Today: DNA!

Deciphering DNA… KNOWN: -The nucleus contains the genetic material of a cell. -The nucleus is composed primarily of proteins and nucleic acids. But which one is the genetic material? PROTEINS seem the obvious choice as they’re very DIVERSE, and highly SPECIFIC. Little is known about nucleic acids.

One of the First, Convincing Pieces of Evidence: Griffith’s Transformation Experiment (1928) Why does this suggest DNA and not PROTEIN is the genetic material?

Oswald Avery spends the next 14 years sifting through all the various chemicals from the heat-killed, pathogenic bacteria. Only DNA can successfully transform bacteria! This announcement (1944) is greeted with much skepticism…

Fortunately for Oswald, Hershey and Chase come along (1952) Hershey and Chase study Bacteriophage Bacteriophage inject their genetic material into host cells to replicate themselves. If Hershey and Chase can figure out what is injected, they can identify the “genetic material”!

The Hershey-Chase Experiment…

Where will it be in batch 2? The Hershey-Chase Experiment… Where will the radioactivity be in batch 1 if DNA is the “genetic material”? Where will it be in batch 2?

The accumulation of evidence: Prior to mitosis (cell division) a eukaryotic cell doubles its DNA content, distributing the DNA equally between the two daughter cells. Somatic cells have twice as much DNA (diploid) as gametes (haploid) Chargaff: DNA composition varies from one species another and in the DNA of any one species the amounts of the four bases are not equal, but do occur in a predictable ratio: (A=T, C=G)

What are the key features of this model? Watson and Crick What are the key features of this model? With help from others, Watson and Crick decipher the structure of DNA! (1953)

The Structure of a DNA Molecule:

The Structure of a DNA Molecule: Key Features: Covalently bonded, negatively charged backbone Complementary pairing of 4 bases Weak hydrogen bonds joining the bases Anti-Parallel

Complimentary Pairing Which is the stronger pairing? Is DNA thermostable?

The “Genetic Material” is DNA in the form of a DOUBLE-HELIX How is this long molecule (~6 cm/chromosome in humans!) packaged and organized inside a cell?? DNA is packaged with PROTEINS to form CHROMATIN Histones (positively charged proteins) are responsible for the first level of organization

Nucleosomes (beads on a string) are then coiled to form a 30 nm fiber

The 30 nm fibers are looped and attached to a scaffold of nonhistone proteins

The looped domains are then themselves folded into a fully condensed chromosome

Only euchromatin may be used by the cell to make proteins! Portions of the chromatin in non-dividing cells stays condensed = Heterochromatin vs Euchromatin “true chromatin” Only euchromatin may be used by the cell to make proteins!

Replicating DNA… Model??

PURINES PYRIMIDINES Pairing of PURINES with PYRIMIDINES is key to both Structure and Replication of DNA

One more important limitation for our model of DNA replication: DNA Molecules are ANTI-PARALLEL 5’3’ = FORWARD STRAND 3’5’ = REVERSE STRAND DNA Polymerases can only add bases to the 3’ end of a DNA Molecule!

The three models of DNA Replication: CONSERVATIVE, SEMICONSERVATIVE and DISPERSIVE.

This model allows each strand to serve as a TEMPLATE for synthesis of a new strand. Watson and Crick’s model is a SEMICONSERVATIVE MODEL (each daughter molecule will have one “parent” strand and one newly synthesized strand)

An Overview of the Mechanics of DNA Replication: Must be FAST and ACCURATE (~1 mistake/billion bases)!

High Energy Covalent Bonds! With the two strands separated in a REPLICATION BUBBLE, a DNA Polymerase can add dNTPs (deoxyribose “N” triphosphate) COUPLED REACTION: hydrolysis of pyrophosphate is exergonic

One more important limitation for our model of DNA replication: DNA Molecules are ANTI-PARALLEL 5’3’ = FORWARD STRAND 3’5’ = REVERSE STRAND DNA Polymerases can only add bases to the 3’ end of a DNA Molecule!

The LEADING STRAND is a CONTINUOUS molecule. Because DNA Polymerase can only attach nucleotides to the 3’ end of a growing DNA molecule, the two strands of Parental DNA are replicated differently. The LEADING STRAND is a CONTINUOUS molecule. The LAGGING STRAND is composed of OKAZAKI FRAGMENTS “stitched” together by DNA LIGASE.

Other Key Players: Another enzyme, HELICASE, untwists the double helix while SINGLE-STRAND BINDING PROTEINS hold them apart so that DNA Replication can proceed.

Let’s Watch! How do your drawings hold up? http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/micro04.swf::DNA%20Replication%20Fork

DNA Replication: A more Complete View

One more Complication: DNA POLYMERASE can’t initiate DNA Replication! It can only add nucleotides to an existing chain. Thus DNA Replication requires a PRIMER, and an enzyme, PRIMASE, to make it!

Catching & Repairing Mistakes DNA Polymerases err at a rate of 1/10,000 base pairs. “Proofreading” reduces the error rate to 1/1 billion base pairs. Cells can repair many errors; Humans have 130 known DNA repair enzymes!

The Importance of DNA Repair The ability to repair damaged DNA is critical to long-term survival. Individuals with xeroderma pigmentosum produce defective nucleotide excision repair enzymes. Without these repair enzymes, mutated DNA in skin cells (UV) is not repaired, leading to skin cancers.

Another Challenge: The End Replication Problem As eukaryotes replicate their DNA, they are unable to “fill-in” the 5’ end of new molecules. Thus, over time, the daughter molecules become shorter and shorter.

The Solution: Telomeres- noncoding multiple repeats of a short nucelotide sequence (TTAGGG) Protects genes (coding sequences) from being eroded. (Erosion triggers programmed cell death!) Telomeres are regenerated by telomerases in germ-line cells and most cancers.

Was Dolly old before her time? Dolly the Sheep may have been susceptible to premature ageing, new research suggests. A team from the biotechnology firm PPL Therapeutics in Scotland examined Dolly's telomeres. They report in the journal Nature that the structures are slightly shorter than would be expected in a sheep of her age born normally. Source: BBC NewsThursday, May 27, 1999

A Thought Exercise: Work with your neighbor(s) to diagram normal DNA replication. Then, come up with a list of INGREDIENTS and CONDITIONS you would need to replicate DNA in a test tube!

The Polymerase Chain Reaction (PCR) DNA Replication in a tube! Exponential process used to generate large quantities of specific regions of DNA for use in forensics, DNA sequencing, research, medical diagnostics, etc.

Let’s Watch This, Too… http://www.sumanasinc.com/webcontent/animations/content/pcr.html