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

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

2 1 3

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

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

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??)

Holds DNA Active site (2 metal ions)

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

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

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

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

Polymerases undergo conformational changes  3. Shape selectivity

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

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

thermodynamically uphill rxn nucleophilic attack but no leaving group

Mechanism of DNA ligase

Bacterial helicase (PcrA) ssDNA binding ATP binding and hydrolysis

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

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

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!)

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

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

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

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

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

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

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

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

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

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?)

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

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

!!! Topo I or 2?

Bi-directional DNA synthesis

Cell cycle Cyclins Cyclin-dependent protein kinase (CDK)

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

!!! Chromosome shortening after each round of DNA replication

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

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

High levels of telomerase in dividing cells tumor and aging

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