DNA (Deoxyribonucleic Acid)

Scientific History n The march to understanding that DNA is the genetic material –T.H. Morgan (1908) –Frederick Griffith (1928) –Avery, McCarty & MacLeod (1944) –Erwin Chargaff (1947) –Hershey & Chase (1952) –Watson & Crick (1953) –Meselson & Stahl (1958)

Transformation of Bacteria

What carries hereditary information? n By the 1940s, scientists knew that chromosomes carried genes. n They also knew that chromosomes were made of DNA and protein. n They did NOT know which of these molecules actually carried the genes. n Since protein has 20 types of amino acids that make it up, and DNA only has 4 types of building blocks, it was a logical conclusion. n Most Scientists thought protein carried genes

Chromosomes are made of DNA and protein

Transformation of Bacteria

DNA is the “Transforming Principle” n Avery, McCarty & MacLeod –purified both DNA & proteins separately from Streptococcus pneumonia bacteria which will transform non-pathogenic bacteria? –injected protein into bacteria no effect –injected DNA into bacteria transformed harmless bacteria into virulent bacteria 1944 What ’ s the conclusion? mice die

Avery’s Experiment 1. Avery repeated Griffith’s experiments with an additional step to see what type of molecule caused transformation. 3. When Avery added enzymes that destroy DNA, no transformation occurred. 2. Avery used enzymes to destroy the sugars and transformation still occurred—Sugar did not cause transformation. Avery used enzymes to destroy lipids, RNA, and protein one by one. Every time transformation still occurred—none of these had anything to do with the transformation. So…he knew that DNA carried hereditary information!

Oswald AveryMaclyn McCartyColin MacLeod Avery, McCarty & MacLeod n Conclusion –first experimental evidence that DNA was the genetic material 1944 | ??!!

n The experiment involved viruses to see if DNA or protein was injected into the bacteria in order to make new viruses. n One group of viruses was infected with radioactive protein and another group with radioactive DNA. n Then the viruses attack the bacteria. n Radioactive DNA shows up in the bacteria, but no radioactive protein. Hershey-Chase Experiment

Chargaff n DNA composition: “Chargaff’s rules” –varies from species to species –all 4 bases not in equal quantity –bases present in characteristic ratio humans: A = 30.9% T = 29.4% G = 19.9% C = 19.8% 1947 That ’ s interesting! What do you notice? Rules A = T C = G

Rosalind Franklin n Took X-ray pictures of DNA. n The photos revealed the basic helix, spiral shape of DNA.

Maurice Wilkins n Worked with Rosalind Franklin. n Took her x-ray photos and information to Watson and Crick

Watson and Crick n Used Franklin’s pictures to build a series of large models. n Stated that DNA is a double-stranded molecule in the shape of a double helix, or twisted ladder. n Won the Nobel Prize for their work in 1962.

Semiconservative replication, n when a double helix replicates each of the daughter molecules will have one old strand and one newly made strand. n Experiments in the late 1950s by Matthew Meselson and Franklin Stahl supported the semiconservative model, proposed by Watson and Crick, over the other two models. (Conservative & dispersive)

Double helix structure of DNA “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

Basic DNA Structure S P A C S P S P T n A nucleotide is the monomer of DNA n A nucleotide is made of –a sugar called deoxyribose –a phosphate –and a base (ATCG)

Directionality of DNA n You need to number the carbons! –it matters! OH CH 2 O PO 4 N base ribose nucleotide This will be IMPORTANT!!

Deoxyribose n Simple sugar molecule like glucose that has 5 carbons n The five carbons are numbered clockwise starting from the first one after the oxygen

Phosphate n The negatively charged phosphate bonds to the 5’ Carbon of the deoxyribose.

Bases n The base bonds to the 1’ Carbon. Base

Bases n There are two main types of bases purines and pyrimidines. –Purines have two rings in their structure. Adenine and guanine are purines. –Pyrimidines only have one ring. Thymine and Cytosine are pyrimidines. Pyrimidines Purines

Basic DNA Structure S P A C S P S P T n To form one strand of DNA, the phosphate of one nucleotide covalently bonds to the 3’ Carbon of the deoxyribose from another nucleotide.

S P A C S P S P T G S P S P AT S P n The two strands of DNA are held together by hydrogen bonds

Anti-parallel strands n Nucleotides in DNA backbone are bonded from phosphate to sugar between 3 & 5 carbons –DNA molecule has “direction” –complementary strand runs in opposite direction

Base Pairs n The nucleotides that bond together by their bases are called base pairs. –Adenine only bonds to Thymine –Guanine only bonds to Cytosine

Does each of your cells have the same DNA? n YES

DNA Replication n Before a cell divides, DNA must make a copy of itself so that each new cell has a complete set of DNA.

Step 1-Unzip DNA n An enzyme called helicase untwists the ladder and breaks the hydrogen bonds between the bases and “unzips” DNA down the middle. Helicase Enzyme

Step 2-Prime the DNA n An enzyme called DNA primase put a few nucleotides of RNA on the DNA. n This is only to create a starting place and these will later be removed.

Step 3-Elongation n The two strands of the Parent DNA become templates for the new strands. n New nucleotides are added by an enzyme called DNA polymerase.

Step 3-Elongation n DNA polymerase only adds nucleotides in the 5’ to 3’ direction on both strands beginning at the RNA primer.

Step 4 – Fine tuning n RNA primer is removed and any gaps are sealed by an enzyme called ligase. n DNA polymerase proof reads the new copy and fixes any mistakes.

Helicase unwinds and unzips DNA S P A C S P S P T T G S S P P S P A

DNA Polymerase Adds New Nucleotides T G S S P P S P A S P A C S P S P T T S P G S P S P A S P AC S P S P T

n Are the two copies of DNA the same? n Why would it be important for the two copies of DNA to be the same?

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 III Replication fork / Replication bubble leading strand lagging strand leading strand lagging strand leading strand growing replication fork growing replication fork lagging strand 5 3

DNA polymerase III RNA primer  built by primase  serves as starter sequence for DNA polymerase III Limits of DNA polymerase III  can only build onto 3 end of an existing DNA strand Starting DNA synthesis: RNA primers growing replication fork primase RNA

DNA polymerase III RNA primer  built by primase  serves as starter sequence for DNA polymerase III Limits of DNA polymerase III  can only build onto 3 end of an existing DNA strand Starting DNA synthesis: RNA primers growing replication fork primase RNA

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

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 Houston, we have a problem!

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

Replication fork 3’ 5’ 3’ 5’ 3’ 5’ helicase direction of replication SSB = single-stranded binding proteins primase DNA polymerase III DNA polymerase I ligase Okazaki fragments leading strand lagging strand SSB

Length of DNA n The length of the DNA from one cell is –3 meters n "Unravel your DNA and it would stretch from here to the moon"

DNA Packing DNA double helix (2-nm diameter Histones “Beads on a string” Nucleosome (10-nm diameter) Tight helical fiber (30-nm diameter ) Supercoil (200-nm diameter ) Metaphase chromosome 700 nm

Nucleosomes n “Beads on a string” –1 st level of DNA packing –histone proteins 8 protein molecules positively charged amino acids bind tightly to negatively charged DNA 8 histone molecules

DNA packing as gene control n Degree of packing of DNA regulates transcription –tightly wrapped around histones no transcription genes turned off  heterochromatin darker DNA (H) = tightly packed  euchromatin lighter DNA (E) = loosely packed H E

The End!