Lecture 9 DNA Structure & Replication

What is a Gene?  Mendel’s work left a key question unanswered:  What is a gene?  The work of Sutton and Morgan established that genes reside on chromosomes  But chromosomes contain proteins and DNA  So which one is the hereditary material  Several experiments ultimately revealed the nature of the genetic material

The Griffith Experiment  In 1928, Frederick Griffith discovered transformation while working on Streptococcus pneumoniae  The bacterium exists in two strains  S  Forms smooth colonies in a culture dish  Cells produce a polysaccharide coat and can cause disease  R  Forms rough colonies in a culture dish  Cells do not produce a polysaccharide coat and are therefore harmless

Thus, the dead S bacteria somehow “transformed” the live R bacteria into live S bacteria How Griffith Discovered Transformation

The Avery and Hershey-Chase Experiments  Two key experiments that demonstrated conclusively that DNA, and not protein, is the hereditary material  Oswald Avery and his coworkers Colin MacLeod and Maclyn McCarty published their results in 1944  Alfred Hershey and Martha Chase published their results in 1952

 Avery and his colleagues prepared the same mixture of dead S and live R bacteria as Griffith did  They then subjected it to various experiments  All of the experiments revealed that the properties of the transforming principle resembled those of DNA 1.Same chemistry and physical properties as DNA 2.Not affected by lipid and protein extraction 3.Not destroyed by protein- or RNA-digesting enzymes 4.Destroyed by DNA-digesting enzymes The Avery Experiments

 Viruses that infect bacteria have a simple structure  DNA core surrounded by a protein coat The Hershey-Chase Experiment  Hershey and Chase used two different radioactive isotopes to label the protein and DNA Thus, viral DNA directs the production of new viruses  Incubation of the labeled viruses with host bacteria revealed that only the DNA entered the cell  Therefore, DNA is the genetic material

Nitrogenous base Discovering the Structure of DNA 5-C sugar  Chemically, DNA is made up of nucleotides  Each nucleotide has a central sugar, a phosphate group and an organic base  The bases are of two main types:  Purines Large bases:  Adenine (A)  Guanine (G)  Pyrimidines Small bases:  Cytosine (C)  Thymine (T)

More Key Observations About DNA  Erwin Chargaff made key DNA observations that became known as Chargaff’s rule Rosalind Franklin’s X-ray diffraction experiments revealed that DNA had the shape of a coiled spring or helix Purines = Pyrimidines A = T C = G Rosalind Franklin ( )

The Structure of DNA Revealed  In 1953, James Watson and Francis Crick deduced that DNA was a double helix James Watson (1928- ) Francis Crick ( ) They came to their conclusion using Tinker toy models and the research of Chargaff and Franklin

Dimensions suggested by X-ray diffraction The two possible base pairs The DNA Double Helix  If the sequence on one strand is ATACGCAT TATGCGTA  The other’s sequence must be  Each chain is a complementary mirror image of the other  So either can be used as template to reconstruct the other  The two DNA strands are held together by weak hydrogen bonds between complementary base pairs  A and T  C and G

Original DNA molecule is preserved Daughter DNAs contain one old and one new strand Old and new DNA are dispersed in daughter molecules 3 possible methods for DNA replication

Thus, DNA replication is semi-conservative Evidence for Semi-Conservative Replication  These three mechanisms were tested in 1958 by Matthew Meselson and Franklin Stahl

Overview of How DNA Copies Itself  DNA helices begin unwinding from the nucleosomes  Helicase untwists the double helix and exposes complementary strands  The enzyme primase puts down a short piece of RNA termed the primer  DNA polymerase reads along each naked single strand adding the complementary nucleotide  The site of replication is the replication bubble  Each nucleotide strand serves as a template for building a new complementary strand DNA Replication Overview PLAY

Template strandNew strand Sugar- phosphate backbone C G T A A T C G A A HO 3’ O O O O O O O O O O 5’ 3’ OH 5’ P P P P P P P P P P T O OH P P P DNA polymerase How nucleotides are added in DNA replication

DNA polymerase can only build a strand of DNA in one direction The leading strand is made continuously from one primer The lagging strand is assembled in segments created from many primers Summary of How DNA Copies Itself  The process of DNA replication can be summarized as such  The enzyme helicase first unwinds the double helix  The enzyme primase puts down a short piece of RNA termed the primer  DNA polymerase reads along each naked single strand adding the complementary nucleotide

DNA Replication  Since DNA polymerase only works in one direction:  A continuous leading strand is synthesized  A discontinuous lagging strand is synthesized  DNA ligase splices together the short segments of the discontinuous strand  Two new telomeres are also synthesized  This process is called semiconservative replication

RNA primers are removed and replaced with DNA Ligase joins the ends of newly-synthesized DNA Mechanisms exist for DNA proofreading and repair DNA Replication (cont.) DNA Replication PLAY

Transcription  The path of genetic information is often called the central dogma DNARNAProtein A cell uses three kinds of RNA to make proteins Messenger RNA (mRNA) Transfer RNA (tRNA) Ribosomal RNA (rRNA)

Transcription  Gene expression is the use of information in DNA to direct the production of proteins  It occurs in two stages

Transcription  The transcriber is RNA polymerase It binds to one DNA strand at a site called the promoter It then moves along the DNA pairing complementary nucleotides It disengages at a stop signal

Working with DNA  Key techniques used by today’s genetic engineers include  PCR amplification  Used to increase the amounts of DNA  cDNA formation  Used to build genes from their mRNA  DNA fingerprinting  Used to identify particular individuals