DNA Replication

1. DNA Replication Biology 12

2. Cells can contain 6-9 feet of DNA. If all the DNA in your body was put end to end, it would reach to the sun and back over 600 times. • DNA in all humans is 99.9 percent identical. It is about one tenth of one percent that makes us all unique, or about 3 million nucleotides difference. • DNA can store 25 gigabytes of information per inch and is the most efficient storage system known to human. So, humans are better than computers!! • In an average meal, you eat approximately 55,000,000 cells or between 63,000 to 93,000 miles of DNA. • It would take a person typing 60 words per minute, eight hours a day, around 50 years to type the human genome.

3. So What Do We Know? DNA is composed of units called NUCLEOTIDES, which are composed of three sub-molecules: 1. Pentose Sugar (deoxyribose) 2. Phosphate 3. Nitrogen Base (purine or pyrimidine)

4. N N C N C C C N N C Two Kinds of Bases in DNA N • Pyrimidines are single ring bases. • Purines are double ring bases. N C O C C N C

5. So What Do We Know? DNA is composed of two complimentarystrands of nucleotides joined by hydrogen bonds: Adenine with Thymine (A-T or T-A) They join with 2 hydrogen bonds Cytosine with Guanine (C-G or G-C) They join with 3 hydrogen bonds DNA twists into a double helix

6. Chargraff’s Rule: Adenine and Thymine always join together A T Cytosine and Guanine always join together C G

7. 10 Bonding 1 Adenine Thymine Guanine Cytosine The bases always pair up in the same way to form base pairs. Adenine forms a bond with Thymine and Cytosine bonds with Guanine

8. 11 Bonding 2 PO4 PO4 thymine adenine PO4 PO4 cytosine guanine PO4 PO4 PO4 PO4

10. Replication Copying the genetic material is REPLICATION. Replication occurs prior to cell division, because the new, daughter cell will also need a complete copy of cellular DNA. Replication

11. DNA Replication Problem Solving • On the surface…. The replication of DNA is pretty simple. Just unzip, plug in the the spare parts by complementary base pairing, and stitch up the new backbone. • There are a lot of irritating details and problems with this process. • The solutions to these problems involve a list of vital enzymes.

12. DNA Replication Complexities: • DNA is a very stable molecule but its stability depends on its double stranded nature. Single stranded DNA is vulnerable to a number of kinds of damage. So…..to take a huge DNA molecule and seperate its two strands for its entire length....is a very bad idea. • So how can long DNA molecules be replicated without making them single stranded for long periods of time?

13. DNA is very long. VERY long. How can a very long DNA molecule get itself replicated in a relatively short period of time?

14. The primary DNA replication enzyme, DNAPolymerase, is highly specific in a number of ways. One of those specificities is that it can only add new nucleotides to an already existing growing strand of nucleotides. This is a problem because it means that DNA polymerase is not capable of actually starting the process itself. How does replication get started?

15. DNA polymerase is also highly specific in that it can only build new polynucleotide strands in the 5' to 3' direction--new strands must always run from 5' to 3'. But the two strands of DNA are antiparallel to each other--one runs 3' to 5', the other from 5' to 3'. Both sides have to be replicated. How is this puzzle solved in DNA replication? What's the name of the scientist credited with this discovery?

16. DNA is double stranded, and the two strands twist around each other. These two strands need to be pulled apart, thus tightening the twist between Origin points. This would lead to accidental breakage of the polynucleotide strands as the twists got compressed into smaller and smaller lengths. How is this problem avoided in DNA replication? What is the name of the enzyme needed to solve this problem (it has several; any of them will do)?

17. Finally, the solutions to the previous problems leave us with an embarrassing problem: single stranded breaks in the polynucleotides of our new DNA molecules. How are these breaks repaired? Again, you'll need to identify the enzyme involved.

18. DNA Replication Overview • Enzyme breaks weak hydrogen bonds • DNA strands open up • Free nucleotides (from our food) fill in the open side (free nucleotides are a significant component of the nucleoplasm in any cell. • Using complimentary base pairing • End Result: • 2 identical DNA strands

19. How Does DNA Replicate? • Three Hypotheses: Conservative Semi-Conservative Dispersive 2 strands of the parent stay together, daughter gets new two strands New DNA is made of a random Mixture of parent and daughter DNA 2 strands of parent separate, daughter gets 1 strand of DNA from parent

20. Meselson-Stahl Parental (old) DNA molecule • Discovered the ‘semi-conservative’ model • Experimented with bacteria • Replication fork (site of replication) Daughter (new) strand Daughter DNA molecule (double helices) Figure 10.6

21. Semi-Conservative Replication Origin of name: • Original parent strands conserved BUT are not still attached together End Result: • Each new daughter DNA strand is ½ “old” and ½ “new”

22. Three Steps In DNA Replication • 1. Initiation – Replication begins at a location on the double helix known as “oriC” to which a certain initiator proteins bind and trigger unwinding. Enzymes known as helicases unwind the double helix by breaking the hydrogen bonds between complimentary base pairs, while other proteins keep the single strands from rejoining. The topoisomerase proteins surround the unzipping strand and relax the twisting that migh damage the unwinding DNA.

23. 2. Elongation • With the primer as the starting point for the leading strand, a new DNA strand grows one base at a time. The old (existing) strand is the template for the new strand. The enzyme DNA polymerase controls elongation, which can only occur in the leading direction. • The lagging strand unwinds in small sections that DNA polymerase replicates in the leading direction. The resulting “Okazaki fragments” can contain between 100 to 200 bases. The fragments terminate in an RNA primer that is later removed so that enzymes stitch the back together into one long strand.

24. 3. Termination • After the elongation is completed, two new double helices have replaced the original one. • During termination the last primer must be removed from the end of the lagging strand. • Enzymes proofread the new double helix and remove mispaired bases.

25. Step #1 Separating DNA Strands Gyrase Relieves tension by unwinding Helicase Breaks hydrogen bonds Unzips the DNA Terminates at fork • - Enzyme breaks the weak hydrogen bonds • Splitting the parent DNA strand • Leaving two separated strands

26. Separating DNA Strands SSBs(Single-stranded binding proteins) • Bind to the exposed DNA • Keep from “annealing” • Reattaching with complimentary base pair

27. The two sides of the molecule are separated for a short distance. Since DNA is most stable (and least vulnerable to damage) in its double stranded configuration, as little of it as possible will be single stranded at once.

28. DNA Replication • Priming: 1. RNA primers: before new DNA strands can form, there must be small pre-existing primers (RNA)present to start the addition of new nucleotides (DNA Polymerase). 2. Primase: enzyme that polymerizes (synthesizes) the RNA Primer.

29. DNA polymerase performs only one job, following the complementary base pairing rule. It adds the new free nucleotides in the new strand of a replicating DNA molecule. This also means that DNA polymerase cannot actually start the process of replication. An enzyme called primase (an RNA polymerase) actually begins the replication process. It builds a short piece of RNA called a primer. This primer is later removed by RNAse H and replaced by DNA nucleotides.

30. Step #2 Building Complimentary Strands Replication proceeds along Both sides of the replication fork. • Free nucleotides with the assistance of a protein called DNA polymerase, attach to original parent DNA strand which serves as a template. • Nucleotides in the new strand are selected using complimentary base pairing • A with T and C with G

31. DNA polymerase is only capable of building a new strand from the 5’ end to the 3’ end. This is a problem because the two sides of the DNA are antiparallel. • One side of the new strand (called the leading strand) can be directly and continuously constructed from its 5’ end to its 3’ end.

32. 5’ 3’ 5’ RNA Primer DNA Polymerase Nucleotide DNA Replication • Synthesis of the new DNA Strands: 1. DNA Polymerase: with a RNA primer in place, DNA Polymerase (enzyme) catalyze the synthesis of a new DNA strand in the 5’ to 3’ direction.

33. 5’ 3’ 5’ RNA Primer DNA Polymerase Nucleotides DNA Replication 2. Leading Strand: synthesized as a single polymerin the 5’ to 3’ direction.

34. Figure 5 Single-stranded binding proteins Parent strand Replication fork III

35. The other strand (called the laggingstrand) must be constructed in short segments, built backwards. • These short strands of new DNA are called Okasakifragments after the man who discovered them. • The Okasaki fragments will eventually be connected by an enzyme named DNA ligase. DNA ligase specializes in healing single stranded nicks in DNA. It simply seals the bond between one nucleotide and the neighboring nucleotide.

36. Leading Strand 5’ 3’ 3’ 5’ DNA Polymerase RNA Primer 5’ 3’ 5’ 3’ Lagging Strand DNA Replication 3. Lagging Strand: also synthesized in the 5’ to 3’ direction, but discontinuously against overall direction of replication.

37. DNA Polymerase Okazaki Fragment RNA Primer 5’ 3’ 5’ 3’ Lagging Strand DNA Replication 4. Okazaki Fragments: series of short segments on the lagging strand.

38. DNA ligase Okazaki Fragment 1 Okazaki Fragment 2 5’ 3’ 3’ 5’ Lagging Strand DNA Replication 5. DNA ligase: a linking enzyme that catalyzes the formation of a covalent bond from the 3’ to 5’ end of joining stands. Example: joining two Okazaki fragments together.

39. DNA ligase • Joins Okazaki fragments together • Completes backbone • Lagging strand only!

40. Building Complimentary Strands Leading strand Built continuously From parent 3’ end toward replication fork

41. Building Complimentary Strands Lagging strand • Built in short Okazaki fragments between RNA primers • Discontinuous • From parent 3’ end away from replication fork

42. Single-stranded binding proteins Okazaki fragments Parent strand Replication fork III

43. Step 1 Step 2 Step 3

44. DNA is extremely long. It would take a very long time to replicate the whole molecule from end to end using only a single replication fork. • Each of these long molecules have many sites called Origins. This forms a configuration called a replicationbubble. Each replication bubble actually has two forks, one on each end of the bubble and travelling in opposite directions.

45. Fork vs. Bubble Origin of replication Origin of replication Replication Bubble: • Begins at multiple sites • Produces multiple forks • Proceeds in both directions • Result: • Faster replication Origin of replication Parental strand Daughter strand Bubble Two daughter DNA molecules Figure 10.8

47. DNA cannot be double stranded and not twist. Unless there is a way to relax the twists between replication bubbles, the helical twists would compress putting increasing stress on the molecule causing random breaking and damage. This problem is solved by the creation of single stranded breaks (swivels) between origin sites. These nicks are made by an enzyme called DNA topoisomerase (previously called “unwindase”, then “swivelase”. After the replication bubbles meet, ‘the single strands are healed by DNALigase.

48. Proofreading DNA polymerase I and III • Check for proper base pairing • If mistake found: • Removes • Replace with proper nucleotide

49. End Result • Results in two identical daughter DNA strands • ½ new and ½ old

50. Leading vs. Lagging