replication

1. Chapter 5 & 6
DNA & DNA Replication

2. History
 DNA
 Comprised of genes
 In non-dividing cell nucleus
as chromatin
 Protein/DNA complex
 Chromosomes form during
cell division
 Duplicate to yield a full set in
daughter cell

3. DNA is Genetic Material

4. From Chapter 2
 Nucleic acids are polymers
 Monomers are called nucleotides
 Nucleotides = base + sugar + phosphate
 Base = purine or pyrimidine
 Purines = adenine, guanine
 Pyrimidines = thymine, cytosine, uracil
 Sugar = deoxyribose or ribose
 Phosphate, a single phosphate in DNA
 Sugar of nt 1 is linked to the phosphate of nt
2 by a phosphodiester bond

5. Panel 2-6

6. Chapter 2
– cont’d

7. DNA is a Double Helix
 Nucleotides
 A, G, T, C
 Sugar and phosphate
form the backbone
 Bases lie between the
backbone
 Held together by
H-bonds between the
bases
 A-T – 2 H bonds
 G-C – 3 H bonds

8. H - Bonds  Base-pairing rules
 AT only (AU if DNA-
RNA hybrid)
 GC only
 DNA strand has
directionality – one end is
different from the other end
 2 strands are anti-parallel,
run in opposite directions
 Complementarity results
 Important to replication

9. Helical Structure

10. Nucleotides as Language
 We must start to think of the nucleotides –
A, G, C and T as part of a special
language – the language of genes that we
will see translated to the language of
amino acids in proteins

11. Genes as Information Transfer
 A gene is the sequence of nucleotides
within a portion of DNA that codes for a
peptide or a functional RNA
 Sum of all genes = genome

12. DNA Replication
 Semiconservative
 Daughter DNA is a
double helix with 1
parent strand and 1
new strand
 Found that 1 strand
serves as the
template for new
strand

13. DNA Template
 Each strand of the parent DNA is used as a
template to make the new daughter strand
 DNA replication makes 2 new complete double
helices each with 1 old and 1 new strand

14. Replication Origin
 Site where replication
begins
 1 in E. coli
 1,000s in human
 Strands are separated to
allow replication machinery
contact with the DNA
 Many A-T base pairs
because easier to break 2
H-bonds that 3 H-bonds
 Note anti-parallel chains

15. Replication Fork
 Bidirectional movement of the DNA replication machinery

16. DNA Polymerase
 An enzyme that
catalyzes the addition of
a nucleotide to the
growing DNA chain
 Nucleotide enters as a
nucleotide tri-PO4
 3’–OH of sugar attacks
first phosphate of tri-
PO4 bond on the 5’ C of
the new nucleotide
 releasing pyrophosphate
(PPi) + energy

17. DNA Polymerase
 Bidirectional synthesis of the DNA double
helix
 Corrects mistaken base pairings
 Requires an established polymer (small
RNA primer) before addition of more
nucleotides
 Other proteins and enzymes necessary

18. How is DNA Synthesized?
 Original theory
 Begin adding nucleotides at origin
 Add subsequent bases following pairing rules
 Expect both strands to be synthesized simultaneously
 This is NOT how it is accomplished

19. Why DNA
Isn’t
Synthesized
3’5’
Correction: Refer to
Figure 6-15 on page 205
of your textbook for
“corrected” figure. This
figure fails to show the
two terminal phosphate
groups attached on the 5’
end of the nucleotide
strand located at the top
of this figure.

20. How is DNA Synthesized?
 Actually how DNA is synthesized
 Simple addition of nucleotides along one
strand, as expected
 Called the leading strand
 DNA polymerase reads 3’  5’ along the leading
strand from the RNA primer
 Synthesis proceeds 5’  3’ with respect to the
new daughter strand
 Remember how the nucleotides are
added!!!!! 5’  3’

21. How is DNA Synthesized?
 Actually how DNA is synthesized
 Other daughter strand is also synthesized
5’3’ because that is only way that DNA can
be assembled
 However the template is also being read
5’3’
 Compensate for this by feeding the DNA strand
through the polymerase, and primers and make
many short segments that are later joined (ligated)
together
 Called the lagging strand

22. DNA Replication Fork Fig 6-12

23. Mistakes during Replication
 Base pairing rules must be maintained
 Mistake = genome mutation, may have
consequence on daughter cells
 Only correct pairings fit in the polymerase
active site
 If wrong nucleotide is included
 Polymerase uses its proofreading ability to cleave
the phosphodiester bond of improper nucleotide
 Activity 3’  5’
 And then adds correct nucleotide and proceeds
down the chain again in the 5’  3’ direction

24. Proofreading

25. Starting Synthesis
 DNA polymerase can only ADD nucleotides
to a growing polymer
 Another enzyme, primase, synthesizes a
short RNA chain called a primer
 DNA/RNA hybrid for this short stretch
 Base pairing rules followed (BUT A-U)
 Later removed, replaced by DNA and the
backbone is sealed (ligated)

26. Primers – cont’d
 Simple addition of primer
along leading strand
 RNA primer synthesized 5’
 3’, then polymerization
with DNA
 Many primers are needed
along the lagging strand
 1 primer per small
fragment of new DNA
made along the lagging
strand
 Called Okazaki fragments

27. Removal of Primers
 Other enzymes needed to excise
(remove) the primers
 Nuclease – removes the RNA primer
nucleotide by nucleotide
 Repair polymerase – replaces RNA with DNA
 DNA ligase – seals the sugar-phosphate
backbone by creating phosphodiester bond
 Requires Mg2+ and ATP

28. Other Necessary Proteins
 Helicase opens double helix and helps it
uncoil
 Single-strand binding proteins (SSBP) keep
strands separated – large amount of this
protein required
 Sliding clamp
 Subunit of polymerase
 Helps polymerase slide along strand
 All are coordinated with one another to
produce the growing DNA strand (protein
machine)

29. Components of the DNA Replication

30. Polymerase & Proteins Coordinated
 One polymerase complex apparently synthesizes
leading/lagging strands simultaneously
 Even more complicated in eukaryotes

31. DNA Repair
 For the rare mutations occurring during
replication that isn’t caught by DNA
polymerase proofreading
 For mutations occurring with daily
assault
 If no repair
 In germ (sex) cells  inherited diseases
 In somatic (regular) cells  cancer

32. Effect of Mutation

33. Uncorrected Replication Errors
 Mismatch repair
 Enzyme complex recognizes mistake and excises
newly-synthesized strand and fills in the correct
pairing

34. Mismatch Repair – cont’d
 Eukaryotes “label”
the daughter strand
with nicks to
recognize the new
strand
 Separates new from
old

35. Depurination or Deamination
 Depurination – removal of a purine base from
the DNA strand
 Deamination is the removal of an amine group
on Cytosine to yield Uracil
 Could lead to the insertion of Adenine rather than
Guanosine on next round

36. Chemical Modifications

37. Thymine Dimers
 Caused by exposure to UV light
 2 adjacent thymine residues become
covalently linked

38. Repair
Mechanisms
 Different enzymes
recognize, excise
different mistakes
 DNA polymerase
synthesizes proper
strand
 DNA ligase joins new
fragment with the
polymer