DNA Replication Ch 16 Unit Test: Ch 16-20

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)

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?

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

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

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?

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.

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

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

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

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)

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

Overall direction of replication 3 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

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)

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: http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#

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

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

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

Laying Down RNA Primers

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

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 16.19 p. 319 Ex. A 70 yr old person’s cells divide approx. 20-30X vs an infant which will divide 80-90X

Telomeres

Suggested Videos http://www.youtube.com/watch?v=OnuspQG0Jd0 (bioflix) https://www.youtube.com/watch?v=27TxKoFU2Nw (3d medical) http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html# http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#