DNA

1. DNA • Consists of • Deoxyribose sugar • Phosphate group • A, T, C, G • Double stranded molecule (Double Helix) • Two strands of DNA run antiparallel to each other (opposite direction) • 5’ to 3’ • 5’ is the end with the phosphate group • 3’ is where deoxyribose sugar is located • Nitrogenous bases • Held together by hydrogen bonds • A pairs with T ( forms double bond) • C pairs with G (forms a triple bond)

2. DNA • 46 chromosomes • 6 billion base pairs • ~25,000 genes

3. Four Requirements for DNA to be Genetic Material • 1. Must carry information • Cracking the genetic code • 2. Must replicate • DNA replication • 3. Must allow for information to change • Mutation • 4. Must govern the expression of the phenotype • Gene function

4. DNA Replication S phase G1 G2 interphase Mitosis -prophase -metaphase -anaphase -telophase • Process of duplication of the entire genome prior to cell division • Biological significance • extreme accuracy of DNA replication is necessary in order to preserve the integrity of the genome in successive generations • In eukaryotes , replication only occurs during the S phase of the cell cycle.

5. Rules of Replication • Semi-conservative • Parent strands serve as templates for daughter strands • Starts at the ‘origin’ • Site(s) of parent strand separation • Synthesis always in the 5-3’ direction • Problems with anti-parallel strands • Semi-discontinuous • Solution to anti-parallel strands • RNA primers required

6. Mechanism of DNA Replication 1. Strand Separation • Proteins bind to DNA and open up double helix • Prepare DNA for complementary base pairing 2. Building Complementary Strands • Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA 3. Dealing With Errors During DNA Replication • Proteins release the replication complex

7. DNA Replication is Semi-Conservative • Separating the two parent strands and building new complementary strand for each • New DNA has one new strand and one old strand

8. Strand Separation • Double Helix • Unwound at replication origins (many origins on DNA) • Helicase • binds to origins and unwinds the two strands creating replication bubbles • Two strands separating creates a replication fork

9. Enzyme Enzyme DNA Strand Separation Problems 1. Unwinding DNA creates tension (supercoil) • Topoisomerases • relieves tension by cutting strands near the replication fork 2. Single strands want to join back together (annealing) • Single-stranded binding proteins (SSBs) • attachtothe DNA strands stabilizing them

10. Strand Separation • Multiple replication origins decrease the overall time of DNA replication to about 1 hour

11. Building Complementary Strands • RNA primasebegins the replication process • Builds small complementary RNA segments on strand at beginning of replication fork (RNA primers) • DNA polymerase III • Builds new strand using nucleoside triphosphates • Adds nucleotides to the 3’ end of a parent strand • New strands are always assembled 5’ to 3’

12. DNA polymerase III 3 3 3 3 3 3 3 3 3 3 3 growing replication fork growing replication fork 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Building Complementary Strands • Leading Strand • DNA that is copied in the direction toward the replication fork • Lagging Strand • DNA that is copied in the direction away from the replication fork leading strand lagging strand leading strand lagging strand leading strand lagging strand

13. Building Complementary Strands • Anti parallel strands replicated simultaneously • Leading strand synthesis continuously in 5’– 3’ • Lagging strand synthesis in fragments in 5’-3’ • semi-discontinuous

14. Leading Strand • Single RNA primer is used to start strand • DNA polymerase III moves towards replication fork 5’ to 3’ direction • Continuous

15. Lagging Strand • DNA polymerase III moves away from replication fork • Discontinuous • Okazaki fragments are used to solve problem • 1000 – 2000 base pairs long • Multiple RNA primers are used • DNA polymerase I • removes RNA primers and replaces with DNA nucleotide • Fills the gaps

16. Building Complementary Strands • DNA ligase • Links last nucleotide to Okazaki fragment • Formation of phosphodiester bond

17. Dealing With Errors • DNA polymerase III • Proofread and correct errors • Errors are usually base pair mismatches • After replication • Average of 1 error per million base pairs • DNA polymerase II • Repairs damage after strands have been synthesized

19. 3 3 3 3 5 5 5 5 Chromosome Erosion • DNA polymerases can only add to 3 end of an existing DNA strand • Loss of bases at 5 ends in every replication • DNA polymerase I cannot replace final RNA primer DNA polymerase I growing replication fork DNA polymerase III

20. Does it Create a Problem?

21. Telomeres 3 3 3 3 5 5 5 5 • Repeating, non-coding sequences at the end of chromosomes • Protective cap • TTAGGG • Limit to ~50 cell divisions • Telomerase extends telomeres • Can add DNA bases at 5’ end • Shorten gradually after each cell division growing replication fork telomerase TTAAGGG TTAAGGG TTAAGGG

22. Cells Aging Process • Cell senescence • Cells loses ability to function properly as a person ages • Hayflick limit • Telomeres length decrease with age • No longer provide protection for the chromosome • Possibly links to age-related diseases • Dementia, atherosclerosis, macular degeneration • Gene expression changes • Key to living forever?

23. Packing of Eukaryotic DNA • Organization • Negative DNA wraps around positive histones • Nucleosome – cluster of 8 histones • Solenoids – coiled strings of nucleosomes (chromatin fibres)