1. Chapter 5: DNA Replication, Repair, and Recombination

2. Goals Illustrate how structure of DNA affects its function
Describe enzymes involved in replication
Summarize their functions
Explain how DNA is repaired and why it needs to be repaired

3. DNA Structure (A Review)
DNA consists of two strands
Each strand is a polymer of nucleotides
Strand has orientation due to nucleotide structure: 5’ and 3’ ends
The two strands are antiparallel

4. DNA Function (A Review)
DNA function is information storage
Sequence of strand stores info
Genes are copied into RNA (transcription)
“Control elements” regulate protein interactions with DNA
DNA passed on to descendant cells
Accurate copying
Repair of any damage to avoid changes
Accurate subdivision
Stored info determines cell’s functions; therefore best if transmitted error-free
Copied DNA split between cells- also important, probably not covered (much?)

7. Functions Determine Form
Double strands of DNA allow “easy” replication
Rules for obligate pairing
Each strand acts as template for the other
Multiple proteins involved
Act in concert
Act as complexes
Recognize DNA by shape of bases
Sequence of nucleotides is what must remain unchanged, so that new daughter cells get correct instructions on genes to express and when and how much (as well as sequence of genes themselves)
Theme of series is involvement of multiple proteins in carrying out DNA function, including “self-replication”- they must act together in appropriate order and often do so as large clusters of proteins interacting together

8. Paired DNA Strands
Nucleotides on inside with phosphate and deoxyribose on outside
There are hydrogen bond donors and acceptors not involved in base pairing
These as well as several of donor/acceptors involved in base pairing can interact with proteins in the major and minor grooves

9. Replication Overview Replication complex binds to replication “origin”
Double-stranded DNA is “melted”
Each strand is used as a template for DNA synthesis

10. Mechanism of DNA Replication
General Features of DNA Replication
Semiconservative
Complementary Base Pairing
DNA Replication Fork is Assymetrical
Replication occurs in 5’ ’ Direction

11. Semi-conservative Replication

12. Semi-conservative Replication

13. DNA Replication

14. DNA Replication

15. Tools of Replication Enzymes are the tools of replication:
DNA Polymerase - Matches the correct nucleotides then joins adjacent nucleotides to each other
Primase - Provides an RNA primer to start polymerization
Ligase - Joins adjacent DNA strands together (fixes “nicks”)

16. More Tools of Replication
Helicase - Unwinds the DNA and melts it
Single Strand Binding Proteins - Keep the DNA single stranded after it has been melted by helicase
Gyrase - A topisomerase that Relieves torsional strain in the DNA molecule
Telomerase - Finishes off the ends of DNA strands

19. In the Beginning… Problems due to structure
DNA is double-stranded
Template must be single-stranded
Strands must be separated
Separation is difficult due to structure
Melting strands causes tension elsewhere
If unrelieved tension can snap DNA strand

20. Topoisomerase (Gyrase)
Relieves stress caused by melting DNA
Cleaves DNA and spins around itself to unwind helix
Type I cleaves one strand, type II cleaves both
Reseals DNA strands after relaxation achieved
Use example of two ropes twined around each other, held at one end; stick finger into hole of a loop then pull two apart at on end- tension increases and finger gets squeezed

21. DNA Replication DNA Helicase
Hydrolyze ATP when bound to ssDNA and opens up helix as it moves along DNA
Moves 1000 bp/sec
2 helicases: one on leading and one on lagging strand
SSB proteins aid helicase by destabilizing unwound ss conformation

22. Single Strand Binding Protein
Binds to DNA with no sequence preference
Binds tighter to single strand than double
Keeps separated strands from rejoining

23. DNA Replication
SSB proteins help DNA helicase destabilizing ssDNA

24. Primase Creates a primer for DNA polymerase Template-dependent
An RNA polymerase
Active briefly at beginning of strand synthesis

25. DNA Replication DNA Primase
uses rNTPs to synthesize short primers on lagging Strand
Primers ~10 nucleotides long and spaced ~ bp
DNA repair system removes RNA primer; replaces it w/DNA
DNA ligase joins Okazaki fragments

27. DNA Polymerase Enzyme that synthesizes a DNA strand
Uses existing strand as template
Requires a free “3’ end” to add new nucleotides
Has several catalytic functions
Several forms exist

28. Eukaryotic DNA Polymerases
Enzyme Location Function
Pol  (alpha) Nucleus DNA replication
includes RNA primase activity, starts DNA strand
Pol  (gamma) Nucleus DNA replication
replaces Pol  to extend DNA strand, proofreads
Pol  (epsilon) Nucleus DNA replication
similar to Pol , shown to be required by yeast mutants
Pol  (beta) Nucleus DNA repair
Pol  (zeta) Nucleus DNA repair
Pol  (gamma) Mitochondria DNA replication

29. Maintenance of DNA Sequences
DNA Polymerase as Self Correcting Enzyme
Correct nucleotide has greater affinity for moving polymerase than incorrect nucleotide
Exonucleolytic proofreading of DNA polymerase
DNA molecules w/ mismatched 3’ OH end are not effective templates; polymerase cannot extend when 3’ OH is not base paired
DNA polymerase has separate catalytic site that removes unpaired residues at terminus

30. Maintenance of DNA Sequences
High Fidelity DNA Replication
Error rate= 1 mistake/109 nucleotides
Afforded by complementary base pairing and proof-reading capability of DNA polymerase

31. DNA Replication DNA Polymerase held to DNA by clamp regulatory protein
Clamp protein releases DNA poly when runs into dsDN
Forms ring around DNA helix
Assembly of clamp around DNA requires ATP hydrolysis
Remains on leading strand for long time; only on lagging strand for short time when it reaches 5’ end of proceeding Okazaki fragments

35. DNA Replication Okazaki Fragments
RNA that primed synthesis of 5’ end removed
Gap filled by DNA repair enzymes
Ligase links fragments together

37. Extension - Okazaki Fragments
DNA
Pol. 3’ 5’
RNA Primer
Okazaki Fragment
DNA Polymerase has 5’ to 3’ exonuclease activity. When it sees an RNA/DNA hybrid, it chops out the RNA and some DNA in the 5’ to 3’ direction. 3’ 5’
RNA Primer
DNA
Pol.
RNA and DNA Fragments
DNA Polymerase falls off leaving a nick. 3’ 5’
RNA Primer
Ligase
Nick
The nick is removed when DNA ligase joins (ligates) the DNA fragments.

38. DNA Replication Initiation and Completion of DNA Replication in Chromosomes
Bacteria
Single Ori
Initiation or replication highly regulated
Once initiated replication forks move at ~ bp/sec
Replicate 4.6 x 106 bp in ~40 minutes

39. DNA Replication Initiation and Completion of DNA Replication in Chromosomes
DNA Replication Begins at Origins of Replication
Positions at which DNA helix first opened
In simple cells ori defined DNA sequence bp
Sequence attracts initiator proteins
Typically rich in AT base pairs

40. DNA Replication Initiation and Completion of DNA Replication in Chromosomes
Eukaryotic Chromosomes Have Multiple Origins of Replication
Relication forks travel at ~50 bp/sec
Ea chromosome contains ~150 million base pairs
Replication origins activate in clusters or replication units of ori’s
Individual ori’s spaced at intervals of 30, ,000 bp

41. DNA Replication Initiation and Completion of DNA Replication in Chromosomes
Mammalian DNA Sequences that Specify Initiation of Replication
1000’s bp in length
Can function when placed in regions where chromo not too condensed
Human ORC required for replication initiation also bind Cdc6 and Mcm proteins
Binding sites for ORC proteins less specific

44. The Eukaryotic Problem of Telomere Replication
RNA primer near end of the chromosome on lagging strand can’t be replaced with DNA since DNA polymerase must add to a primer sequence.

45. Different types of Nucleotide Polymerases
DNA polymerase
Uses a DNA template to synthesize a DNA strand
2) RNA polymerase
Uses a DNA template to synthesize an RNA strand
(= transcription)
3) Reverse transcriptase
Uses an RNA template to synthesize a DNA strand
Found in many viruses
Telomerase is a specialized reverse transcriptase

46. Telomerase Two components of the human telomerase:
the human RNA subunit(hTR)
5’-CUAACCCUAAC-3’
The human telomerase
reverse transcriptase(hTERT)

47. Telomerase Function: Specialized reverse transcriptase
Prevents “shortening ends problem” problem by adding telomeres to the end
Copies only a small segment of RNA that it carries by itself
Requires a 3’ end as a primer
Synthesis proceeds in 5’ – 3’ direction
Synthesizes one repeat then repositions itself
When active provides cell immortality

48. Telomerase is composed of both RNA and protein

49. What are telomeres? Telomeres are…
Repetitive DNA sequences at the ends of all human chromosomes
They contain thousands of repeats of the six-nucleotide sequence, TTAGGG
In humans there are 46 chromosomes and thus 92 telomeres (one at each end)

50. Telomeres Repeated G rich sequence on one strand in humans: (TTAGGG)n
Repeats can be several thousand basepairs long. In humans,
telomeric repeats average 5-15 kilobases
Telomere specific proteins, eg. TRF1 & TRF2 bind to
the repeat sequence and protect the ends
Without these proteins, telomeres are acted upon by DNA
repair pathways leading to chromosomal fusions

51. What do telomeres do? They protect the chromosomes.
They separate one chromosome from another in the DNA sequence
Without telomeres, the ends of the chromosomes would be "repaired", leading to chromosome fusion and massive genomic instability.

52. Telomere function, cont’.
Telomeres are also thought to be the "clock" that regulates how many times an individual cell can divide. Telomeric sequences shorten each time the DNA replicates.

54. Telomerase
Telomerase binds to the telomer and the internal RNA component aligns with the existing telomer repeats.
2. Telomerase synthesizes new repeats using its own RNA component as a template
3. Telomerase repositions itself on the chromosome and the RNA template hybridizes with the DNA once more.

55.  and Primase

56. Structure of Telomeres
Elongated strand of telomere repeats are rich in guanine nucleotides
5’-TTTTGGGGTTTTGGGGTTTTGGGG-3’
They have the capacity to hydrogen bond to one another in the form of G-quartets.
These create three-dimensional structures.

57. How Does Telomerase Work?
Telomerase works by adding back telomeric DNA to the ends of chromosomes, thus compensating for the loss of telomeres that normally occurs as cells divide.
Most normal cells do not have this enzyme and thus they lose telomeres with each division.

58. How Does Telomerase Work?
In humans, telomerase is active in germ cells, in vitro immortalized cells, the vast majority of cancer cells and, possibly, in some stem cells.

59. How Does Telomerase Work?
Research also shows that the counter that controls the wasting away of the telomere can be "turned on" and "turned off". The control button appears to be an enzyme called telomerase which can rejuvenate the telomere and allow the cell to divide endlessly. Most cells of the body contain telomerase but it is in the "off" position so that the cell is mortal and eventually dies.

60. How Does Telomerase Work?
Some cells are immortal because their telomerase is switched on
Examples of immortal cells: blood cells and cancer cells
Cancer cells do not age because they produce telomerase, which keeps the telomere intact.

61. Telomerase and Cancer
There is experimental evidence from hundreds of independent laboratories that telomerase activity is present in almost all human tumors but not in tissues adjacent to the tumors.

62. Telomerase and Cancer
Thus, clinical telomerase research is currently focused on the development of methods for the accurate diagnosis of cancer and on novel anti-telomerase cancer therapeutics

63. Experimentation
Many experiments have shown that there is a direct relationship between telomeres and aging, and that telomerase has the ability to prolong life and cell division.