DNA Replication I Biochemistry 302 Bob Kelm January 26, 2004

Overview of Information Metabolism • • • •

Replication (copying) Transcription (readout) Translation (decoding) Template-dependent (DNA or RNA) – Initiation (point of regulation, generally) – Chain elongation – Termination

Fig. 4-23

Watson Crick prediction: Each stand of parent DNA serves as a template for synthesis of a new complementary daughter strand

Fig. 4.12

Plausible models of gene replication

with respect to parental duplex DNA

Fig. 4.13

Proof of semiconservative DNA replication

heavy

Meselson-Stahl, 1958 Differential isotopic light labeling of E. coli DNA coupled with density gradient centrifugation to equilibrium hybrid ¾L ¼H

Fig. 4.14

DNA Replication: the early years …What was learn and not learned • Watson and Crick model predicted semiconservative DNA replication. • Meselson-Stahl experiment biochemically confirmed this notion. – Did not answer whether replication was sequential (with DNA unwinding) and ordered (in terms of direction). – Could not answer whether DNA replication was initiated at only one or more fixed points (or origins) on a chromosome.

Biochemical & genetic approaches led to notion of bidirectional replication from a fixed origin •

Fig. 24.14

•

•

Figure shows multiple sequential initiation with solid line radiolabeled.

First studied by whole chromosome radiolabeling experiments. John Cairns visualized nascent E. coli DNA synthesis. Subsequent work utilized denaturation mapping to visualize bidirectional replication of λ phage DNA. Other genetic experiments relied on the copy number of inherited marker genes to establish bidirectionality.

Termination and re-initiation occur on opposite sides of E. coli chromosome

Fig. 24.12

Higher specific activity [3H]thymidine added at end of replication cycle

radioautogram

Basic features of DNA replication • Semi-conservative • Ordered and sequential – Starts at a fixed point. – Synthesis of new daughter strands is in 5′→3′ direction and follows parental DNA unwinding.

• Utilizes “activated” substrates (dNTPs) • Discontinuous – Daughter strands grow in opposite directions to maintain anti-parallel polarity w/ template strand.

• Extremely accurate

Enzymes and other proteins involved in DNA replication (some multi-subunit complexes) • • • • • •

DNA Polymerases ssDNA-binding proteins Helicases Primase Topisomerases DNA Ligase

Basic chemistry of DNA synthesis • Incoming dNTP is positioned by base-pairing with template nucleotide. • DNA Pol catalyzes reaction between the terminal 3′ OH on the primer strand and the 5′ α PO3 of dNTP to form 3′− 5′ PDE bond. • Release and hydrolysis of pyrophosphate drives the reaction energetically. • Consequently, chain growth can only occur in one direction. Fig. 24.2

Requirement for 5 ′ to 3′ synthesis imposes a topological problem • Two strands of DNA duplex are anti-parallel but both parental strands are replicated in the same “fork.” • If fork movement is to occur in a concerted manner, there must be a mechanism for replicating each strand in 3′→ 5′ direction. • Solution: Two DNA polymerase molecules (Pol III, E. coli) one catalyzing forward (leading strand) synthesis and the other backward (lagging strand) synthesis.

Anatomy of a replication fork: leading and lagging strand synthesis Unwinding continuous

Priming 5′

3′

3′

5′ discontinuous Figure from Kornberg and Baker “DNA Replication”

Overall growth of lagging strand pieces (Okazaki fragments) is 3′→5′.

Extending

Editing

Stitching Fig. 24.3

Directionality of DNA synthesis: a very simple model of a replication fork Fork movement primasome RNA primer DNA polymerase III, replicates as leading and lagging strand dimer Leading strand Fig. 24.1

Lagging strand

Lagging strand synthesis requires repeated RNA priming due to physical constraint imposed by Pol III dimerization at the fork.

Chromosomes from different cell types exhibit different replication parameters • • • • • •

Bacterial chromosomes: single origin (E. coli ~4.6 x 106 bp) Eukaryotes: multiple origins on a single chromosome (103104 total /cell) Replication in bacteria is 10-fold faster than in eukaryotes: 850 nt/sec/fork vs 60-90 nt/sec/fork Doubling time of bacteria ∼20-30 minutes vs 24 hours in a “typical” eukaryotic cell (HeLa) E. coli can completely replicate its genome in 40 min, 8 hours for a HeLa cell (S phase of interphase) Bacteria re-initiates more frequently than the doubling time – Daughter cell receives a chromosome well into its next round of replication to adapt to rapid growth. – “Replicon firing” earlier in the cell cycle implicates initiation as the key control point in DNA replication.

Basic features of E. coli DNA replication continued…. • Replication is an enzymatic reaction. – Substrates: dNTPs (also need primer-template) – Enzymes: Polymerases (Pol III: rapid synthesis and Pol I: editing) along with topoisomerases, helicases, primase, and DNA ligase

• Three main phases: – Initiation: formation of specific protein:DNA complex at specific origins (ori): highly regulated – Elongation: the actual copying process by polymerase and other machinery – Termination: separation of daughter chromosomes at Ter sites by Tus protein

DNA Pol I: the first polymerase (discovered by A. Kornberg, 1950s Nobel prize 1959) • Three activities: – polymerization – 3′ to 5′ exonucleolytic proofreading & repair – 5′ to 3′ exonuclease (“nick translation”, RNA primer removal)

• Basic polymerase reaction – (dNMP)n + dNTP → (dNMP)n+1 + PPi

• Pol I is not processive enough for genome replication

Kornberg & Baker

DNA Pol I structure: 103 kDa peptide. Limited tryptic digestion → small N-term 35 kDa fragment and larger C-term 68 kDa fragment (Klenow, used for DNA synthesis in vitro)

DNA Polymerase I 5′→3′ exo activity: Nick Translation − concerted NMP removal from 5′ end of RNA primer & dNMP addition to 3′ end of Okazaki fragment

Fig. 24.5

Closed right hand structure common among Klenow family of DNA Pols (active site lies in palm domain)

Fig.24.17

Bacteriophage RB69, a Doublie et al. (1998) Nature 391:251 cousin of phageT4, 1st one Bacteriophage T7 DNA Pol

Molecular basis of primer-template recognition and proofreading Doublie et al. (1998) Nature 391:251

N3

O2

floor of active site Arg429 and Gln615 are strictly conserved.

Extensive contacts between the T7 DNA Pol enzyme and DNA minor groove explains Pol DNA-binding w/o sequence specificity, detection of base misincorporation. O2 of Pyr and N3 of Pur are universal Hbond acceptors of the minor groove. Aform helix has wider minor groove.

DNA polymerase active site opens and closes

530

530

Doublie et al. (1998) Nature 391:251

O Helix = dynamic finger subdomain

Two metal ion mechanism of catalysis by DNA polymerases (T. Steitz model) T7 DNA Pol

E. coli Klenow

3′

5′

water Doublie et al. Nature 1998

Fig. 24.18

Metal ions stabilize a multivalent transition state between phosphate oxygens and two conserved Asp residues which 1) polarizes the primer 3′ OH and 2) facilitates the leaving of pyrophosphate.

Lesson learned from structural analyses • Structural similarities among DNA polymerases – Klenow family including RB69 and T7 DNA Pol – Pol β family: palm, fingers, and thumb but different fold – Common evolutionary origin and mechanism

• High resolution T7 structure with bound ddGTP – Multiple contacts between enzyme and DNA minor groove – DNA near primer-template terminus is A-form facilitating minor groove interactions. – Incoming dNTP fits into a pocket or slot favoring correct base-pairing. – Position of bound metal ions support a catalytic model proposed by T. Steitz based on Klenow fragment structure.

Summary of DNA Replication I • DNA is replicated by a semi-conservative mechanism in a fixed 5→3 direction and with a defined chemistry. • The initiation, speed, and timing of DNA replication differs in different organisms. • The “palm, fingers, & thumb” architecture is characteristic of all DNA Pol enzymes, along with active site residues (Asp) and the two metal ion mechanism of catalysis.