1. DNA Replication
Ch 16
Unit Test: Ch 16-20

2. Experiments and People to Know: *Read your book and take notes!
Griffith (and Avery)- Transformation (non-virulent S. pneumoniae strain transformed)
Hershey and Chase- Used phages and proved DNA was hereditary mat’l (not protein)
Chargaff- %A=%T; etc
Watson and Crick- double helix
Rosalind Franklin- x-ray diffraction (W&C “borrowed” her work)
Meselson and Stahl- How DNA replicates (used N15 and N14)

3. The Structure of DNA What we know… Double helix
Each nucleotide is made up of:
Deoxyribose (sugar)
phosphate group
Base (purines: G,A; pyrimidines: C,T).
**What is a nucleoside?

4. The Structure of DNA Base-Pairing Rules (Chargaff)
DNA strands are antiparallel
*3 H bonds between C and G; 2 H bonds between A and T
3’ end- OH; 5’ end-PO4

5. What are differences in DNA and RNA? (*You fill in!)

6. Models of replication 1.Conservative 2.Semi-conservative 3.Dispersive
Two parental strands came back together after replication
2.Semi-conservative
One new and one old strand in a newly synthesized double stranded DNA
3.Dispersive
All 4 strands post-replication have a mixture of old and new DNA
*Which is correct?

7. Meselsohn and Stahl labeled the old strand with N15 (heavy)
Meselsohn and Stahl labeled the old strand with N15 (heavy). New strands will have N14.
Centrifugation after replication supports the semi-conservative model.

8. Semi-Conservative Replication
Each new DNA molecule has one old strand and one new strand

9. DNA Replication: how it works
Occurs in the nucleus
A new and identical molecule of DNA is made, using the old one as a template
Occurs in opposite directions on each strand. One strand runs 5’->3’; other runs 3’->5’; triphosphate nucleotides can only be added to 3’ end of growing (daughter) strand. New strand grows 5’->3”
*n’tide triphosphate releases 2 PO4’s (exergonic) for polymerization of new strands

10. How Nucleotides add to the old Strand
5’ end
3’ end
5’ end

11. DNA Replication a special sequence of DNA
begins at the origin of replication
a special sequence of DNA
2 strands are separated by helicase, forming a replication bubble
Replication fork is formed at each end of the replication bubble
Prokaryotes use same mechanism, but have a circular chromosome (p. 313)

12. Origin of Eukaryotic replication (many “bubbles”): pg. 313

13. Overall direction of replication3
REPLICATION FORKS:
DNA polymerase molecule
5 end 5
Daughter strand synthesized continuously
Parental DNA 3’
How DNA daughter strands are synthesized 5 5’ 3
Daughter strand synthesized in pieces 3 P 5 3’ 5’
The daughter strands are identical to the parent molecule 5 3’ P 3 5’ 3’
DNA ligase 5’
Overall direction of replication
Figure 10.5C

14. Key Enzymes Required for DNA Replication: pg. 317
Helicase - catalyzes the untwisting of the DNA at the replication fork
DNA Polymerase III - catalyzes the elongation of new DNA and adds new nucleotides on the 3’ end the growing strand.
DNA Polymerase I- replaces the RNA with DNA nucleodtides.
SSBP’s - single stranded binding proteins, prevents the double helix from reforming
Topoisomerase – Breaks the DNA strands and prevents excessive coiling
RNA primase – synthesizes the RNA primers and starts the replication first by laying down a few nucleotides initially.
Ligase- attaches okazaki fragments
** DNA Polymerase II is a prokaryotic enzyme (editing)

15. DNA Replication-New strand Development
Leading strand: synthesis is toward the replication fork
(only in a 5’ to 3’ direction from the 3’ to 5’ master strand)
-Continuous
Lagging strand: synthesis is away from the replication fork
-Only short pieces are made called “Okazaki fragments”
- Okazaki fragments are 100 to 2000 nucleotides long (longer in prokaryotes)
-Each piece requires a separate RNA primer
-DNA ligase joins the small segments together
(must wait for 3’ end to open; again in a 5’ to 3’ direction)
View video clip:

16. Leading Strand
DNA polymerase can only add nucleotides to the 3’ end of a DNA strand
In the leading strand, DNA polymerase III follows the replication fork

17. Lagging Strand
DNA polymerase works in the opposite direction of the replication fork
Short segments of DNA are made
Okazaki fragments
Okazaki fragments are joined by DNA ligase

18. RNA Primers
Initiates the Replication process and begins the building of the newly formed strands.
Laid down by RNA primase
Consists of 5 to 14 nucleotides
Synthesized at the point where replication begins
Will be laid down on both template strands of the DNA

19. Laying Down RNA Primers

21. Issues with Replication
Prokaryotes: (ex. E. coli)
Only one origination point
E. coli: ~4.6 million Nucleotide base pairs
Rate for replication: 500 nucleotides/sec
Eukaryotes:
Each chromosome is one DNA molecule
Has free ends
Humans (46) has approx. 6 billion base pairs
Rate for replication: 50 per second (humans)
Telomeres on ends for protection
Errors:
Rate is one every 10 billion nucleotides copied
Proofreading is achieved by DNA polymerase I. Fig 16.18

22. Telomeres Short, non-coding pieces of DNA
Contains repeated sequences (ie. TTGGGG many times)
Can lengthen with an enzyme called Telomerase
Lengthening telomeres will allow more replications to occur.
Telomerase is found in germ cells; but not in normal somatic cells. Has been observed in cancer cells
Artificially giving cells telomerase can induce cells to become cancerous
Shortening of telomeres may contribute to cell aging and Apotosis
See Fig p. 319
Ex. A 70 yr old person’s cells divide approx X vs an infant which will divide 80-90X

23. Telomeres

24. Suggested Videos http://www.youtube.com/watch?v=OnuspQG0Jd0 (bioflix)
(3d medical)