1. DNA Replication
AHMP 5406

2. Objectives: Outline the mechanisms of eukaryotic DNA replication
Describe the cellular mechanisms that help avoid error generation during DNA synthesis
Describe the possible pathways of DNA repair
Relate chromatin density and the cell cycle to DNA replication

3. DNA Replication
The process of copying DS DNA by templated polymerization
In Eukaryotes occurs only during S phase
Overall replication scheme similar to prokaryotes

4. DNA Replication
Base pairing is responsible for DNA replication and repair
Multiple initiation points
Linear chromosome (Proks. circular)
Many polymerases and accessory factors required

5. Chromosome Size and Topology
Species
Genome Size (bp)
Haploid Chromosome Number
Chromosome Size (bp)
Chromosome Shape
Escherichia coli
5 x 106 1
circular
Saccharomyces cerevisiae
1.4 x 107 16
8.8 x 105
linear
Homo sapiens
3 x 109 23
1.3 x 108

6. DNA replication is semi-conservative
During one round of replication
One strand used as template

7. Repl. begins at specific chromosomal sites
Replication origins
Regardless of organism are:
unique DNA segments with multiple short repeats
recognized by multimeric origin-binding proteins
usually contain an A-T rich stretch

8. Most DNA replication is bidirectional

9. Eukaryotic Chromosome Replication
DNA replication are very similar in proks and euks
Differences:
Euks have many chromosomes
one in prokaryotes
The problem with nucleosomes
euk DNA is “packaged”
wrapped around histones
In eukaryotes DNA and histones must be doubled with each cell division

10. Eukaryotic Replication
DNA synthesis
In eukaryotes
small portion of the cell cycle (S)
continuously in prokaryotes
Eukaryotes have more DNA to replicate
How is this accomplished?
Multiple origins of replication
prokaryotes one origin – OriC
Two different polymerases

11. Problems that must be overcome for DNA polymerase to copy DNA
DNA polymerases can’t melt duplex DNA
Must be separated for copying
DNA polymerases can only elongate a preexisting DNA or RNA strand (the primer)
Strands in the DNA duplex are opposite in chemical polarity
All DNA polymerases catalyze nucleotide addition at 3-hydroxyl end
Strands can grow only in the 5 to 3 direction

12. Structure of DNA Rep. Fork
Both daughter strands polymerized in
5’-3’ direction
Lagging strand DNA synth. in short segments
Okazaki fragments

13. Proteins at the fork form a replication machine
Mammalian replication fork

14. Specialized enzymes Helicases separate two parental DNA strands
Polymerases synthesize primers and DNA
Accessory proteins promote tight binding of enzymes to DNA
Increase polymerase speed and efficiency (sliding clamp)
Editing exonucleases work with polymerases
Topoisomerases convert supercoiled DNA to the relaxed form

15. DNA Helicase Hexameric ring Separate DNA strands
Use ATP hydrolysis for Energy

16. Primase Activated by helicase Synthesizes short RNA primer
Uses DNA as template

17. Sliding clamp Keeps DNA polymerases attached to DNA strand
Assisted by clamp loader through ATP hydrolysis
Will disassociate if DNA pol reaches DS DNA

18. Single stranded binding proteins
Bind tightly and
cooperatively to SS DNA
Do not cover bases
Remain available for templating
Aid in stabilizing unwound
DNA
Prevent hairpin structures

19. Mammalian DNA polymerases
Synthesize new DNA strand
Requires primer
DNA Pol a
Associated with primase
DNA Pol d
Elongates

20. Mammalian DNA Polymerases
a : Repair and Replication and primase function
b: Repair function
g : Mitochondrial DNA polymerase
d : Replication with PCNA (processivity factor)
e : Replication

21. Topoisomerase Some proteins change topology of DNA
Helicase can unwind the DNA duplex
induce formation of supercoils
Topoisomerases catalyze addition or removal of supercoils

22. Topoisomerase
Type I topoisomerase relax DNA by nicking and closing one strand of duplex DNA
Covalently attach to DNA phosphate
Allow rotation

23. Topoisomerase
Type II topoisomerase change DNA topology by breaking and rejoining double stranded DNA

24. Action of E coli Topoisomerase I

25. Type II topoisomerases (gyrases) change DNA topology by breaking and rejoining double-stranded DNA

26. Replicated circular DNA molecules are separated by type II topoisomerases
Linear daughter chromatids also are
separated by type II topoisomerases

27. The eukaryotic replication machinery is generally similar to that of E
The eukaryotic replication machinery is generally similar to that of E. coli

28. More on Telomeres

29. Telomeres
Further evidence of a relationship b/w telomere length and aging in humans
Disorder called progerias (premature aging)
Hutchinson-Gilford Syndrome (severe) – death in the teen years
Werner Syndrome (less severe) – death usually in the 40s

30. Telomere Replication Regions of DNA at each end of a linear chromosome
Required for replication and stability of that chromosome.
Human somatic cells (grown in culture) divide only a limited number of times (20-70 generations)

31. Telomere Replication
Correlation between telomere length and the number of cell divisions preceding senescence and death
Cells with longer telomeres survive longer (more divisions) than cells with short telomeres

32. Problem with Telomeres
DNA polymerase require free 3’OH end
cannot replace the RNA primer
at the terminus of the lagging strand.
If not remedied, the DNA would become shorter and shorter
Telomerase resolves the terminal primer problem

33. Telomerase Telomerase = enzyme made up of both protein and RNA
RNA component is base sequence complementary to telomere repeat unit
Catalyzes synthesis of new DNA using RNA as template

34. End-Replication Problem5 3 5 3 + 5 3
Process Okazaki Fragments 5 3 + 5 3

35. Telomere Structure Telomeres composed of short (6-10 bp) repeats5 3
G-rich
C-rich
Telomeres composed of short (6-10 bp) repeats
G-rich in one strand, C-rich in other

37. Telomerase Germ-line cells possess telomerase activity
Most human somatic cells lack telomerase activity
Cultured immortal cell lines have been shown to have telomerase activity
Possible cancer therapy may be to control telomerase activity in cancer cells