Chapter 25 DNA MetabolismReplication, Repair and

Recombination• Semiconservative

DNA replication

• Each strand of DNA acts as a template for synthesis of a new strand

• Daughter DNA contains one parental and one newly synthesized strand

Meselson-Stahl Experiment• 1953 Watson-Crick Structure• 1957 This experiment proves the semi-conservative

model of DNA replication• Nitrogen-15 isotope used (heavy)• Nitrogen-14 isotope (most common, light)

E. Coli grown on 15N first

Transferred to 14N medium

Samples withdrawn at different generations

Subjected to CsCl gradient centrifugation

Chromosomal DNA Replication is Bidirectional• E. coli chromosome is circular, double-stranded

DNA (4.6x103 kilobase pairs)

• Replication begins at a unique site (origin)

• Proceeds bidirectionally until the two replication complexes meet (termination site)

• Replisome - protein machinery for replication (one replisome at each of 2 replication forks)

• Duplication in about 38 minutes

• Bidirectional DNA replication in E. coli

• New strands of DNA are synthesized at the two replication forks where replisomes are located

Eukaryotic replication• Eukaryotic chromosomes are large linear, double-

stranded DNA molecules

• Fruit fly large chromosomes ~5.0x104 kb (~10x larger than E. coli)

• Replication is also bidirectional

• Multiple sites of initiation of DNA synthesis (versus one site in E. coli)

Replicating DNA in the fruit fly • Large number

of replication forks at opposite ends of “bubbles” of duplicated DNA

DNA Polymerase• E. coli contains at least 5 DNA polymerases

• DNA polymerase I - repairs DNA and participates in DNA synthesis of one strand

• DNA polymerase II - role in DNA repair

• DNA polymerase III - the major DNA replication enzyme, responsible for chain elongation

Chain Elongation Is a Nucleotidyl-

Group-Transfer Reaction • Base pair between incoming deoxynucleotide 5'

triphosphate (blue) and a residue of the parental strand

• Terminal 3' OH attacks a-phosphorous of incoming nucleotide to form new phosphodiester linkage

• Strand growth always in the 5' → 3' direction

• Irreversible due to formation or PPi which is quickly hydrolyzed to 2Pi by pyrophosphatase

DNA PolIII Remains Bound to the Replication Fork

• DNA polymerase III is a processive enzyme (remains bound to the replication fork until replication is complete)

• Certain subunits form a sliding clamp which surrounds the DNA molecule

• Two -subunits associate to form a head-to-tail dimer in the shape of a ring that completely surrounds the DNA

• Remaining subunits of DNA pol III are bound to this structure

Bacteriophage DNA polymerase bound to

DNAProofreading Corrects Polymerization Errors• Recall enzymes that degrade polynucleotides

•Exonucleases – degrade from chain ends•Endonucleases – cut at recognized interior sites

• DNA polymerase III holoenzyme also possesses 3’ 5’ exonuclease activity

• Pol III can catalyze both chain elongation and degradation

• Recognizes distortion in the DNA caused by incorrectly paired bases

• Exonuclease activity removes mispaired nucleotide before polymerization continues

DNA Polymerase Synthesizes Two Strands

Simultaneously • DNA pol III catalyzes chain elongation only in the

5' 3'direction (antiparallel DNA strands)

• Leading strand - synthesized by polymerization in the same direction as fork movement

- continuous replication -polynucleotide (from

origin to the termination site) • Lagging strand - synthesized by polymerization in

the opposite direction of fork movement

-discontinuous replication in short pieces (Okazaki fragments)

- Pieces are joined by a separate reaction

Two core complexes of DNA pol III, one for leading,

one for lagging strand

RNA Primer Begins Each New Strand, including each Okazaki

Fragment• Primosome is a complex containing primase enzyme which synthesizes short pieces of RNA at the replication fork (complementary to the lagging-strand template)

• DNA pol III uses the RNA primer to start the lagging-strand DNA synthesis

• Replisome - includes primosome, DNA pol III

• Okazaki fragments are joined to produce a continuous strand of DNA in 3 steps:

(1) Removal of the RNA primer (pol I)(2) Synthesis of replacement DNA (pol I)(3) Sealing of adjacent DNA fragments (DNA ligase)

DNA polymerase I activities• The 5‘ 3' activity of DNA pol I removes the

RNA primer at the beginning of each Okazaki fragment

• Synthesizes nick translation: polymerase activity synthesizes DNA in place of RNA

Nick – Break in the DNA backbone

• Klenow (large) fragment of DNA pol I, lacks 5'→3' exonuclease activity

• Used for DNA synthesis

DNA ligase activity • Catalyzes the formation of a phosphodiester linkage

between 3’-hydroxyl and 5’-phosphate of adjacent Okazaki fragments

• Eukaryotic enzymes require ATP cosubstrate

• E. coli DNA ligase uses NAD+ as a cosubstrate

Model of the Replisome• Replisome contains: a primosome, DNA

polymerase III holoenzyme, additional proteins

• DnaB helicase is part of the primosome and facilitates unwinding of the DNA helix

• Topoisomerases relieve supercoiling ahead of the replicating fork (not part of the replisome)

• Single-stranded binding proteins (SSBs) stabilize single-stranded DNA

3 Stages of DNA Replication in E. coli

1. Initiation 2. Elongation 3. Termination

Initiation• Regulated for once per cell cycle• Replisome assembles at origin site (oriC)• Origin site is a highly conserved sequence and

contains two series of short repeats

• DnaA is first initiation protein• binds at four 9 bp repeat sequences• causes denaturation at three 13 bp repeats• requires ATP and HU (histone-like protein)

• Hexamers of DnaB (aided by DnaC) unwind DNA

Elongation

• DNA helicases – unwinding ahead of fork• SSB’s stabilize single strands• Primase – synthesizes RNA primers

-1 for leading strand- 1 for each Okazaki fragment

• Core – catalyzes polymerization reaction• subunits – clamp for processivity• Lagging strand

- subunits load template onto clamp- New one every Okazaki fragment

• ~1000 nucleotides/s• Process completed by DNA pol I and DNA ligase

Termination• Terminator utilization substance (Tus) binds to the

ter site• Tus inhibits helicase activity and thus prevents

replication forks continuing through this region• ter sites act as replication “block”

• Last few hundred bp made by unknown mechanism• Topoisomerase IV frees catenated DNA’s

Catenanes - circles wound around each other

DNA Replication in Eukaryotes• Mechanism similar to that in prokaryotes:

leading strand continuous synthesis, lagging strand discontinuous synthesis

• Replication forks move more slowly, but many replication forks (~50 nucleotides/s)

• Okazaki fragments are shorter in eukaryotes (~100-200 residues)

• At least 5 different DNA polymerases

Accessory proteins associated with the replication fork

• PCNA (proliferating cell nuclear antigen) forms structure resembling -subunit sliding clamp (E. coli DNA polymerase III)

• RPC (replication factor C) similar to complex of DNA pol III

• RPA (replication factor A) similar to prokaryotic SSB

• Helicases also present to unwind DNA