Enzymology of Replication 2.pptvhhvhfhfhf

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1. Section 2
Enzymology
of DNA Replication

2. Enzymes and protein factors
protein M
r # function
DnaA protein 50,000 1recognize origin
DnaB protein 300,000 6open dsDNA
DnaC protein 29,000 1assist DnaB binding
DNA pol Elongate the DNA
strands
DnaG protein 60,000 1synthesize RNA primer
SSB 75,600 4single-strand binding
DNA topoisomerase 400,000 4release supercoil
constraint
2

3. •The first DNA-
dependent DNA
polymerase(short for
DNA-pol I) was
discovered in 1958 by
Arthur Kornberg who
received Nobel Prize in
physiology or medicine
in 1959.
§2.1 DNA Polymerase
DNA-pol of prokaryotes
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4. •Later, DNA-pol IIand DNA-pol IIIwere
identified in experiments using
mutated E.colicell line.
•All of them possess the following
biological activity.
1. 53polymerizing
2. exonuclease
4

5. DNA-pol of E. coli
5

6. DNA-pol I
•Mainly
responsible for
proofreading
and filling the
gaps, repairing
DNA damage
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7. DNA-pol II
•Temporary functional when DNA-pol I
and DNA-pol III are not functional
•Still capable for doing synthesis on
the damaged template
•Participating in DNA repairing
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8. DNA-pol III
•A heterodimer enzyme composed of
ten different subunits
•Having the highestpolymerization
activity (10
5
nt/min)
•The true enzyme responsible for the
elongationprocess
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9. Structure of DNA-pol III
α：has5´→3´
polymerizing activity
ε：has3´→5´
exonuclease activity
and plays a key role to
ensure the replication
fidelity.
θ: maintain
heterodimer structure
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10. 10

11. 11

12. DNA-pol of eukaryotes
DNA-pol :elongation DNA-pol III
DNA-pol :initiate replication
and synthesize primers
DnaG,
primase
DNA-pol :replication with
low fidelity
DNA-pol :polymerization in
mitochondria
DNA-pol :proofreading and
filling gap
DNA-pol I
repairing
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13. §2.2 Primase
•Also called DnaG
•Primaseis ableto synthesize primers
using free NTPsas the substrate and
the ssDNAas the template.
•Primers are short RNA fragments of a
several decades of nucleotides long.
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14. 14

15. •Primers provide free 3´-OH groupsto
react with the -P atom of dNTP to
form phosphoester bonds.
•Primase, DnaB, DnaC and an origin
form a primosomecomplexat the
initiation phase.
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16. §2.3 Helicase
•Also referred to as DnaB.
•It opens the double strand DNAwith
consuming ATP.
•The opening process with the
assistance of DnaA and DnaC
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17. §2.4 SSB protein
•Stand for single strand DNA binding
protein
•SSB protein maintains the DNA
templatein the single strand form in
order to
•prevent the dsDNA formation;
•protect the vulnerable ssDNA from
nucleases.
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18. §2.5 Topoisomerase
•Opening the dsDNA will create
supercoil ahead of replication forks.
•The supercoil constraint needs to be
released by topoisomerases.
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19. 19

20. •The interconversion of topoisomers
of dsDNA is catalyzed by a
topoisomerase in a three-step
process:
•Cleavage of one or both strands
of DNA
•Passage of a segment of DNA
through this break
•Resealing of the DNA break
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21. •Also called -proteinin prokaryotes.
•It cutsa phosphoester bond on one
DNA strand, rotates the broken DNA
freely around the other strand to relax
the constraint,and reseals the cut.
Topoisomerase I (topo I)
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22. •It is named gyrasein prokaryotes.
•It cutsphosphoester bonds on both
strandsof dsDNA, releases the
supercoil constraint, and reforms the
phosphoester bonds.
•It can change dsDNA into the
negative supercoilstate with
consumption of ATP.
Topoisomerase II (topo II)
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23. 23

24. 3'
5'
5'
3' RNAase
POH
3'
5'
5'
3' DNApolymerase
P
3'
5'
5'
3'
dNTP DNAligase
3'
5'
5'
3'
ATP §2.6 DNA Ligase
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25. •Connect two adjacent ssDNA strands
by joining the 3´-OHof one DNA
strand to the 5´-Pof another DNA
strand.
•Sealing the nick in the process of
replication, repairing, recombination,
and splicing.
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26. §2.7 Replication Fidelity
•Replication based on the principle of
base pairing is crucial to the high
accuracyof the genetic information
transfer.
•Enzymes use two mechanisms to
ensure the replication fidelity.
–Proofreading and real-time correction
–Base selection
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27. •DNA-pol I has the function to correct
the mismatched nucleotides.
•It identifiesthe mismatched
nucleotide, removesit using the 3´-
5´exonuclease activity,adda correct
base, and continuesthe replication.
Proofreading and correction
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28. 3´→5´
exonuclease
activity
excise mismatched
nuleotides
5´→3´
exonuclease
activity
cut primer or
excise mutated
segmentC T T C A G G A
G A A G T C C G G C G
5' 3'
3' 5'
Exonuclease functions
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