Examining the genetic materialExamining the genetic materialI.I. Criteria for genetic materialCriteria for genetic materialII.II. Discovering the genetic materialDiscovering the genetic materialIII.III. Reviewing the structure of DNAReviewing the structure of DNA

A.A. NucleotidesNucleotidesB.B. PolynucleotidesPolynucleotidesC.C. Structure of DNA holds the key to understanding its Structure of DNA holds the key to understanding its

function!function!

IV.IV. DNA Replication ReviewDNA Replication ReviewA.A. Semiconservative replication, Replication forkSemiconservative replication, Replication forkB.B. DNA Polymerase & InitiationDNA Polymerase & InitiationC.C. Elongation: Antiparallel strands: continous & Elongation: Antiparallel strands: continous &

discontinuousdiscontinuous

I. Criteria for genetic materialI. Criteria for genetic material1.1. Must Must store the informationstore the information necessary to necessary to

construct an entire organism construct an entire organism (BLUEPRINT)(BLUEPRINT)

2.2. During reproduction, the genetic material During reproduction, the genetic material usually must usually must be replicatedbe replicated, passing from parent , passing from parent to offspring, and each cell must be supplied with to offspring, and each cell must be supplied with a copy. a copy. (FAITHFUL REPLICATION)(FAITHFUL REPLICATION)

3.3. Must Must have variationhave variation that can account for the that can account for the know phenotypic variation within each species know phenotypic variation within each species (POTENTIAL FOR MUTATION)(POTENTIAL FOR MUTATION)

4.4. Must be able to be activated in order to create productsMust be able to be activated in order to create products (EXPRESSION OF INFORMATION)(EXPRESSION OF INFORMATION)

DNA - - - the genetic materialDNA - - - the genetic material DNA stores information DNA stores information Can be faithfully replicatedCan be faithfully replicated Undergoes mutationUndergoes mutation Is expressedIs expressed

II. Discovering the genetic materialII. Discovering the genetic material

Experiments implied that the Experiments implied that the substance responsible for genetic substance responsible for genetic transformation was the DNA of the celltransformation was the DNA of the cell

1.1. GriffithGriffith

2.2. Avery, MacLoed & McCartyAvery, MacLoed & McCarty

3.3. Hershey & ChaseHershey & Chase

Griffith’s experiment:

Avery, MacLeod & McCarty’s follow-up experiment:

The Hershey & Chase experiment:

Verified that DNA was the genetic material – Bacteriophage added to E.coli in radioactive medium (35S – protein, 32P – DNA)

III. Reviewing the structure of DNAIII. Reviewing the structure of DNA

DNA - function based upon molecularDNA - function based upon molecularStructureStructureA. Nucleotides:A. Nucleotides:

Composed of 3 kinds of molecules:Composed of 3 kinds of molecules:1.1. Nitrogenous compound (pyrimidine/purine)Nitrogenous compound (pyrimidine/purine)2.2. Five-carbon sugar (ribose/deoxyribose)Five-carbon sugar (ribose/deoxyribose)3.3. Phosphate Phosphate Linked together via phosphodiester bond with 5’ to 3’ Linked together via phosphodiester bond with 5’ to 3’

directionality directionality

Nucleotide triphosphate (NTP)

B. Polynucleotides

C. Structure of DNA holds the key C. Structure of DNA holds the key to understanding its function!to understanding its function!

1)1) Base composition studies (Chargraff)Base composition studies (Chargraff) Amount of adenine residues is proportional to the Amount of adenine residues is proportional to the

amount of thymine residues in the DNA of any amount of thymine residues in the DNA of any species…species…

(A+G) = (C+T)(A+G) = (C+T)

2)2) X ray diffraction analysisX ray diffraction analysis

3)3) Watson-Crick, Franklin ModelWatson-Crick, Franklin Model

complementaryantiparallel

base pair = unit of length 1,000 bp = 1kb

Note the directionality -

Secondary structure

Alternative forms of DNAAlternative forms of DNA

DNA structure & functionDNA structure & function

How does DNA fulfill the requirements of How does DNA fulfill the requirements of genetic material?genetic material? Replicate?Replicate? Encode info?Encode info? Mutate?Mutate? Get expressed?Get expressed?

IV. DNA Replication REVIEWIV. DNA Replication REVIEW

Transfer of genetic info from parent to Transfer of genetic info from parent to progeny by faithful replication of the progeny by faithful replication of the parent DNAparent DNA

Faithful but not perfect… and this is a good Faithful but not perfect… and this is a good thing because???thing because???

Complex process, not completely Complex process, not completely understoodunderstood

http://www.wehi.edu.au/education/wehi-tvhttp://www.wehi.edu.au/education/wehi-tv/dna/replication.html/dna/replication.html

A. DNA is reproduced by semiconservative replicationA. DNA is reproduced by semiconservative replication

Meselson-Stahl Meselson-Stahl ExperimentExperiment

Bacteria labeled with heavy isotope (15N) transferred to medium w/light isotope (14N) – the new DNA would be “lighter” than the parent DNA & could be distinguished via centrifugation.

The replication forkThe replication fork

Strands of helix unwind at Strands of helix unwind at the replicon (origin of the replicon (origin of replication: replication: oriori))

Bidirectional, continues Bidirectional, continues until it reaches the until it reaches the termination region: termination region: terter

Speed – in humans is Speed – in humans is about 50 nucleotides per about 50 nucleotides per second per replication forksecond per replication fork

B. DNA Polymerase & InitiationB. DNA Polymerase & Initiation

Polymerase Polymerase catalyzes the synthesiscatalyzes the synthesis of the daughter of the daughter DNA strand DNA strand

Uses the Uses the parent strand as a templateparent strand as a template

chain elongation proceeds in the chain elongation proceeds in the 5’ to 3’ direction5’ to 3’ direction - - New dNTPs are added to the 3’OH endNew dNTPs are added to the 3’OH end

DNA POL I, POL II, POL III (all can elongate and DNA POL I, POL II, POL III (all can elongate and existing DNA strand)existing DNA strand)

3' to 5' exonuclease functions as a "3' to 5' exonuclease functions as a "proofreaderproofreader".".

5' to 3' exonuclease activity to 5' to 3' exonuclease activity to remove RNA primersremove RNA primers.. POL I & POL II DNA repairPOL I & POL II DNA repair POL III ReplicationPOL III Replication

POL III: 10 different subunitsActive form = holoenzyme

InitiationInitiation1)1) Unwinding of HelixUnwinding of Helix

DNA helix unwinds at the originDNA helix unwinds at the origin

HelicasesHelicases unwind Helix at the unwind Helix at the oriori H-bonds “melt”H-bonds “melt”

Single strand binding proteins (Single strand binding proteins (SSBPsSSBPs) hold the ) hold the strands apartstrands apart

2). Primer2). Primer

PRIMER = Sequence of RNA synthesized on PRIMER = Sequence of RNA synthesized on the templatethe templatePrimer terminus generated within Duplex Primer terminus generated within Duplex DNA, a nick generates free 3’-OH end of the DNA, a nick generates free 3’-OH end of the RNA that is extended by PolymeraseRNA that is extended by Polymerase

RNA primers are synthesized by RNA primers are synthesized by RNA RNA polymerasepolymerase or by a or by a primaseprimase (which is made (which is made up of additional proteins)up of additional proteins)

C. Elongation=synthesis of a new strand of DNA by C. Elongation=synthesis of a new strand of DNA by the addition of a new base, (one base at a time).the addition of a new base, (one base at a time).

http://www.contexo.info/DNA_Basics/replication%20move.htm

Antiparallel strands: continuous & Antiparallel strands: continuous & discontinuousdiscontinuous

Antiparallel structure: problem, as replication fork Antiparallel structure: problem, as replication fork advances-daughter strands must be synthesized on advances-daughter strands must be synthesized on both parental single strands. both parental single strands. New nucleotides only New nucleotides only added on 3’-OH endadded on 3’-OH endLeading strandLeading strand: one strand serves as template for : one strand serves as template for continuouscontinuous DNA synthesis DNA synthesisLagging strandLagging strand: oriented in the 3’-5’ direction, so : oriented in the 3’-5’ direction, so short fragments synthesized “backwards” in the 5’ to short fragments synthesized “backwards” in the 5’ to 3’ direction 3’ direction Series of fragments joined together via Series of fragments joined together via DNA ligaseDNA ligase

http://nobelprize.org/medicine/educational/dna/a/replication/lagging_ani.html

Eukaryotic replicationEukaryotic replication

Similar, but more complexSimilar, but more complex

Multiple replication originsMultiple replication origins

Six different DNA polymerasesSix different DNA polymerases

TelomersTelomers