DNA AND ITS ROLE IN HEREDITY

DNA is the genetic material: a short history - DNA was found in the nucleus by Miescher (1868) Early in the 20th century, the search for genetic material led to DNA – T. H. Morgan’s group (1908): genes are on chromosomes – Frederick Griffith (1928): experiments on S. pneumoniae – Oswald Avery (1944): confirmed Griffith’s experiments – Hershey and Chase (1952): DNA is the genetic material – Erwin Chargaff (1947): The amount of thymine = adenine – Watson and Crick (1953): Structure of DNA – Rosalind Franklin (1951): X-Ray Structure of DNA – Meselson and Stahl (1958): DNA Replication

DNA was found in chromosomes using dyes that bind specifically to DNA.

Oswald AveryMaclyn McCartyColin MacLeod Avery, McCarty & MacLeod Conclusion – first experimental evidence that DNA was the genetic material injected protein into bacteria - no effect injected DNA into bacteria - transformed harmless bacteria into virulent bacteria

Evidence That Viral DNA Can Program Cells Bacteriophages (or phages), are viruses that infect bacteria T2 Phage

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

DNA Structure Reflects Its Role as the Genetic Material After identifying DNA as the genetic material, scientists hoped to answer two questions about the structure: 1.How is DNA replicated between cell divisions? 2.How does it direct the synthesis of specific proteins?

Structure of DNA How does the structure of DNA account for its role in genetic inheritance? Maurice Wilkins and Rosalind Franklin – used X-ray crystallography to study molecular structure

Fig Sugar–phosphate backbone 5 end Nitrogenous bases Thymine (T) Adenine (A) Cytosine (C) Guanine (G) DNA nucleotide Sugar (deoxyribose) 3 end Phosphate DNA STRUCTURE Erwin Chargaff reported (1947) 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

Hydrogen bond 3 end 5 end 3.4 nm 0.34 nm 3 end 5 end 1 nm Watson and Crick

Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data Watson and Crick reasoned that the pairing was specific, dictated by the base structures

DNA in the Nucleus and in the Cell Cycle

DNA Is a Double Helix

Base Pairs in DNA Can Interact with Other Molecules Cytosine (C) Adenine (A)Thymine (T) Guanine (G)

But how is DNA copied? Replication of DNA – base pairing suggests that it will allow each side to serve as a template for a new strand “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

Models of DNA Replication Alternative models – become experimental predictions conservativesemiconservativedispersive 1 2 P

DNA Replication Semiconservative replication Each half of the double helix acquires a new mate Each new DNA molecule, then, is really half old and half new

DNA has directionality Putting the DNA backbone together – refer to the 3 and 5 ends of the DNA the last trailing carbon OH O 3 PO 4 base CH 2 O base O P O C O –O–O CH

Each New DNA Strand Grows by the Addition of Nucleotides to Its 3′ End

DNA Replication Large team of enzymes coordinates replication

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

Fig Topoisomerase Helicase Primase Single-strand binding proteins RNA primer Replication: 1st step

Fig Topoisomerase Helicase Primase Single-strand binding proteins RNA primer Replication: 2nd step Primase starts an RNA chain from scratch - adds RNA nucleotides one at a time using the parental DNA as a template - 3 end serves as the starting point for the new DNA strand

DNA Polymerase III Replication: 2nd step  Build daughter DNA strand  add new complementary bases to the 3’ end of the RNA primer  DNA polymerase III

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 DNA polymerase What is driving polymerization?

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

Fig The lagging strand is copied away from the fork in short segments, each requiring a new primer. To summarize, at the replication fork, the leading stand is copied continuously into the fork from a single primer.

Template strand The Lagging Strand: A Closer Look

Template strand RNA primer DNA Pol III works in the direction away from the replication fork

Template strand RNA primer Okazaki fragment Okazaki

Template strand RNA primer Okazaki fragment Okazaki

Template strand RNA primer Okazaki fragment Okazaki

Template strand RNA primer Okazaki fragment Overall direction of replication Okazaki

Loss of bases at 5 ends in every replication  chromosomes get shorter with each replication  limit to number of cell divisions? DNA polymerase III All DNA polymerases can only add to 3 end of an existing DNA strand Chromosome erosion growing replication fork DNA polymerase I RNA

Repeating, non-coding sequences at the end of chromosomes = protective cap  limit to ~50 cell divisions Telomerase  enzyme extends telomeres  can add DNA bases at 5 end  different level of activity in different cells  high in stem cells & cancers -- Why? telomerase Telomeres growing replication fork TTAAGGG

Fig µm

Correcting Mistakes more than 130 DNA repair enzymes have been identified in humans An enzyme detects something wrong in one strand of the DNA and removes it Then DNA polymerase copies the information in the intact second strand and creates a new stretch of DNA DNA ligase seals the gap Nobel Prize in Chemistry 2015 Interview

Sunburn Damages DNA

Nucleotide excision repair Nuclease – a DNA cutting enzyme

In mismatch repair, repair enzymes fix incorrectly paired nucleotides. – A hereditary defect in one of these enzymes is associated with a form of colon cancer.

Ghosts of Lectures Past

Frederick Griffith The “Transforming Principle” live pathogenic strain of bacteria live non-pathogenic strain of bacteria mice diemice live heat-killed pathogenic bacteria mix heat-killed pathogenic & non-pathogenic bacteria mice livemice die A.B. C. D.

Semiconservative replication Meselson & Stahl – label “parent” nucleotides in DNA strands with heavy nitrogen = 15 N – label new nucleotides with lighter isotope = 14 N “The Most Beautiful Experiment in Biology” 1958 parentreplication 15 N parent strands 15 N/ 15 N

conservativesemiconservativedispersive 1 2 P 1 P 2

DNA: Count the Carbons! 3’3’ 5’5’ 3’3’ 5’5’ 3’3’

Limits of DNA polymerase III  can only build onto 3 end of an existing DNA strand Leading & Lagging strands Leading strand Lagging strand Okazaki fragments ligase Okazaki Leading strand  continuous synthesis Lagging strand  Okazaki fragments  joined by ligase  “spot welder” enzyme DNA polymerase III  3 5 growing replication fork

DNA polymerase I  removes sections of RNA primer and replaces with DNA nucleotides But DNA polymerase I still can only build onto 3 end of an existing DNA strand Replacing RNA primers with DNA growing replication fork DNA polymerase I RNA ligase