Chapter 16 – The Molecular Basis of Inheritance

Searching for Genetic Material Mendel: modes of heredity in pea plants (1850s) Morgan: genes located on chromosomes (1910s) Frederick Griffith: bacterial work; identified the concept of transformation – a change in genotype and phenotype due to assimilation of an external substance (DNA) by a cell (1928) Oswald Avery: transformation agent was DNA (1944) Before Avery, many scientists believed that protein was the genetic material and not DNA. Can you think of a reason for this theory? Protein is more complex (20 AAs as compared to 4 nucleotides) and a simple DNA strand couldn’t possibly give rise the complexity of humans.

Griffith’s Experiment 2 strains of Streptococcus pneumoniae – 1 with a polysaccharide coat (smooth or S), one without (rough or R) Conducted 4 experiments (fig. 16.2) Injected mice with live S, the mice died, so encapsulated strain was pathogenic (disease causing) Injected mice with live R, the mice stayed healthy, so the strain was non- pathogenic Injected mice with heat-killed S, mice healthy, the polyscaaharide coat was found to not be the disease-causing agent because the coat was still intact in the heat-killed S bacteria Heat killed S mixed with R and injected into mice, the mice died, the blood contained live S strain cells. The R strain “acquired” from S the ability to make a polysaccharide coat. These S cells were cultured and found to produce encapsulated daughter cells, concluded that newly acquired trait was inheritable. The change in phenotype was due to an assimilation of external genetic material by a cell – transformation Concluded that this could not be a protein because heat kills (denatures) proteins

Griffith’s Experiment

Hershey and Chase 1952 – discovered that DNA is the genetic material of a phage called T2 T2 phage can infect E.coli, which is simply DNA enclosed by a protein coat, and could quickly reprogram the E.coli to produce new T2 phages. Conducted experiments to figure out if the DNA or the protein was responsible for this reprogramming phenomenon Phage protein was tagged with 35S, phage DNA was tagged with 32P Allowed phage to infect separate samples of non-radioactive E. coli cells. Centrifuged the samples- the heavier bacteria would fall to the bottom while the light virus would be on top Measured radioactivity Tubes with protein-labeled T2 had radioactivity in supernatant (liquid) Tubes with DNA-labeled T2 had radioactivity in the pellet (bacteria) Phage proteins remained outside the bacterial cells during infection, while phage DNA entered the cells When cultured, bacterial cells with radioactive phage DNA released new phages with some 32P DNA Concluded that DNA is the hereditary material of T2, not protein

Hershey and Chase

DNA Structure Edwin Chargaff (1947) – figured out ratio of nitrogen bases in a DNA molecule, approximate numbers of T=A and C=G, Chargaff’s rule Rosalind Franklin (1950’s) – used X-ray crystallography to determine that DNA is in the form of a double helix

DNA Structure Watson and Crick (1953) – proposed a model for DNA Used x-ray crystallography data from Franklin as well a Chargaff’s rule to propose their model: The structure is a double helix with a sugar-phosphate backbone and nitrogen bases in the center Their model was not widely accepted until they could demonstrate how DNA could be replicated (i.e. its functionality) In 1954, Watson and Crick published a second paper outlining how the process occurs – demonstrated that DNA replication is semiconservative Data supported by Maurice Wilkins in 1956 Watson, Crick, and Wilkins win the Nobel Prize in 1962 (Franklin not awarded prize – deceased, 1958)

DNA Structure

DNA structure (and RNA) DNA structure animation

DNA Replication - semiconservative Proposed by Watson and Crick - strands are complementary; nucleotides line up on template according to base pair rules Each new double helix will have one “old or original” strand and one “new” strand

DNA Replication – semiconservative Meselson and Stahl (late 1950s) designed an experiment to test the theory of semiconservative DNA replication

DNA Replication Bubble Origins of replication – a replication bubble Begins at specific sites where parental strands separate with helicase enzyme and form bubble, stabilized with single-strand binding proteins Each end of the bubble is a replication fork Bubble expands laterally, DNA replication occurs in both directions Eventually bubbles fuse, replication complete

DNA Replication Nucleotide Addition New nucleotides are added to the free 3’ end of the growing strand (DNA formed in the 5’ to 3’ direction) Utilizes a variety of polymerase enzymes to compete this addition DNA is antiparallel, so how replicate both sides, simultaneously, in the same direction?

Leading and Lagging Strands DNA Polymerase III adds new nucleotides to 3’ end Leading strand – continuous replication towards the replication fork Lagging strand – discontinuous replication away from the replication fork, replicated in chunks called Okazaki fragments Animation overview

Lagging Strand All replication begins with primase adding RNA nucleotides to DNA DNA poly III adds nucleotides to the RNA primer sequence DNA poly III releases when reach next RNA primer DNA poly I replaces the RNA with DNA adding to 3’ end DNA ligase forms bond between these two Okazaki fragments to complete DNA chain

Replication Overview

Replication Overview Overview

DNA Repair Enzymes proofread DNA for errors 1 in 10,000 initial errors, 1 in 1 billion end errors Mismatch repair – occurs while being synthesized, uses DNA polymerase Excision repair – corrects accidental changes to DNA, uses nuclease, polymerase, and ligase

DNA Shortening On end of DNA is an RNA primer Since DNA poly can only add to 3’ end, this is a problem Because of this the ends get shorter Telomeres – noncoding regions of DNA at end of chain, in humans repeating chains of TTAGGG Protects genes from being eroded from successive rounds of replication Telomerase catalyzes the length of telomeric DNA restoring the DNA length in germ cells; usually inactive in adult somatic cells