1. DNA Replication and Recombination
Lecture 4
DNA Replication and Recombination

2. DNA Maintenance Mutation rate are extremely low
1 mutation out of 109 nucleotides per generation

3. DNA replication
Separation, Base pair

4. The Chemistry of DNA replication

5. DNA Synthesis by DNA polymerase

6. Nucleotide polymerizing enzyme, first discovered in 1957
DNA Polymerase
Nucleotide polymerizing enzyme, first discovered in 1957

7. DNA replication with two forks

8. This Doesn’t Work!

9. DNA replication Fork

10. DNA Proofreading

11. Structures of DNA polymerase during polymerizing and editing
E: exonucleolytic; P: polymerization

12. Why 5’->3’?
The need for accuracy

13. Site-directed mismatch repair in eucaryotes
In procaryotes, old DNAs are usually methylated on A while newly synthesized ones are not. So Cells can distinguish old and newly synthesized DNAs and mutate mismatches on new ones.

14. DNA Proofreading RNA usually doesn’t have this. Why?
Pairing, correct nucleotide has higher affinity binding to the moving polymerase
Un-Paired nucleotide is easier to be off before covalent ligation, even after binding.
Exonucleotic proofreading
Strand directed mismatch repair

16. DNA Primer synthesis
On Lagging strand
DNA primase

17. DNA Replication at the Lagging strand
RNA primer is 10 nucleotides long

18. DNA ligase joins the 3 end of the new DNA fragment to the 5 end
of the previous fragment
This enzymes seals a broken phosphodiester bond
DNA ligase uses ATP to activate the 5 end at the nick

19. Different Helicase on leading and lagging strand
DNA Helicase
DNA double helix are tightly coupled. High temperature is needed to break them (95oC)
Different Helicase on leading and lagging strand

21. DNA Binding Protein (RPA)
SSB: Single Strand DNA-binding Proteins, also called helix destabilizing proteins

22. Covers total of 8 nucleotides, DNA bases remain exposed DNA complex
SSB Proteins
DNA
Covers total of 8 nucleotides, DNA bases remain exposed DNA complex

23. PCNA

24. Cycle of DNA Polymerase/Clamping Protein loading and unloading
At the lagging strand

25. Protein machinery for DNA replication

26. A Moving Replication

27. Structure of the Moving Complex

28. DNA Topoisomerase prevent DNA tangling
Reversible nucleases binds covalently
to DNA backbone phosphate
Breaking pdiester bond
Reversible and reforms

29. DNA topoisomerase I

30. DNA topoisomerase II

31. Mammalian replication Fork
(eucaryote, DNA polymerase (primase) a synthesize RNA/DNA, DNA polymerase delta is the real polymerase)

32. Summary DNA replication 5’->3’ DNA proof reading
Lagging strand, back-stitching, Okazaki fragment
Proteins involved:
DNA polymerase, primase
DNA helicase and single-strand DNA-binding protein (SSB)
DNA ligase, and enzyme to degrade RNA
DNA topoisomerases

33. DNA Replication in Chromosome

34. Initiating Proteins for DNA replication
1. Initiator protein, 2. helicase binding to initiator protein, 3. helicase loading on DNA, 4. helicase opens the DNA and binds to primase, 5. RNA primer synthesis, 6. DNA polymerase binding and DNA synthesis

35. The four standard phases of a eucaryotic cell
DNA replication occurring at S Phase (DNA synthesis phase)
G1 and G2, gap between S and M

36. Different regions of a chromosome are replicated at different times
Arrows point to the replicating regions at different times

37. Some facts about Replication in eucaryotes
Multiple replication origins occurring inclusters (20-80) (replication units)
Replication units activated at different times
Within replication units, replication origins are separated 30, ,000 pairs apart.
Replication forks form in pairs and create a replication bubbles moving in opposite directions
Different regions on the same chromosome are replicated at distinct times in S phase
Condensed Chromatin replicates late, while less condensed regions replicate earlier

38. Addition of new histones
Chromatin assembly factors (CAFs) help to add and assemble new nucleosomes
Newly H3 and H4 are acetylated on their N-terminal tails after incorporated into chromatin
AG are removed enzymatically

39. Reverse transcriptase with RNA template to bind to DNA strands
Telomerase Structure
Reverse transcriptase with RNA template to bind to DNA strands

40. Telomerase and its function

41. Summary
Specific DNA sequence determine replication origin, recruiting proteins to form replication machinery. relatively complex in eucaryotes
Bacteria has single replication origin. Eucaryotes have multiple origins and less defined.
Replication forks are activated at different times in eucaryotes
Telomere and telomerase