1. DNA replication and repair
Chemistry 256

2. DNA replicates semi-conservatively
Means that each strand of the “parent” molecule is replicated and paired with the newly-synthesized “daughter” complementary strand.
Meselson, M. & Stahl, F. W. (Caltech), “The replication of DNA in Escherichia coli”, Proc. Natl. Acad. Sci. USA, 1958.

3. DNA polymerase I (DNA Pol I)
Arthur Kornberg and group (Washington University) discovered an enzyme in E. coli that seemed to synthesize DNA given a template strand and plenty of dNTPs. Manuscript submitted to JBC in late 1957; rejected with reviewer’s comment: “It is very doubtful that the authors are entitled to speak of the enzymatic synthesis of DNA.”
Fortunately: Bessman, Lehman, Simms, and Kornberg, “Enzymatic Synthesis of Deoxyribonucleic Acid: II. GENERAL PROPERTIES OF THE REACTION”, J. Biol. Chem. (1958)
Kornberg and Ochoa win the Nobel Prize in Physiology or Medicine for this discovery in 1959.

4. DNA polymerases replicate
single-stranded DNA is the template; deoxynucleosides are the substrate
nucleophilic attack by 3’ oxygen on the α-phosphate is the mechanism; PPi hydrolysis is the energy source

5. Replication structures
Replication extends growing complementary strand from 5’ to 3’.
Replication eyes (= theta structures) form where the parent strands separate and daughter strands are attached. Replication fork is where synthesis of complementary strand is occurring.

6. Is replication uni- or bi-directional?
Bi-directional seems more efficient, but one molecule does both strands, so uni-directional.
“Leading” strand is a normal 5’ to 3’ synthesis but the “lagging” strand is replicated in short (100 to 200 base) strands discontinuously. Reiji Okazaki (Nagoya University, Japan) and others, “Mechanism of DNA replication possible discontinuity of DNA chain growth”, Japanese Journal of Medical Science & Biology (1967)

7. Okazaki fragments

8. How to initiate Okazaki fragment growth?
The problem: since this is on the lagging strand, each fragment has to start with a free 5’ end – oddly enough, RNA bases are used to synthesize the first 1 to 60 nucleotides of the fragment, then switch over to dNTPs.
Later, the RNA must be excised and replaced by DNA, then the nick between the fragments sealed.
The enzyme primase synthesizes the RNA “primer”, then the polymerase takes over.

9. endonuclease = hydrolyze within strand
exonuclease = hydrolyze from the end
Note the directionality of this exonuclease – backwards! So-called “proofreading” function.
Figure 25-7

10. This function only occurs where there are nicks; thus, this is perfect for removing the RNA primer on the lagging strand.
Figure 25-8

11. Nick translation
Pol I polymerizes the complement to the lagging strand, the only strand that has the RNA nucleotides added on to the Okazaki fragments.
Uses its 5’  3’ exonuclease function to “chew off” the RNA primer, then uses its polymerase function to synthesize the same bases with dNTPs, translating the nick toward the 3’ end, eventually linking the fragments, using DNA ligase.

12. DNA Pol I Klenow fragment has a distinct structure that is responsible for the nuclease activity
Thomas Steitz (Yale University) and others crystallize the active site of DNA Pol I, including a short segment of template DNA and its complementary primer strand. “Structural Studies of Klenow Fragment: An Enzyme with Two Active Sites”, Cold Spring Harb Symp Quant Biol (1987)

13. DNA Pol I Klenow fragment has a distinct structure
This approximately 600-residue (“large”)fragment contains both the polymerase and the 3’  5’ exonuclease functions.
The DNA enters this fragment at the central cleft, which is lined with positively-charged residues that attract the negatively-charged sugar-phosphate DNA backbone.

14. The action of the Klenow fragment
Since the polymerase (cleft) part of the Klenow fragment is binding the sugar phosphate DNA backbone, it is not specific for a particular DNA sequence.
So how does the enzyme know how to generate the complementary strand?

15. The action of the polymerase
In the Klenow fragment, as the B-DNA (normal) enters the cleft, the three base pairs near the active site switch to the A-DNA conformation; the shallower grooves of this conformation allow access to the bases of those residues.
The enzyme also changes conformation from open (b) to closed (a) only when the correct nucleotide is chosen for the primer strand; this implies the dNTPs are sampled rapidly.

16. Metal ions orient the polymerization
A and B on the diagram are metal ions, usually Mg2+.
A’s function is to pull away the oxygen lone pairs on the 3’ oxygen of the end of the primer strand and allow the nucleophilic attack on the next nucleotide.
B’s function is to stabilize the pyrophosphate group that will be hydrolyzed.

17. Prokaryotes have 3 types of DNA Pol
The synthesis of the leading strand is done by DNA Pol III (called “replicase”). It can’t do nick translation on the lagging strand because it lacks the 5’ to 3’ exonuclease function.
Table 25-1

18. Prokaryote replication protocol
Locate the replication initiation site
Unwind the DNA to separate strands
Prevent single strands from reannealing prior to replication.
Convention: italicized letter sequence (such as oriC) represents a section of DNA; non-italicized letter sequence (such as DnaA) represents a protein.

19. oriC is the initiation site of E. coli replication

20. Helicase DnaB unwinds DNA
DnaB threads the lagging strand in its central void.
Two DnaB are used, one at each end of the melted AT region.
Both DnaB move in a 5’ to 3’ direction.
Uses dNTP hydrolysis to energize the movement, which allows the helicase to change to the open conformation, move and close further 3’-ward.

21. SSB protein prevents reannealing
Single-strand binding (SSB) protein has a cleft that only accommodates ssDNA (not dsDNA).
Also prevents stem loops from forming and nucleases from binding.

22. E. coli DNA replication cartoon
A primosome is a enzyme complex that includes DnaB and RNA-replicating primase (DnaG). Moves in a 5’ to 3’ direction, displacing SSBs along the way.
Primosomes are needed for lagging strand too.

23. E. coli DNA replication cartoon
Two DNA Pol III enzymes (in a replisome) process both strands simultaneously.
For lagging strand to keep up, it has to loop around the complex.
DNA ligase seals the nicks between Okazaki fragments.

24. Processivity of DNA Pol III is enhanced by a “sliding clamp”
DNA Pol III processes 12 nt at a time but the holoenzyme has a processivity of 5000 nt due to a “β clamp” that clamps around the DNA strand and keeps the rest of the holoenzyme (including Pol III) from diffusing away.

25. DNA ligase is initiated by NAD+
NAD+ reacts with a lysine amide to yield a phosphoamide adduct (while losing a nicotine nucleotide). The adduct has an AMP group.
The adduct adds to the free 3’ phosphate on a fragment and this allows the initiation of a nucleophilic attack by the 5’ hydroxy group, which completes the phosphoester linkage.

26. Termination of replication
E. coli contains 7 nearly identical termination sequences called Ter.
3 have one orientation and 4 have the other to make sure both DNA Pols terminate replication – more evidence for bidirectional replication.

27. Tus protein binds to Ter
Mechanism of termination is for the Tus protein to bind to the Ter sequence and prevent the helicase from separating the strands.

28. DNA replication in E. coli is very accurate
Error rate of mispairing of 1 in 108 to 1010 base pairs.
dNTPs are present during replication in nearly equal concentrations.
Polymerase reaction relies on protein conformation change to proceed.
Proofreading function is effective.
DNA sequence is being constantly repaired.

29. Eurkaroytic DNA replication follows similar but more complex mechanisms
Animal cells have 13 different DNA polymerases.
Three main ones: pol α, pol δ and pol ε.

30. Replicase is pol δ
Needs to work in conjunction with “sliding clamp” proliferating cell nuclear antigen (PCNA).
Processivity of pol δ is infinite (does the whole length of template DNA by itself). Pol δ works with pol α to replicate lagging strand; pol ε seems to work on leading strand.
DNA pol γ works to replicate mitochondrial DNA; similar enzyme works on chloroplast DNA.

31. Other similarities Helicase = MCM SSB = RPA
No Pol I exonuclease/polymerase; function fulfilled by Rnase H1 and flap endonuclease-1.

32. Replication starts at multiple origins
Replication fork movement is 10 times slower in eukaryotic DNA compared to prokaryotic.
Initiation sequence is ARS = autonomously replicating sequences. Nucleosomes do not appear to inhibit replication.
Figure 25-22

33. The nagging problem of the 5’ end
The extreme 5’ end of the lagging strand cannot be replicated because polymerases will not recognize this end of the DNA.
So does genomic DNA shorten by a primer length every replication?

34. Solution: telomeres Telomeres occur at the 3’ ends of DNA.
They are sequences rich in G residues.
When replicated, telomeric RNA is added.
Telomerase is a ribonucleoprotein; RNA part is complementary to the telomeric sequence.

35. Telomeres form stabilizing structures
G base can make base “pairing” in the manner shown below: the G-quartet.
The G-quartet may in turn be recoginzed by “capping” proteins.
Since the loss of telomeres can result in replication removing critical genomic material, which is implicated in cell senescence, can restoring telomeres decrease aging effects? Not clear.

36. Mutations = DNA damage Hugo DeVries (1890) – genes “mutate”
Thomas Hunt Morgan (1910; Nobel 1933) – fruit fly mutants related to genes
Herman Muller (1920) – mutations caused by X-rays
How do mutations begin?
Ultraviolet light will cause a cyclobutyl ring to form in thymine or cytosine; these are called pyrimidine dimers.

37. Other mutations involve chemical reactions
Oxidative deamination with nitrous acid – whole new bases (not the standard ones) are created.
Classify mutations under one of the headings: “Point mutation” or “insertion/deletion mutation.”

38. DNA repair In bacteria, DNA photolyases break up pyrimidine dimers.
The “flat hole” in the center of the model shown is the positively-charged active site which attracts the pyrimidine base.

39. Base excision repairs
A damaged base (no longer capable of proper hydrogen bonding) may be removed by a DNA glycosylase, which cleaves the glycosidic bond that attaches the base to the sugar.
Later, the deoxyribose is removed and an exonuclease and ligase complete the repair.
Uracil-DNA glycosylase is effective at removing stray U’s from DNA sequences.

40. Nucleotide excision repair
If a base pair mismatch results in a distortion of the double helix, a large section of the misshapen strand may be removed and replaced.
In humans, tobacco smoke or UV damage repaired this way.

41. Mismatch repair system
May fix insertion or deletions of up to 4 nt.
Occurs soon after replication, so fixes errors that slipped by the proofreading.
Prokaryotes signal an repair needed by methylating a base at the error; eukaryotes don’t do this, so may search for unsealed nicks in daughter strand.

42. Repairs of last resort
DNA Pol η has an error rate of 1 in 30 bp and no proofreading ability; however, it can bypass errors such as pyrimidine dimers and keep copying.
SOS response in prokaryotes activates DNA Pol IV and V which have high error rates and no proofreading; however, they can replicate in the absence of a template strand – lots of errors and few cells survive this. But some cells surviving is better than none!

43. Recombination of DNA allows genetic diversity
Homologous recombination allows similar DNA segments to mix.
The homologous segments must line up and each strand must be nicked (not cut).

44. Homologous recombination
Eventually, a four-stranded structure called a Holliday junction is created, and then separated into two sets of two strands, now with new segments incorporated.

45. DNA can be repaired through recombination
By forming two Holliday junctions, broken dsDNA can be rejoined by homologous end-joining.

46. Transposition can rearrange DNA sequences
Studying maize, Barbara McClintock (Cold Spring Harbor) found transposable elements (“transposons”) that moved bits of sequence around. “Mutable loci in maize”, Carnegie Institution of Washington Yearbook (1951).

47. In bacteria and plasmids, transposons are “insertion sequences”
The sequence comprises a transposase gene and maybe a regulatory gene, which simply allow for more copies of this sequence to be made and moved.

48. More complex transposons
Some transposons also carry genes not related to the transposition process. Antibiotic resistance in bacteria (below, β-lactamase disables the antibiotic ampicillin) is distributed this way.

49. Most complex transposons
Composite transposons are made of two insertion sequences and can move as much DNA as fits between them.