Examining the genetic material I.Criteria for genetic material II.Discovering the genetic material III.Reviewing the structure of DNA A. Nucleotides B. Polynucleotides C. Structure of DNA holds the key to understanding its function! IV.DNA Replication Review A. Semiconservative replication, Replication fork B. DNA Polymerase & Initiation C. Elongation: Antiparallel strands: continous & discontinuous

I. Criteria for genetic material 1.Must store the information necessary to construct an entire organism (BLUEPRINT) 2.During reproduction, the genetic material usually must be replicated, passing from parent to offspring, and each cell must be supplied with a copy. (FAITHFUL REPLICATION) 3.Must have variation that can account for the know phenotypic variation within each species (POTENTIAL FOR MUTATION) 4.Must be able to be activated in order to create products (EXPRESSION OF INFORMATION)

DNA the genetic material DNA stores information DNA stores information Can be faithfully replicated Can be faithfully replicated Undergoes mutation Undergoes mutation Is expressed Is expressed

II. Discovering the genetic material Experiments implied that the substance responsible for genetic transformation was the DNA of the cell 1.Griffith 2.Avery, MacLoed & McCarty 3.Hershey & 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 ( 35 S – protein, 32 P – DNA)

III. Reviewing the structure of DNA DNA - function based upon molecular Structure A. Nucleotides: Composed of 3 kinds of molecules: 1.Nitrogenous compound (pyrimidine/purine) 2.Five-carbon sugar (ribose/deoxyribose) 3.Phosphate  Linked together via phosphodiester bond with 5’ to 3’ directionality

Nucleotide triphosphate (NTP)

B. Polynucleotides

C. Structure of DNA holds the key to understanding its function! 1)Base composition studies (Chargraff) Amount of adenine residues is proportional to the amount of thymine residues in the DNA of any species… Amount of adenine residues is proportional to the amount of thymine residues in the DNA of any species… (A+G) = (C+T) (A+G) = (C+T) 2)X ray diffraction analysis 3)Watson-Crick, Franklin Model

 complementary  antiparallel base pair = unit of length 1,000 bp = 1kb

Note the directionality -

Secondary structure

Alternative forms of DNA

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

IV. DNA Replication REVIEW Transfer of genetic info from parent to progeny by faithful replication of the parent DNA Faithful but not perfect… and this is a good thing because??? Faithful but not perfect… and this is a good thing because??? Complex process, not completely understood tv/dna/replication.html tv/dna/replication.html

A. DNA is reproduced by semiconservative replication

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

The replication fork Strands of helix unwind at the replicon (origin of replication: ori) Bidirectional, continues until it reaches the termination region: ter Speed – in humans is about 50 nucleotides per second per replication fork

B. DNA Polymerase & Initiation Polymerase catalyzes the synthesis of the daughter DNA strand Uses the parent strand as a template chain elongation proceeds in the 5’ to 3’ direction - New dNTPs are added to the 3’OH end DNA POL I, POL II, POL III (all can elongate and existing DNA strand) 3' to 5' exonuclease functions as a "proofreader". 5' to 3' exonuclease activity to remove RNA primers. POL I & POL II DNA repair POL I & POL II DNA repair POL III Replication POL III Replication

POL III: 10 different subunits Active form = holoenzyme

Initiation 1)Unwinding of Helix DNA helix unwinds at the origin Helicases unwind Helix at the ori H-bonds “melt” H-bonds “melt” Single strand binding proteins (SSBPs) hold the strands apart

2). Primer PRIMER = Sequence of RNA synthesized on the template Primer terminus generated within Duplex DNA, a nick generates free 3’-OH end of the RNA that is extended by Polymerase RNA primers are synthesized by RNA polymerase or by a primase (which is made up of additional proteins)

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

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

Eukaryotic replication Similar, but more complex Multiple replication origins Six different DNA polymerases Telomers