The Chemistry of Inheritance DNA Structure and Synthesis
A walk through time What are the instructions that make a plant? Or a mouse? Or a human? 1860s Mendel performs experiments and describes the inheritance of traits in the garden pea. 1870s Scientists observed the nuclei of eggs and sperm fusing during fertilization. 1900s Scientists observed that the number of chromosomes is constant within a species but varies between species. Ex. Humans have 46 chromosomes; fruit flies have 8 So, do the chromosomes, which are found in the nucleus, contain the information that determines inheritance?
Fusion of cytology and genetics Walter Sutton Studying grasshopper cells noticed that chromosomes “come in pairs” pairs split during meiosis (see a picture of dividing plant cells) 1902: Chromosomes obey Mendel’s genetic rules 1903: Chromosomes segregate independently during meiosis, obeying Mendel’s law of independent assortment
DNA or Proteins: The genetic material? 1920s Chromosomes contain proteins and DNA. Most cells of a species contain a constant amount of DNA, but the amount and kind of proteins vary. But, is DNA structurally diverse enough to provide the genetic instructions for an entire organism? Protein structure seems more varied and therefore more likely to be able to contain all of the necessary instructions for heredity…right?
Slimy and Extremely Dangerous Streptococcus pneumoniae Encapsulated, “slimy” strains can cause pneumonia (S or “smooth” strains). Unencapsulated strains do not cause disease (R or “rough” strains). Capsule composed of complex carbohydrate. “R” and “S” strains breed true. Mice injected with “R” strain or dead “S” strain alone do not get sick. Mice injected with live “R” strain and dead “S” strain do get sick, and only “S” strain cells are isolated from the blood of the mice. What is converting the “R” cells to “S” cells?
Avery, MacLeod and McCarty Avery, C.M. et al. 1944. J. Exp. Med.79, 137. Culture of S-strain bacteria Kill cells by heating Mostly R colonies grow; but a few true-breeding S colonies appear Add killed cells to culture of R-strain bacterial Show that the killed cells do not grow.
Avery, MacLeod and McCarty Avery, C.M. et al. 1944. J. Exp. Med.79, 137. Add protease to the dead S-strain cells. Mix the protease-treated dead S-strain bacteria with the R-strain culture. When plated, you see mostly R-strain colonies and a few S-strain colonies. Conclusion: Transforming activity is not protein.
Avery, MacLeod and McCarty Avery, C.M. et al. 1944. J. Exp. Med.79, 137. Add RNase to the dead S-strain cells. Mix the RNase-treated dead S-strain bacteria with the R-strain culture. When plated, you see mostly R-strain colonies and a few S-strain colonies. Conclusion: Transforming activity is not RNA.
Avery, MacLeod and McCarty Avery, C.M. et al. 1944. J. Exp. Med.79, 137. Add DNase to the dead S-strain cells. Mix the DNase-treated dead S-strain bacteria with the R-strain culture. When plated, you see only R-strain colonies. Conclusion: Transforming activity is probably DNA.
Hershey Chase Experiment Phage with labeled DNA direct the production of more phage that also contain labeled DNA. Phage with labeled protein do not direct production of more phage with labeled protein.
Chemical Composition of DNA • DNA is a polymer of nucleotides. Nucleotides consist of: • deoxyribose (5-carbon) sugar • nitrogenous base • phosphate group(s) There are four nitrogenous bases used to make the four types of nucleotides found in a DNA molecule: Adenine, Thymine, Cytosine and Guanine. The nucleotides are linked to form a chain. DNA inside cells consists of two complementary, intertwined chains (double helix). View a schematic of DNA structure. Nucleoside
Chemical Composition of DNA • There are four nitrogenous bases that are found in DNA: adenine, thymine, guanine and cytosine. • Adenine and thymine are purine bases (2-ring structure) • Cytosine and guanine are pyrimidines (single-ring structure)
Chemical Composition of DNA • The phosphodiester bond of the nucleotide chain is formed between the phosphate attached to the 5´ carbon of one sugar and the 3´ carbon of the next. View the structure of a phosphodiester bond. • The 5´ end of the strand bears a phosphate group; the 3´ end bears a hydroxyl (OH) group and is considered “polar”. • The two strands of DNA in a helical molecule are antiparallel to each other. This figure illustrates the polarity of the DNA strands.
Chemical Composition of DNA • Chargaff’s rules Base composition varies among species. Base composition is constant for all cells of an organism and within a species. The amount of adenine equals the amount of thymine. The amount of cytosine equals the amount of guanine. The amount of purine bases equals the amount of pyrimidine bases.
Structure of the DNA Molecule • Within cells the standard structure of DNA is the B form. • The B form structure consists of two antiparallel polynucleotide chains twisted around one another to form a double helix. • The nitrogenous bases form the “rungs” in the center of the helix, with adenine forming hydrogen bonds with thymine and guanosine forming hydrogen bonds with cytosine. • The helix is right-handed, and each chain makes one complete turn every 34 angstroms.
DNA Replication: Meselson and Stahl • Studied DNA replication in E. coli. • Cells were grown in “heavy” medium in which DNA molecules would incorporate 15N (a heavy isotope of nitrogen). • They were transferred to “light” medium, which contained only 14N (“light”) nitrogen. • DNA samples were taken at various time points subjected to centrifugation on a density gradient. • Molecules containing only 15N would run lower on the gradient than molecules containing 15N + 14N or 14N alone.
Evidence for Semi-Conservative Replication • At time 0 all of the DNA banded together as “heavy” DNA • One generation after switching the bacteria to the light medium, half-labeled or “hybrid” molecules were observed in the density gradient. • The gradual appearance of half-labeled and unlabeled molecules on the density gradient led to these conclusions: • The nitrogen of a DNA molecule is divided equally between two subunits…following rpelication each daughter molecule has received one parental subunit…the replicative act results in a molecular doubling. (Meselson and Stahl (1958) Proc. Natl. Acad. Sci. USA44, 671–82.) • A schematic of the experiment is available here.
DNA Polymerase • DNA polymerase catalyzes the chemical reaction that joins the 5´ phosphate with the 3´ OH to form the phosphodiester bond and, hence, the polynucleotide chain. • DNA polymerases require: • The 5´ triphosphates of the four deoxynucleosides (dATP, dGTP, dCTP, dTTP) • A pre-existing single strand of DNA (template) • A primer—short segment of RNA (in cells) that has a free 3´ OH group
Polymerase Proofreading • Not only does DNA polymerase catalyze the addition of new nucleotides to a growing molecule, it can also remove nucleotides. • DNA pol III has a 3´ to 5´ exonuclease activity that allows it to remove incorrectly incorporated nucleotides from the end of the growing molecule. This is the proofreading or editing function of DNA polymerase.
Replication • Several events must happen before DNA polymerase can catalyze the formation of a new DNA molecule based on an existing template. • The double helix must unwind to expose the two template strands. This is usually accomplished by the action of another enzyme, helicase. • Once the helix is unwound it must be “stabilized” in this open state. This is accomplished by single-stranded binding protein. • Before DNA polymerase can extend a strand, it must have a short primer. The primer is synthesized by an RNA polymerase called primase. • The area of the helix that is unwound for replication is the replication fork.
Leading and Lagging Strand Synthesis • DNA polymerase can only add to a free 3´ OH. • The two template strands are antiparallel. • Therefore, only one of the strands can provide a template for continuous 5´ to 3´ new strand synthesis. The strand synthesized from this template is called the leading strand. It is synthesized in one piece in the direction of the replication fork. • The daughter molecule that is synthesized from the other half of the helix is the lagging strand. It is synthesized in short fragments, called Okazaki fragments. These fragments are joined to form the complete daughter molecule.