DNA molecule cannot start new one cannot place first base short RNA primer is built first by primase starter sequences DNA polymerase III can now add nucleotides to RNA primer 2005-2006

Cleaning up primers DNA polymerase I removes sections of RNA primer and replaces with DNA nucleotides 2005-2006

direction of replication Replication fork DNA polymerase III lagging strand DNA polymerase I 3’ Okazaki fragments primase 5’ 5’ ligase SSB 3’ 5’ 3’ helicase DNA polymerase III 5’ leading strand 3’ direction of replication 2005-2006

And in the end… Ends of chromosomes are eroded with each replication an issue in aging? ends of chromosomes are protected by telomeres 2005-2006

Telomeres Expendable, non-coding sequences at ends of DNA short sequence of bases repeated 1000s times TTAGGG in humans Telomerase enzyme in certain cells enzyme extends telomeres prevalent in cancers Why? 2005-2006

Telomeres and Telomerase: Telomeres of Mouse Chromosomes 2005-2006

DNA polymerases DNA polymerase III DNA polymerase I 500 bases/second in bacteria; 50 bases/second in human cells main DNA building enzyme DNA polymerase I 20 bases/second editing, repair & primer removal DNA polymerase III enzyme 2005-2006

Editing & Proofreading DNA 1 in 100,000 wrong bases= lots of typos! DNA polymerase I proofreads & corrects typos repairs mismatched bases excises abnormal bases repairs damage throughout life reduces error rate to 1 in 1 billion bases (Solomon + Berg)

Replication enzymes helicase DNA polymerase III primase ligase single-strand binding proteins 2005-2006

Fast & accurate! It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome divide to form 2 identical daughter cells Human cell copies its 6 billion bases & divide into daughter cells in only few hours remarkably accurate only ~1 error per 1 billion bases ~30 errors per cell cycle 2005-2006

DNA RNA protein The “Central Dogma” transcription translation flow of genetic information within a cell transcription translation DNA RNA protein replication 2005-2006

Replication bubble  Which direction does DNA build? Adds 1000 bases/second!  Which direction does DNA build?  List the enzymes & their role 2005-2006

Structural Basis of DNA Replication: http://highered.mheducation.com/sites/9834092339/student_view0/chapter14/structural_basis_of_dna_replication.html http://sites.fas.harvard.edu/~biotext/animations/replication1.html DNA Replication – W.H Freeman http://bcs.whfreeman.com/webpub/biology/pierce5e/animations/12.3%20Coordination%20of%20Leading%20and%20Lagging-Strand%20Synthesis/1203_leading_lagging.html Nucleotide Excision Repair: http://highered.mheducation.com/sites/9834092339/student_view0/chapter14/nucleotide_excision_repair.html Mechanism of Replication from Dolan DNA Learning Center: https://www.dnalc.org/resources/3d/04-mechanism-of-replication-advanced.html Proofreading Function of DNA Polymerase: http://highered.mheducation.com/sites/9834092339/student_view0/chapter14/proofreading_function_of_dna_polymerase.html Telomerase Function: http://highered.mheducation.com/sites/9834092339/student_view0/chapter14/telomerase_function.html

What is the Role of the Promoter? 2005-2006

Explain why the replication process is a source of few mutations. 2005-2006

A-T-G-G-C-C-T-T-A, what is the complementary strand? If the strand on DNA is A-T-G-G-C-C-T-T-A, what is the complementary strand? 2) What enzymes would be at work and what are their roles to correct the following error(s)? T--G--G--T--T--C--T--A--G l l l l l l l l l A C C T T C A T C 2005-2006

3) DNA polymerase can add new nucleotides only to the… 2005-2006