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Evidence for DNA as the genetic material Early in this century, little known about DNA; regarded as uninteresting junk Proteins were thought to be the only truly complex molecules in cells, and

therefore must be responsible for heredity 1928: Frederick Griffith discovered phenomenon of transformation in

bacteria 1. Used organism Streptococcus pneumoniae. 2. Strep bacillus has two forms:

1. slimy colonies (S strain) forms mucous capsules, survives attack by macrophages in lung, kills mice

2. rough colonies (R strain) lacks capsules, quickly killed by macrophage, no disease

3. When Griffith mixed heat-killed S-strain with live R-strain, resulting organisms killed mice, and lungs were filled with S-strain.

4. View diagram of Griffith experiment 5. Conclusion: some chemical is surviving heat treatment, retains genetic

information, is able to transmit that information to some R-strain bacteria, convert them to S. Griffith didn't know what was responsible.

1944: Avery, McCarty and MacLeod demonstrate that DNA is responsible for transformation in bacteria

1. Fractionated different chemicals in S-strain bacteria, tested each separately to see what would cause transformation.

2. Isolated DNA could transform, but no other isolated fraction could (RNA, protein, lipids, polysaccharides). Conclusion: DNA is transforming principle

3. But critics attacked slight (less than 1%) presence of protein in the DNA extracts used, claimed this might be responsible for transformation

1952: Hershey & Chase prove that only DNA is responsible for bacterial virus infection of host cells.

1. Viruses (called phage if host cells are bacteria) are much simpler than cells, contain only DNA & protein.

2. Hershey & Chase were able to use different radioactive isotopes to distinguish DNA from protein: for DNA, used P-32 (lots of P in DNA, but none in protein); for protein, used S-35 (proteins contain S in certain amino acids, but DNA lacks S).

3. H&C grew phage in hosts with either P or S radioisotope. Then infected different bacteria for short time, vortexed in blendor to separate phage coats from cells, and separated phage (very small) from cells (larger) by centrifugation.

4. Result: only P-32 isotope found in cells. All S-35 could be knocked loose by blending, but cells were still infected and produced new phage. Therefore only DNA, not protein, was responsible for inheritance

Structure of DNA

DNA known to contain purine & pyrimidine bases, deoxyribose, and phosphate -- but how are they arranged?

1947: Chargaff published data showing that % of A, T, C, and G showed certain regularities

1. % of bases varies from organism to organism 2. % A = % T, and % C = % G. This is called Chargaff's rule. What did it

mean? 1952: X-ray pictures of DNA taken by Rosalind Franklin in Wilkins lab in

London showed some kind of helix. 1953: Watson & Crick published a model built from Franklin data = double helix.

Suggested that Chargaff's rule was due to base-pairing of A with T, C with G.

Interact with a DNA molecule: DNA tutorial (requires CHIME plug-in) In linear molecule, one strand has free 3'-end, where the other (complementary)

strand has 5'-end. View 3' and 5' ends of DNA (protected) Two chains of DNA face in opposite directions, called antiparallel (protected)

(defined by which way 3' and 5' sides of sugar molecule are facing). In linear molecule, one strand has free 3'-end, where the other (complementary) strand has 5'-end.

5'-CAGCTAGAGTCATCG-3' 3'-GTCGATCTCAGTAGC-5' W&C also suggested simple model for replication: if double stranded DNA

uncoiled, each strand could serve as template for replication of new DNA. This was an exciting experimental prediction, and many labs set out to try to prove it.

Replication of DNA First enzyme isolated by Kornberg ( Nobel prize): DNA polymerase.

1. Reaction: [dATP, dCTP, dGTP, dTTP] new DNA + P~P (pyrophosphate) reaction requires DNA polymerase, Mg++, template DNA

2. Note 1: Pi ~ Pi is immediately split into 2 Pi (inorganic phosphate ions). 3. Note 2: energy for forming new sugar-phosphate bond comes from

splitting a high-energy phosphate bond as Pi ~ Pi is removed. This always occurs at free 3'-OH group on deoxyribose (and on ribose in RNA synthesis). All nucleic acids grown by addition at 3'-end, not at 5'-end. Often referred to as 5' 3' synthesis.

Eventually discovered that cells have a variety of DNA polymerase enzymes; some serve for DNA repair rather than for new synthesis.

Other enzymes and proteins involved: 1. DNA helicase: unwinds DNA in front of opening replication fork

(otherwise DNA would quickly tangle). Uses ATP, makes single-stranded cut, allows one strand to swivel freely around the other.

2. Single-stranded DNA binding proteins: bind to separated DNA strands, prevent from base-pairing back together

RNA primase: DNA polymerase III cannot start a growing chain from scratch; needs a short primer (a few nucleotides) to add to. This is carried out by DNA-dependent RNA primase, makes very short piece of RNA by base-pairing RNA nucleotides with template DNADNA polymerase : adds new nucleotides at free 3'ends of growing chain, uses base-pairing rules to insert complementary nucleotides (A opposite T, G opposite C, etc.) Can keep on adding indefinitely for millions of nucleotides if not blockage. Also removes RNA primers, fills in gaps by base pairing, inserts new DNA nucleotides to replace RNA primer. (several types of this enzyme)

1. DNA ligase: seals any gaps where adjacent nucleotides on one strand have

not been covalently joined. Note: many gaps result on lagging strand (see below), so lots of need for enzymes

(5) and (6).

Leading and Lagging strands Since two strands in DNA are antiparallel, new DNA must be synthesized in

opposite directions on the two template strands. But overall, DNA must unwind in one direction (at replication fork), overall DNA

synthesis has one direction. No problem for the strand growing in same direction as unwinding = leading

strand. Can make one long, continuous piece of DNA Big problem for strand growing in opposite direction to unwinding = lagging

strand; must grow away from unwinding. As new template is opened up by DNA unwinding, will have to start a new copy.

In fact, just this situation was discovered experimentally by Okazaki; found many short DNA fragments newly synthesized from lagging strand = Okazaki

fragments. Must be joined together by DNA ligase to make continuous DNA strand.