1. DNA REPLICATION & REPAIR Student Edition 9/27/13
Dr. Brad Chazotte
213 Maddox Hall
Pharm. 304 Biochemistry
Fall 2014
Web Site:
Original material only © B. Chazotte

2. GOALS Examine the mechanism of DNA replication.
Learn what steps are involved in replicating DNA.
Examine which enzymes/proteins are involved in DNA replication.
Understand how and why DNA is repaired
Examine the similarities and differences between prokaryotic and eukaryotic DNA replication.
Examine repair mechanisms that insure replication fidelity.

3. PROKARYOTE REPLICATION

4. DNA Replication is Semiconservative The Meselson & Stahl Experiment
Voet, Voet & Pratt 2013 Fig 25.1

5. DNA Replication by DNA Polymerase
DNA is replicated using a single strand as the template.
The template strand is read in the 3’ → 5’ direction
The synthesis of the new (replicated) complementary strand by DNA polymerase occurs only in the 5’ → 3’
The new strand forms a double helix with the template strand that is antiparallel.
Voet, Voet & Pratt 2013 Fig 25.2
3’ OH group of sugar
5’ PO4 group on sugar
Incoming dNTP
Voet, Voet & Pratt 2013 Fig 3.3

6. Replication of the E. Coli Chromosome
“θ structure”
DNA synthesis occurs at a replication fork in the replication eye (θ structure).
There may be one fork (unidirectional replication) or two forks (bidirectional replication.
Found that θ replication in bacteria is almost always bidirectional.
Voet, Voet & Pratt Fig 25.3; & 2008 Fig. 25.4

7. DNA Replication is Semidiscontinuous
DNA synthesis is ALWAYS 5’ →3’ direction.
Leading strand synthesis is ALWAYS 5’→3’ as well and is able to be CONTINUOUS.
Lagging strand synthesis is STILL 5’→3’ direction but must be DISCONTINUOUS. Lagging strand synthesis proceeds with 1K to 2K size fragments called Okazaki fragments that are subsequently linked together by another enzyme, DNA ligase.
Voet, Voet & Pratt 2013 Fig 25.4

8. DNA Replication Requires an RNA Primer
DNA polymerase requires a free 3’OH on the sugar moiety.
RNA primers synthesized complementary to template strand provide a free 3’OH group for the DNA polymerase.
In E. Coli PRIMASE synthesizes the RNA primers 1-60 nucleotides long (primer length is species dependent).
RNA primer is replaced by DNA later.
The leading strand needs one primer the lagging strand needs multiple primers (for each Okazaki fragment).
Voet, Voet & Pratt 2013 Fig 24.5

9. Major Steps of Prokaryote Replication
Initiation
Find the designated replication initiation site
Separate the strands
Unwind the DNA
Prevent the single strands from reannealing
Synthesis (Elongation)
Replisome (has 2 polymerases) moves along the template strands and synthesizes DNA. DNA Polymerase III is E. Coli’s replicase.
Check fidelity of replication (proofreading)
Joining of Okazaki fragments on the lagging strand (DNA ligase) after removing RNA primer
Termination
Replication terminus has a number of terminator sites

10. Sequences in E. Coli Replication Origin “oriC”
Enzymes need to “know” where to start replication process.
Want a start site that has familiar features “consensus” that a protein and/or enzyme can recognize plus certain chemical/physical properties.
Note that the sequence on the left is AT-rich.
Lehninger (Nelson & Cox) 2005 Fig 25.11

11. Initiation of Replication
In E. Coli replication begins at a region know as oriC – highly conserved sequence in gram(-) bacteria.
Multiple copies of protein DnaA bind to oriC. Cause ~45 bp AT-rich sequence (13 bp repeats) to separate into single strands. (“melting”).
Hu histone-like protein binds and stimulates initiation
Helicase enzyme DnaB recruited by DnaA to oriC. One DnaB binds to each melted strand to further separate the strands. (Helicases act to unwind DNA by traveling along one strand of the double helix.)
Single Strand binding protein (SSB) electrostatically binds to DNA behind the advancing helicase to prevent reannealing of the strands.
DNA Gyrase (a topoisomerase II) relieves the stress on DNA molecule due to helicase driven unwinding
Lehninger (Nelson & Cox) 2005 Fig 25.12

12. DNA Synthesis (Elongation)
Basic Concept
Involves two related, yet distinct, operations: Lagging and Leading strand syntheses.
Accomplished via the replisome, a single multiprotein particle, that includes TWO polymerase III enzymes
Problem? - DNA polymerase synthesis is 5’→3’ and strands are antiparallel. How can you synthesize the lagging strand?
Berg, Tymoczko & Stryer 2012 Fig 28.9

13. PRIMING for DNA Synthesis
Primosome – a protein assembly that creates a RNA primer complementary to the template strand so DNA polymerase can synthesize DNA.
In E. Coli the primosome includes the DnaB helicase and DnaG an RNA-synthesizing primase.
Primosome moves in 5’→3’ direction along the LAGGING strand, i.e. towards the replication fork, while displacing SSB.
The primosome must initiate EACH Okazaki fragment on the lagging strand. (The leading strand needs only one primer.)

14. Okazaki Fragment Syntheses the Lagging Strand
DNA Topoisomerase relieves strain while unwinding DNA. Type II cuts both strands and then reseals.
Helicase separates dsDNA. Binds at initiator site and uses energy to separate strands
SSB keep single strands separate
Primase synthesizes RNA primer
DNA polymerase III synthesizes DNA
Has to add to a free 3’ OH group
Lehninger (Nelson & Cox) 2005 Fig 25.13

15. E. Coli DNA Polymerase I Klenow Fragment with helical dsDNA
Polymeri-zation adds to 3’OH
Voet, Voet & Pratt 2013 Fig 25.9
Voet, Voet & Pratt 2006 Fig 24.10

16. Comparison of E. Coli DNA Polymerases
Main DNA Synthesis
Replaces RNA primers
Voet, Voet & Pratt 2013 Table 25.1

17. β-Clamp of E. Coli DNA Polymerase III around a DNA Molecule and Processivity
Processivity: the number of consecutive residues an enzyme can synthesize.
The β-clamp of the polymerase III holoenzyme keeps the enzyme synthesizing for >5,000 residues versus 12 in its absence for the Polymerase III core enzyme.
DNA molecule
Voet, Voet & Pratt 2008 Fig 24.16b

18. Leading & Lagging Strand DNA Synthesis I
helicase .
Topoisomerase II not shown
Lehninger (Nelson & Cox) 2005 Fig 25.14a

19. Leading & Lagging Strand DNA Synthesis II
Lehninger (Nelson & Cox) 2005 Fig 25.14b

20. Leading & Lagging Strand DNA Synthesis III
Lehninger (Nelson & Cox) 2005 Fig 25.14c

21. Leading & Lagging Strand DNA Synthesis IV
Lehninger (Nelson & Cox) 2005 Fig 25.14d

22. Leading & Lagging Strand DNA Synthesis V
Lehninger (Nelson & Cox) 2005 Fig 25.14e

23. Simplified Schematic Overview of the Overall DNA Replication Process
The helicase unwinds the two parental strands
SSB proteins prevent the parental strands from rejoining
The primase puts on an RNA primer; multiple primers on the lagging strand on the right.
DNA polymerase IIIs on the replicase complex synthesize the leading and lagging strands
DNA polymerase I on the lagging strand removes the RNA primer and replaces it with DNA
DNA ligase seals the nick left by the DNA polymerase I on the lagging strand.
Berg, Tymoczko & Stryer 2002 Fig 27.32

24. E. Coli Chromosome with Termination Sites
Replication terminus - A 350 kb region flanked by 7 nearly identical nonpalindromic terminator sites ~25 bp each.
Termination sites act as one way valves – insures bidirectional replication forks will meet in replication terminus
Counter clockwise
TUS protein specifically binds to a Ter site and prevents strand separation by Dna B helicase
clockwise
Voet, Voet & Pratt 2013 Fig 25.19

25. Accurate Replication is Important
Cells maintain a balance of dNTP’s
Polymerase itself uses a two stage process. dNTP complementary binding followed by polymerization
3’→5’ polymerase (I & III) exonuclease activity detects and removes occasional errors.
Other cellular repair mechanisms for errors in new DNA or later damage to DNA exist.
Using an RNA primer that is later replaced with DNA reduces errors that can arise when the amount of bases for cooperative base-pairing is low.

26. EUKARYOTIC REPLICATION

27. Eukaryotic DNA Polymerases - Properties
PCNA proliferating cell nuclear antigen – a sliding clamp protein
Polymerases and names are different in eukaryotes.
Voet, Voet & Pratt 2008 Table 25.2

28. Eukaryotic Initiation & Elongation
Multiple origins in eukaryotic chromosomes, but each replicon is replicated only once during a cell cycle
Eukaryotic DNA is packaged in nucleosomes (more complex chromosome structure) – histones disassemble immediately ahead of replication fork then reassociate with daughter duplexes
STEPS:
Helicase needed to prepare DNA for replication
Separated strands are coated with protein to prevent reassociation replication protein A (viz. SSB).
Along with accessory proteins DNA Pol α/Primase start new strand.
Pol δ replace Pol α and extends the strand until another replication fork is met (no termination sequence viz. Ter). [

29. Eukaryotic Primer Removal
Two enzymes involved in primer removal:
RNase H1
Flap endonuclease-1 (FEN-1)
Voet, Voet & Pratt Fig 25.23

30. Avoiding a Bad End: Telomeres and Telomerase
Problem: DNA polymerase cannot synthesize the extreme 5’ end of lagging strand. Could result in shorter chromosome each replication cycle
Solution: Special enzyme, telomerase, to synthesize DNA at end of chromosome, the region called a telomere.
Telomeric DNA – tandem repeats of short G-rich sequence on 3’ ending strand of each chromosome end.
Voet, Voet & Pratt 2013 Fig 25.25

31. Synthesizing Telomeric DNA
Telomerase – a ribonuclear protein
Telomerase contains an RNA sequence complementary to the repeating DNA sequence (i.e. its own template)
Repeatedly translocates to the new 3’ end of the DNA – adds multiple telomeric sequences.
Normal cellular “machinery” for lagging strand synthesizes the complementary DNA strand to the telomeric sequence leaving a 3’ overhang on the G-rich strand
Voet, Voet & Pratt 2013 Fig 25.26

32. Extension of Chromosome Ends by Telomerase and Polymerase
a) Telomerase hybridizes with G-rich 3’ end of telomere strand.
b) Adds TTG (complmentary to AAC on telomerase) to 3’ end of daughter strand
c) Then add GGGTTG sequence using RNA template to 3’ end.
{Steps a – c can repeat many times}
d) When 3’ strand much longer a RNA primer is synthesized by primase complementary to G-rich strand
e) DNA pol. Uses primer to fill remaining gap in C-rich strand
f) Primer removed leaving nt G-rich strand 3’ overhang
Weaver 2005 Fig 21.34

33. NEED FOR DNA REPAIR
Undesirable changes to DNA if not fixed can become part of the permanent genome.
Mutation – a heritable alteration of the genetic information. Notable when they occur in germ-line cells of multicellular organism
Changes result from:
Point mutations
Transition: a purine (or a pyrimidine) replaced by another
Transversion: a purine replaced by a pyrimidine or vice versa
Insertion/Deletion mutations: one or more nucleotide pairs are inserted or deleted.

34. Types of Repair Direct Reversal
There are several enzymes that recognize and repair several types of DNA damage – DNA photolyases for pyrimidine dimers. Alkyltransferases for base methylation.
Base Excision Repair (BER) – remove bases that cannot be directly repaired, e.g. by DNA glycosylases.
Nucleotide Excision Repair (NER) – correct pyrimidine dimers and other DNA lesions where bases are displaced from their normal position or have bulk substituents.
Mismatch Repair (MMR) - correct replication mispairing and insertions or deletions up to 4 nucleotides. Bad MMR system then higher incidence of cancer!

35. DNA Repair by Base-Excision Repair Pathway
A glycosylase cleaves the glycosidic bond of corresponding type of altered nucleotide removing the base
An AP endonuclease removes the apurinic or apyrimidinic site.
DNA Pol I – synthesizes replacement DNA
DNA ligase seals nick
Voet, Voet, &Pratt Fig 25.33
Lehninger (Nelson & Cox) 2005 Fig 25.23

36. Nucleotide Excision Repair of Pyrimidine Dimers
Repair prompted by distortion of the helix structure. Pathway similar in all organisms.
In E. Coli.
ATP-dependent process cleaves 11nt sequence.
Cleaved nt displaced by UvrD (Helicase II)
Pol I and DNA ligase repair
Voet, Voet & Pratt 2013 Fig 25.35

37. Mismatch Repair (MMR) - E. Coli
MutS binds to mismatched pair or unpaired base.
MutS dimer then binds MutL.
MutS2MutL2 complex translocates along DNA in both directions to form DNA loop.
Parent strand distinguishable due to existing methylation that occurs later to new daughter strand.
MutH recruited and nicks strand. UvrD helicase separates strands.
DNA polymerase III replaces daughter strand sequence.
Voet, Voet & Pratt 2013 Fig 25.36

38. DNA Methylation
Voet, Voet & Pratt 2013 Box 25.4

39. End of Lectures