Structure (chapter 10, pages 266 – 278) and Replication of DNA (chapter 12, pages 318 – 334)

Structure of DNA Designate the Nucleotides –Purines Guanine = G Adenine = A –Pyrimidines Thymine = T Cytosine = C

Structure of DNA Nucleotides join together, forming a polynucleotide chain, by phosphodiester bonds –The phosphate attached to the 5’ carbon on one sugar –Attaches to the 3’ hydroxyl (OH) group on the previous nucleotide

5’-phosphate of last nucleotide chemically bonded to the 3’-hydroxyl of the next-to-last nucleotide A phosphodiester bond

Structure of DNA DNA is a double helix (two strands) held together by hydrogen bonds –Adenine (A) and thymine (T) are paired –Guanine (G) and cytosine (C) are paired –Always a purine pairs with a pyrimidine

The two polynucleotide strands (the backbones) in the double helix run in opposite directions, and are said to be anti- parallel 5’-end 3’-end 5’-end (free 5’- phosphate) 3’-end (free 3’-OH)

Because of the pairing (A-T; G-C), one polynucleotide chain is always complementary to the base sequence of the other strand 5’-end 3’-end 5’-end (free 5’- phosphate) 3’-end (free 3’-OH)

It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. J. D. Watson and F. H. C. Crick, 1953

Matthew Meselson and Franklin Stahl, 1958 entirely new AND entirely old DNA molecules present ALL DNA molecules are made up of both old and new DNA entirely new DNA molecules present BUT not entirely old DNA molecules

Meselson and Stahl Experiment –Grew E. coli in a growth medium containing only 15 N (heavy nitrogen) (note: Normal isotope is 14 N lighter nitrogen) –Did this for many generations so that all of the bacterial DNA would be “heavy”

Meselson and Stahl Experiment –Then grew the bacteria with 15 N incorporated in their DNA in medium containing only 14 N (would be incorporated into the new DNA) This way they could differentiate the original DNA from newly incorporated DNA

Meselson and Stahl Experiment –At each generation Isolated the DNA Looked at the density of the DNA in a CsCl gradient

Matthew Meselson and Franklin Stahl, 1958

What Meselson and Stahl expected if semiconservative replication

entirely new DNA molecules present BUT not entirely old DNA molecules

First generation results helped them rule out one of the three possible mode of replication

Matthew Meselson and Franklin Stahl, 1958 entirely new AND entirely old DNA molecules present ALL DNA molecules are made up of both old and new DNA entirely new DNA molecules present BUT not entirely old DNA molecules

Clincher evidence! Why? In dispersive model lighter DNA band should not have formed

Great test question: Predict what the cesium chloride gradients would look like for conservative and dispersive replication!

Should be able to draw something like this for conservative and dispersive

Meselson and Stahl showed that the semiconservative pattern of replication is what was found So the DNA double helix unwinds and each strand acts as a template for replication of the new half

Replication General features: 1. There is a specific site where replication begins (origin) which must be recognized 2. The two strands of DNA must be separated 3. The original strand becomes the template for the new DNA strand 4. A primer molecule must be added on which the new DNA chain can be built 5. New nucleotides must be added complementary to the template strand 6. The newly synthesized DNA must be edited and joined into one continuous molecule

Replication Origin of replication –Where synthesis of new DNA begins –A specific location with a specific sequence of nucleotides

In some organisms a specific location that can be mapped

Multiple and random origins in eukaryotes

In bacteria and viruses one origin of replication In eukaryotes there can be thousands of replication origins Origins of replication

Initiator Proteins Recognizes the Origin of Replication Start to denature the DNA so each strand can act as a template

Replication DNA is unwound by a helicase –Separates the double helix by breaking the hydrogen bonds

Helicase

The separated (single strand DNA) is combined with single-strand binding proteins Protects DNA from degradation Keeps the complementary strands from rejoining

Replication As DNA is unwound it will tangle and knot, called supercoiling (from the unwinding of the helix)

The supercoiling must be relaxed (the DNA unknotted) This is done by a class of enzymes called topisomerases (gyrase)

Review: Proteins and their function in the early stages of replication 1 = initiator proteins 2 = single strand binding proteins 3 = helicase 4 = topoisomerase (gyrase)

Replication DNA polymerases –Enzymes that synthesize new DNA

5’ triphosphates of the four nucleotides must be present (dATP, dGTP, dTTP, dCTP) Two of the phosphates are cleaved- off, providing energy to run the reaction

The preexisting single strand of DNA is the template strand Complementary base to the template strand Phosphates cleaved to provide energy for the reaction Nucleotide monophosphates are then joined to the 3’OH group

Replication DNA Polymerase –Must have a free 3’-OH group onto which to add the new nucleotides –No known DNA polymerase is able to initiate chains; thus, requires a primer to start synthesis

DNA is always polymerized in a 5’ to 3’ direction and antiparallel to the template strand

KNOW ALL TERMS!

Replication DNA Polymerase –Must have a free 3’-OH group onto which to add the new nucleotides –No known DNA polymerase is able to initiate chains; thus, requires a primer to start synthesis

Must have a primer (which is an RNA molecule) The primer is synthesized by the enzyme primase (RNA polymerase)

Replication Two DNA polymerase enzymes are necessary for replication in E. coli –DNA polymerase I –DNA polymerase III Both have polymerase and exonuclease activities (functions)

Replication Polymerase: –Synthesize new DNA in the 5’  3’ direction Exonuclease: –Remove nucleotides from the end of a chain (proofreading and editing functions) 5’  3’ (removes primers) 3’  5’ (editing, removes incorrect bases)

5’  3’ exonuclease activity

Replication DNA Polymerase I –Synthesize new DNA in the 5’  3’ direction Only synthesizes short sequences of new DNA –3’  5’ exonuclease activity (proofreading) –5’  3’ exonuclease activity (remove primers) DNA Polymerase III –Synthesize new DNA in the 5’  3’ direction Synthesizes long sequences of new DNA –3’  5’ exonuclease activity (proofreading)

Replication The phosphodiester backbone of DNA must be joined This is done by the enzyme ligase

The phosphodiester backbone of DNA must be joined