DNA STRUCTURE & REPLICATION . Last class. We walked through the historical time line of the discovery of DNA We ended with James Watson, Francis Crick, and Rosalind Franklin Franklin was the first to capture an image of DNA
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Last class... • We walked through the historical time line of the discovery of DNA • We ended with James Watson, Francis Crick, and Rosalind Franklin • Franklin was the first to capture an image of DNA • But it was Watson and Crick who finally discovered the true structure
Watson and Crick’s structure • Double helix that is comparable to a ladder. • The uprights are made of: • DEOXYRIBOSE sugars connected by PHOSPHATE groups • Held together by phosphodiester linkages • The ladder rungs are made of: • NITROGENOUS bases
A Closer Look... Phosphate • Same as you have seen before • 1 phosphorus atom • 4 oxygen atoms • Overall –ve charge • DNA is –ve!!! Deoxyribose Sugar • 5 carbons • 2’ C is missing an oxygen • 3’ C and 5’ C are most important
A Closer Look... Adenine Thymine Guanine Cytosine
A Closer Look... Adenine Thymine Guanine Cytosine PURINES The AGgies are PURe. PYRAMIDINES
A Closer Look... • A purine will always bond with a pyramidine • How nucleotide bases bond together: • A & T share 2 H-bonds C & G share 3 H-bonds • **The H-bonding gives DNA its stability!
A Closer Look... *** Thymine can not bond with Guanine due to lack of H-bonding*** *** Adenine can not bond with Cytosine due to lack of H-bonding***
Antiparallel... • DNA is said to run ANTI-PARALLEL • One strand runs in the 5’ 3’ direction • The other runs in the 3’ 5’ direction • The 5’ refers to the 5th carbon on the deoxyribose sugar • The 3’ refers to the 3rd carbon on the deoxyribose sugar • The 5’ end terminates with an phosphate group • The 3’ end terminates with an -OH group
DNA REPLICATION The Basics: • Cells can reproduce (as you have seen in mitosis) • Genetic information is divided equally from parent cell into daughter cells • Identical genetic information from parent to daughter cell is important to maintain identical cellular function • From their structure, Watson and Crick could tell: • H-bonds between nucleotides could break • DNA could ‘unzip’ • Each strand could act as a template to build a complementary strand
DNA REPLICATION Meselson & Stahl – important experiment • In 1958, suggested that DNA replication is SEMI-CONSERVATIVE • Each daughter cell receives one strand of parental DNA • Conservative = one daughter receives both strand
DNA REPLICATION Procedure • Grew E. Coli in a nutrient rich medium in 15N isotope • Allowed to replicate 17 times • thus all DNA should contain 15N • Next, bacteria with 15N were transferred to a medium of 14N • Now, 14N should be found in daughter DNA • One strand should be heavier than the other • As replication continues, more 14N should be found • All samples were centrifuged to separate by density • Results....
DNA REPLICATION Results • Tube A = DNA of cells before switching to 14N • Tube B = DNA after 1st replication of 15N in 14N sol’n • Both 14N and 15N • Tube C = DNA after 2nd replication • Intermediate band (14N+15N) and a light band (14N only) Tube A Tube B Tube C
DNA REPLICATION Conclusion • Original strands are still present after many replications, thus semi-conversative • They must act as a template • NOT conservative or we would see one heavy band and one light band Tube A Tube B Tube C
DNA REPLICATION The Roster • Many enzymes are used in this process • DNA helicase • DNA gyrase • DNA polymerase III • DNA polymerase I • DNA ligase • RNA primase • All have a very specific function in this process *Proteins can’t exist without DNA, but DNA has no function without proteins*
DNA REPLICATION The Process 1. DNA Helicase unwinds DNA by breaking H-bonds • Obviously, they are going to want to rebound (anneal) • Luckily, single-stranded binding proteins (SSBs) bind to exposed DNA blocking H-bonding 2. DNA Gyrasehelps the unwinding process by relieving any excess tension • DNA can’t be unwound all at once as it is too big. • The length of DNA in 1 chromosome is 1cm • The diameter of a cell 0.00005cm!! • To avoid this we need to replicate in regions
DNA REPLICATION The Process 3. DNA replication proceeds in the direction of the replication fork • Replication bubbles occur when two replication forks are close to one another 4. DNA polymerase III starts to build complementary strand
DNA REPLICATION The Process • Before DNA polymerase III can initiate a new strand by itself • It needs a PRIMER 5. RNA PRIMASE allows an RNA primerto be added • RNA primer marks the startof the initiation sequence
DNA REPLICATION The Process 6. Elongation - DNA polymerase: • Synthesizes DNA from 5’ 3’ direction • Adds free nucleotides to the 3’ end • Elongation occurs easily on the 3’ 5’ strand • LEADING STRAND • The 5’ 3’ strandcreates short fragments • LAGGING STRAND
DNA REPLICATION The Process • RNA primers must be continually added to the 5’3’ parent strand to create lagging strand • This allows DNA polymerase III to build short fragments called OKAZAKI FRAGMENTS
DNA REPLICATION The Process 7. DNA polymerase I • Removes RNA primers • Replaces them withappropriate nucleotides 8. DNA Ligase • Joins one Okazaki fragment to another • Creates a phosphodiester bond
DNA REPLICATION The Process 9. When mistakes occur, DNA polymerase III & DNA polymerase I act as exonucleases • They can backtrack to remove the incorrect nucleotide and replace it with the correct one • A form of proof-reading
DNA REPLICATION Recap