dna structure and replication

MOLECULAR BIOLOGYFOS 730

DNA REPLICATION

CHEMISTRY OF DNA SYNTHESIS

DNA synthesis requires- four deoxynucleoside triphosphates, dGTP, dCTP, dATP,dTTP

- primer:template junction- particular arrangement of ssDNA ans dsDNA- primer must have a terminal 3'OH


- DNA is synthesized in the 5'-3' direction- phosphodiester bond is formed in an SN2 reaction3'OH of the primer attacks the "-phosphoryl group of theincoming nucleoside triphosphate

- the leaving group is pyrophosphate - energy released in the hydrolysis of pyrophosphate bypyrophosphatase drives the reaction

XTP + (XMP)n6(XMP) n+1 + 2Pi ()G = -7kcal.mole)

- DNA synthesis is irreversible (Keq -105)


DNA POLYMERASE

- single active site catalyzes the addition of any of the four dNTPs- geometry of A:T and G:C base pairs nearly identical- monitors the ability of the incoming dNTP to form and A:T or G:C base pair

- kinetic selectivity- correct base pairing produces an optimal placement of the primer3'OH and the "-phosphate of the incoming dNTP for catalysis

- incorrect base pairing produces a catalytically unfavorablealignment of the substrates

- rate of incorporation of incorrect nucleotide is 10,000-fold slowerthan for the correct base

DNA POLYMERASE

- selectivity - distinguish between rNTPs and dNTPs- steric exclusion of rNTPs from active site- discriminator amino acids in nucleotide binding pocket - pocket too small to accommodate the 2'OH

DNA POLYMERASE

- three domains- thumb- not intimately involved in catalysis - interacts with most recently synthesized DNA- maintains correct position of the primer and active site- maintains processivity of DNA

- palm- $ sheet- catalytic site- binds two divalent metal ions (Mg2+ or Zn2+) that alter thechemical environment around the correctly base paired dNTP andthe 3'OH of the primer

- monitors the accuracy of base-pairing - fingers- closed form of the polymerase upon correct base pair formation- stimulates catalysis

- bends template (-90° turn phosphodiester backbone)exposes only first template base after the primer

- reopen after formation of the phosphodiester bond - primer:template junction moves over by one base pair

DNA POLYMERASE: ACTIVE SITE METAL IONS

template

primer

Cofactors- metal ions (typically Mg2+ or Zn2+)

- held in place by interactions with two highly conserved aspartate(Asp, D) residues

- one metal ion (A in diagram) interacts with the 3'OH of the primer- reduces the association between the H and O - produces a nucleophilic 3'O- which attacks the "- phosphate of theincoming dNTP

- the other metal ion (B in the diagram) coordinates the negativecharges of the $- and (- phosphates of the incoming dNTP andstabilizes the pyrophosphate produced during catalysis

DNA POLYMERASE: OPEN & CLOSED FORMS

- closed form stimulates catalysis by moving the incoming nucleotidecloser to the catalytic metal ions

- residues in the O-helix position the dNTP- tyrosine (Tyr, Y) makes stacking interactions with the base - lysine (Lys, K) and arginine (Arg, R) associate with thetriphosphate

PROOFREADING

incorrect nucleotide reducesaffinity of 3' end of primer foractive site of DNA polymerase

mismatched 3' end has anincrease affinity for theproofreading exonucleaseactive site

mismatch nucleotide removedprimer:template junction slidesback into polymerase active site

- DNA polymerase inserts one incorrect nucleotide for every 105

nucleotides added- due to tautomeric “flickering” of the bases- T:G base pair formed instead of an T:A

- proofreading exonuclease remove incorrectly base-pairednucleotides- domain of DNA polymerase

- degrade DNA in a 3'65' direction- polymerase can add the correct nucleotide- error rate reduced to 1 mistake in every 107 base pairs added- note rate is further reduced to 1010 post-replication mismatchrepair

TAUTOMERIC “FLICKERING” OF BASES

- bromouracil base analogue of thymine- Br in lieu of CH3

- keto tautomer base pairs with adenine by forming two hydrogenbonds

- enol tautomer base pairs with guanine by forming three hydrogenbonds

- extrapolate example to thymine and the other bases- misincorporation of dNTPs during replication

REPLICATION FORK

Replication fork- junction between the newly separated template strands and theunreplicated DNA duplex

- both stands of DNA are synthesized in the 5'63' direction- leading strand continuously- lagging stand discontinuously- Okazaki fragments- 1,000 to 2,000 nucleotides in bacteria - 100 to 400 nucleotides in eukaryotes

PRIMERS

- DNA polymerases cannot synthesize DNA de nuovo - require a primer with a free 3'OH

- primase- RNA polymerase synthesizes a short (5-10 nucleotides) RNAprimer on a ss DNA template- does not require specific DNA sequences for initiation- activated by association with other DNA replication proteins(DNA helicase)

- leading strand one primer- lagging strand one primer for each Okazaki fragment

PRIMER REMOVAL

- RNA primers must be removed and replaced with DNA- RNAse H recognizes and removes most of each RNA primer- specific for RNA:DNA hybrids- removes all of RNA primer except the ribonucleotide directlylinked to the DNA end- enzyme can only cleave bonds between ribonucleotides

- 5' exonuclease removes final ribonucleotides- degrades RNA or DNA from the 5' end

- gap filled in by DNA polymerase- every nucleotide based paired and phosphodiester bonds formed except for a “nick” (break) in the backbone between the 3'OH and

the 5' phosphate of the repaired strand- DNA ligase seals the nick- creates a phosphodiester bond between the 5' phosphate and 3'OH using ATP as a cofactor

DNA LIGASE

DNA HELICASES

DNA helicases- catalyze the separation of two strands of duplex DNA- hexameric proteins- bind to and move directionally along ssDNA

- polarity of either 5'63' or 3'65' - energy for directional movement provided by hydrolysis of ATP

- ATP binding necessary for assembly - binding of helicase

SINGLE-STRANDED BINDING PROTEIN

Single-stranded binding protein (SSB)- bind to and stabilize separated DNA strands- sequence-independent - electrostatic interactions with the phosphate backbone- stacking interactions with the bases

- cooperative binding- binding of one SSB facilitates the binding of another SSB to theadjacent DNA

TOPOISOMERASES

break two covalent bonds

Topoisomerases- as helicase unwinds the DNA the dsDNA in front of the replicationfork becomes positively supercoiled (overwound)- if no phosphodiester bonds are broken the linking number remainsconstant in a topologically constrained molecule- ccc DNA, long linear molecules, or linear DNA complexed withproteins (histones)

- topoisomerase catalyze the breaking of either one (Type I) or two (Type II, require ATP for conformational changes not cleavage) of the DNA strands, passing the DNA through the break, and reseal the backbone

DNA POLYMERASES

E.coli- DNA Pol III holoenzyme- replication- elongation- highly processive- tens of thousands of nucleotides added per binding event

- complex- two copies of DNA Pol III core enzyme- one copy of the (-complex (sliding clamp loader)- two copies of J-protein

- proofreading

- DNA Pol I- removal of RNA-DNA linkage resistant to RNAse H

- 5'63' exonuclease (proofreading)- DNA synthesis across the ssDNA gap- not highly processive- adds 20-100 nucleotides per binding event

DNA POLYMERASES

Eukaryotes- multiple DNA polymerases- more than 15 per typical cell

- three essential for duplication of the genome- DNA Pol "/primase- 4 subunits- two-subunit primase which synthesizes RNA primer- two-subunit DNA Pol " initiates DNA synthesis

- low processivity- DNA polymerases * and , replace DNA Pol "/primase- elongation

POLYMERASE SWITCHING

SLIDING DNA CLAMPS

Sliding clamps- proteins composed of multiple identical units that assemble in theshape of a “doughnut”

- encircle and slide along DNA without dissociating- bind tightly to DNA polymerases at the replication forks- responsible for processivity

- once a ssDNA template is completely copied DNA polymerase must be released- change in affinity between the polymerase and the sliding clamp- DNA polymerase bound to a primer:template junction has a highaffinity for the clamp

- when a DNA polymerase reaches the end of a ssDNA template aconformational change in the polymerase reduces its affinity forthe clamp- polymerase released- clamp remains bound to DNA - other proteins bind to and interact with clamps - Okazaki repair in prokaryotes and eukaryotes - chromatin assembly in eukaryotes

CLAMP LOADERS

AssemblyATP binds to clamp loader clamp loader binds clamp andopens ring at the interfacebetween one of the subunits

Clamp loader binds to DNA atprimer:template junction

Disassemblybinding of the ternary complex toDNA stimulates ATP hydrolysiswhic causes the clamp loader torelease the clamp and dissociatefrom the DNA

Sliding clamp loaders ((-complex in E. coli; replication factor C (RF-C in eukaryotes)- catalyze the opening and placement of sliding clamps- couple ATP hydrolysis to the placement of the sliding clamparound primer:template junctions in DNA

- clamp loading occurs any time a primer:template junction is present- same region of the sliding clamp interacts with the clamp loader,DNA polymerase, nucleosome assembly factors, Okazaki repairfactors and other DNA repair proteins

- clamp removed from DNA only if not being used by anotherenzyme- after all the enzymes that bind to the clamp have competed theirfunctions

COORDINATE REPLICATION-“TROMBONE MODEL”

REPLISOME

Replisome- combination of all the proteins that function at the replication fork

- J-subunit interacts with DNA polymerase and helicase - stimulates helicase activity tenfold -unwinds DNA at same rate that polymerase synthesizes DNA

- primase associates with helicase and SSB protein about once per second- weak interaction between helicase and primase - stimulates primase activity about 1, 000-fold- regulates length of the Okazaki fragments

INITIATION OF DNA REPLICATION

REPLICON MODEL OF INITIATION

Origin of replication (ori)- site(s) at which DNA unwinds and replication is initiated

Replicon- all the DNA replicated from a single origin- initiation of replication controlled by- replicator- set of cis-acting DNA sequences sufficient to direct initiation ofDNA replication- contains the ori

- initiator protein- protein that recognizes a DNA element in the replicator andactivates the initiation of replication- only sequence-specific DNA binding protein involved ininitiation of replication

- bind ori



Replicator sequences- binding site for initiator protein- A:T rich

green: initiator binding sites

blue: unwinding

red: site of first DNAsynthesis

E. coli- replicator oriC- four 9-mer initiator (Dna A) binding sites- three 13-mer repeated elements that are the sites for initial DNAunwinding

- overall structures of replicators in eukaryotic viruses and S.cerevisiae similar to that in E. coli

- replicators in multicellular eukaryotes are larger (>1,000 bp) andpoorly understood



DNA helicase

Initiator proteins- bind specific DNA sequence within the replicator- distort or unwind a region of DNA adjacent to their binding site- recruit additional factors to the replicator

E. coli- initiator protein Dna A binds to the repeated 9-mer elements in oriC- sequence specific binding- regulated by ATP-Dna A/ATP interacts with DNA in the 13-mer repeats- melts DNA over more than 20 bp within the 13-mer region- unwound DNA template for additional proteins- protein-DNA interactions that are sequence independent - protein-protein interactions

- replication of chromosome is bidirectional from the single ori



multiple DnaA•ATP proteins bindto 9-mers

DnaA•ATP binding leads to strand

separation within 13-mers mediated by ssDNA bindingdomain in DnaA•ATP

DnaB (helicase) and helicase loader(DnaC) associate with DnaA boundoriginhelicase loaders catalyze opening ofhelicase ring around ssDNA atorigin loading of helicase leads todissociation of loader

helicases each recruit primasehelicase movement removes anyremaining DnaA

new primers recognized by clamploaders sliding clamps assembled on eachRNA primerleading strand synthesis is ititiated

after each helicase moves -1000bases a second RNA primer issynthesized on lagging strandsliding clamp loadedlagging stand synthesis is initiated

REGULATION OF REPLICATION

E. coli- DnaA•ATP levels - SeqA- binds to hemimethylated GATC sequences and inhibits- methylation at A by Dam methyl transferase- DnaA•ATP binding


Eukaryotes- set of oris must be activated once per round of cell division

- steps in initiation of replication separated temporally- replicator selection in G1- identify sequences that will direct the initiation of replication

- origin activation in S phase- trigger replicator-associated protein complex to initiate DNAunwinding and DNA polymerase recruitment


Eukaryotes: Formation of the pre-replication complex (pre-RC)

Pre-RC formation in G1 - composed of four proteins which assemble in an ordered fashion at

the replicator- ORC, the initiator, recognizes the replicator- helicase loading proteins, Cdc6 and Cdt1- putative replicator fork helicase, Mcm 2-7 complex


Eukaryotes: Activation of the pre-replication complex (pre-RC)

Pre-RC activation in S - protein kinases, Cdk and Ddk, are activated in S phase

- phosphorylate (activate) the pre-RC and other replicationproteins- three DNA polymerases (DNA Pol "/primase; Pol * and ,)

PCNA= poliferatingcell nuclear antigen


Eukaryotes: Cdks

Cdks activity - required for pre-RC activation- inhibits formation of new pre-RCs

G1

S, G2, M


Eukaryotes: Cdks

FINISHING REPLICATION

Circular chromosomes- Type II topoisomerase separate catenanes


Linear chromosomes- no inherent topological linkage like in prokaryotes but folding and

attachment of DNA to protein scaffolds in large linear moleculescreates problems during chromosome segregation that require theactivity of type II topoisomerases

- end replication problem- lagging strand- inability to synthesize DNA at the 5' terminus

- shortening of chromosomes


TELOMERASE: 3' ends

telomeres- ends of eukaryotic chromosomes

- head to tail repeats of a TG-rich DNA sequence- in humans 5'-TTAGGG3'

- 3' end ssDNA- act as ori

telomerase- extends the 3' ends of chromosomes- ribonucleoprotein (RNA + protein)- protein is an RNA directed DNA polymerase (reverse transcriptase)- template for DNA sequence is the RNA component of the enzyme

- contains 1.5 copies of the complement of the telomere sequence (humans 5' TAACCCTAA3') What’s wrong with this ?

- 3' end of DNA serves as the primer- has an RNA•DNA helicase activity

What’s wrong with this ?


5' ends

5' ends extended- lagging stand replication machinery

- the extended 3' end provided by telomerase serves as the template

- 3' end still has a ss region- proteins bind telomeres

- inhibit telomerase (limit length of telomeres) - protect ends from recombination and degradation

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dna structure and replication