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Berg • Tymoczko • Stryer

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1 Berg • Tymoczko • Stryer
Biochemistry Sixth Edition Chapter 28 DNA Replication, Repair, and Recombination Part II: DNA replication Copyright © 2007 by W. H. Freeman and Company


3 Semi-conservative model of DNA replication
Parental strand serves as “template”





8 2 1 3

9 Problems (because of the double helix):
Antiparallel strands (opposite strands) Double strands Supercoiling, unwinding

10 E. coli DNA polymerase I (Klenow fragment) Polymerase unit
3’->5’ exonuclease unit (proofreading/correction)

11 DNA polymerases catalyze the formation of polynucleotide chains
Template-directed enzyme (base pair-dependent) Catalyzes nucleophilic attack by 3’-OH on the “a” phosphate Requires a primer with free 3’-OH (RNA polymerase??)

12 Holds DNA Active site (2 metal ions)

13 participate in polymerase
Two bound metal ions participate in polymerase Activates 3’-OH Stabilizes (-) charge

14 Is hydrogen bond-base pairing enough for adding the right nucleotide?

15 Fails to form H bonds but
can still direct addition of T  1. Shape complementarity

16 major minor Residues of the enzyme form H bonds with minor
ruler Structural study Residues of the enzyme form H bonds with minor groove side of the base pairs in the active site  2. Minor groove interactions


18 Polymerases undergo conformational changes
 3. Shape selectivity

19 Where does this primer come from?
(5 nt) Removed by hydrolysis


21 Replication fork ~1000 nt Primer Remove and fill in (DNA polymerase I) DNA ligase

22 thermodynamically uphill rxn nucleophilic attack but no leaving group

23 Mechanism of DNA ligase

24 Bacterial helicase (PcrA)
ssDNA binding ATP binding and hydrolysis

25 Separation of strands requires helicase and ATP
Both A1 and B1 bind DNA ATP  closure, A1 releases DNA ATP hydrolysis  open, B1 releases Move in 3’  5’ direction

26 Helicase: large class (5’->3’, RNA, oligomers) Conserved residues among helicase ATP-induced conformational change Hexameric helicase (euk)  ATPase (AAA family)

27 DNA replication must be rapid!
Ex. E. coli: 4.6x106 bp, replicate in <40 mins  2000bp/sec Differences in eukaryotic: Multiple origins Additional enzyme for telomeres Polymerases: catalytic potency, fidelity and processivity (catalysis without releasing substrates) (processive vs. distributive enzymes!)

28 b2 subunit of DNA polymerase III How does DNA get in this?
Keep polymerase associated with DNA  Sliding DNA clamp How does DNA get in this?  clamp loader (requires ATP) 35 Å


30 Topoisomerase II (add – supercoils) (DnaB) (single-strand-binding)


32 DNA polymerase “holoenzyme”
Interacts with SSB 3‘5’ proofreading DNA polymerase “holoenzyme”

33 Add 1000 nt before releasing & new loop DNA pol I Remove primers Fill in

34 Where does replication begin??
In E. coli: a unique site “origin of replication” is called oriC locus

35 DnaAoriC: Preparation for replication Bind to each others’ ATPase domains; Breaking apart when ATP hydrolyzed

36 Preparation for replication
DnaAoriC: Preparation for replication DnaB (hexameric helicase) + DnaC (helicase loader) SSB “Prepriming complex” DnaG (primase) 1 2

37 DNA pol III holoenzyme + Prepriming complex ATP hydrolysis within DnaA
Breakup of DnaA (preventing addition round of replication!) 3

38 Eukaryotic replication, why more complex?
Size of DNA (6 billion bp) Number of chromosome (23 vs. 1) Linear vs. circular

39 Eukaryotic replication, why more complex? Size of DNA
Number of chromosome 30,000 origins! But no defined sequence ORCs (origin of replication complexes) = prepriming complex Replicon: replication unit (how many does E. coli have?)

40 Eukaryotic DNA replication
ORCsorigins Preparation for replication Cdc6, Cdt1  MCM2-7 (licensing factors  formation of initiation complex) Replication protein A (=SSB) Two distinct polymerases: Pol a (initiator) Pol d (replicative) 1 2 3 Polymerase switching

41 Eukaryotic DNA replication
Pol a (initiator): Primase + polymerase (20nt) Replication factor C (RFC): displaces pol a recruits PCNA (b2 of pol III) Replication until replicons meet (Primers, ligase) 3 4 5


43 !!! Topo I or 2?

44 Bi-directional DNA synthesis



47 Cell cycle Cyclins Cyclin-dependent protein kinase (CDK)

48 Eukaryotic replication, why more complex?
Size of DNA (6 billion bp) Number of chromosome (23 vs. 1) Linear vs. circular

49 !!! Chromosome shortening after each round of DNA replication

50 Chromosome ends = telomeres
Telomere-binding protein (AGGGTT)n Loop for protection

51 Telomeres are replicated
by telomerase Telomerase has: RNA template Reverse transcriptase

52 High levels of telomerase
in dividing cells tumor and aging

53 Summary: DNA replication
Mode Enzymology Polymerization steps Eukaryotes vs. prokaryotes Telomeres

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