DNA Polymerase & Replication

Kornberg Experiments: Discovery of RNA Polymerase

• grow E.coli (we know we that E.coli has everything it needs to replicate DNA; so the polymerase must be in there)

• break open cells

• prepare a soluble extract

• fractionate extract to resolve different proteins (and repeat)

• look for incorporation of radioactivity in polymerized DNA (which has a different solubility than free nucleotides)

Purification Strategy

• take a complex mixture → cells are broken open → membrane components spun down thru centrifugation

  • left w/ soluble fraction (many diff proteins in this mixture)

    • now can be separated based on different biophysical properties (e.g size, hydrophobicity, charge)

• purifications: size, solubility, surface charge, binding capacity, etc.

Kornberg Activity Assay

• used radioactive thymidine b/c it is found in DNA, but not RNA → allowed them to isolate DNA polymerase

• checking whether any mixtures allow for the synthesis of a polymer of labeled thymidine into DNA

  • need to separate the labeled nucleotide from polymerized DNA

• after reaction: added acid to DNA → polymerized DNA precipitates

  • unincorporated labeled nucleotide would remain free in solution

  • radioactivity in the insoluble fraction indicates DNA synthesis

• DNase breaks up polymers of DNA into single nucleotide

  • does it switch from pellet into soluble fraction

Kornberg Activity Assay FIGURE

Kornberg Activity Assay: Next Steps

• rxns confirmed presence of protein by DNAase (still very many) → repeat steps (eg. fractionation) until they can isolate a homogenous solution of DNA polymers

DNA Polymerase Reaction Mechanism

• incoming (antiparallel) triphosphate nucleotide forms W-C (canonical interaction) w/ template strand

  • phosphodiester bond b/w 3’OH of most recently added base and ⍺-phosphate of incoming nucleotide

• magnesium stabilizes 3’OH and ⍺-phosphate (intermediate state; temporary (-) charge on 3’OH; allows for nucleophilic attack)

• β and γ phosphate is released as PPi new phosphodiester bond is formed

• as long as template strand is still available a new substrate is generated for another round of polymerization

DNA Polymerase Mechanism Figure

Requirement of DNA-templated DNA polymerase

• single-stranded template

• deoxyribonucleotides w/ 5’ triphosphate (dNTPs)

• Mg²⁺ ions (essential co-factor for polymerase): coordinate the reaction and neutralize (-) charge

• annealed primer (often RNA) w/ a free 3’OH: or else can’t make phosphodiester bond and no new nucleotides can be added

  • the free 3’OH is necessary b/c of 5’→3’ directionality

Single-Stranded Template

• polymerization is guided by a template DNA strand according to W-C base pairing rules

  • first step is template guided; ensures we’re getting a correct copy during replication (b/c antiparallel complementarity is preserved)

• single-stranded template in vivo is generated by a helicase (ATP-dependent specialized protein that unwinds double-stranded nucleic acid polymers)

Single-stranded Binding Protein

• once unwound, single-stranded DNA is protected by single-stranded binding protein

  • temporarily binds binding protein

• requirements to work:

  • needs to be able to interact w/ any type of single-stranded DNA (can’t be sequence-specific)

  • must easily slide out the way (DNA replication is very fast)

Contribution of Base-Pair Geometry to Fidelity of DNA Replication

Shape discrimination

• formation of W-C interactions: highly favorable b/c of complementary H-bonding capacity + active site that fits W-C interactions perfectly

• the active site has enough flexibility to enable catalysis of properly matched bases

• incorrect base pairing results in contortions of the binding pocket to form H-bonds b/w the template and the incoming nucleotide (much less favorable)

Why is replication accurate?

• formation of H-bonds

• geometry of the active site

• exonuclease editing capcity

• mismatch repair

Why replication is possible

• DNA must be replicated every cell cycle

• its double-stranded complementary structure suggests a copying mechanism

• unraveling the two parental strands results in the production of two identical DNA molecules

Error Correction by DNA Polymerase I

• translocation of the enzyme is inhibited when an incorrect nucleotide is added

  • makes non W-C interactions w/ template

  • phosphodiester backbone is formed

  • doesn’t fit in active site

• many DNA polymerases have intrinsic 3’→5’ exonuclease proofreading activity

  • permits the enzyme to remove a newly added nucleotide

  • mismatched pairs fit well in the editing site (more favorable)

  • makes a break in the phosphodiester bond

Proposed Models of DNA Replication

Conservative: replication of both strands, but parental strands would come tgt and daughter strands come tgt

Semi-conservative: new daughter strand stays attached to parental strand (2 new strands formed from 1 daughter + 1 parent strand)

Dispersive: DNA is broken up → replication → reanneal back tgt

Meselson-Stahl Experiment

• grew E.coli in the presence of heavy nitrogen (¹⁵N) to label the bacteria’s DNA

  • grew for many generations → know all the genome is heavy

  • bacteria copy their entire complement of DNA, or genome, before every cell division

• then moved the bacteria to a normal ¹⁴N-containing medium

  • allowed the cells to divide once

  • separated the DNA by density

• the results supported semi-conservative replication

Heavy vs Light DNA on Cesium Chloride Density Gradient

• heavier DNA travels further

• an ability to distinguish easily b/w heavy and light DNA means that after replication in light DNA differences in total density should be observed

Meselson-Stahl Experiment: Semi-convservative replication results

• after 1 generation (1 doubling of the E. coli – means all the DNA has been duplicated) the DNA from these samples is a mixture of heavy and light (intermediate density)

  • rules out conservative replication mode

• after 2 generations, the DNA is either all light, or a mixture of heavy and light

  • supports the semi conservative model

Bidirectional Replication

• replication begins at an origin and proceeds bidirectionally

• replication forks = dynamic points where parent DNA is being unwound and separated strands replicated

• both DNA strands are replicated simultaneously

• both ends of the bacterial chromosome have active replication forks (bidirectional replication)

Primase

• adds RNA primer that DNA polymerase can use as a substrate

• short DNA sequence (RNA polymerase-like rxn is mechanistically similar)

  • 1st nucleotide (has 5’ phosphate) added just by making W-C interactions

• RNA primer can’t be added by DNA polymerase; required RNA polymer-like enzyme (in E.coli, added by pol-⍺)

Primase Figure

Okazaki Fragments

• short DNA segments created on the lagging strand during DNA replication

  • lagging strands synthesis requires discontinuous, piece-by-piece production that DNA polymerase then joins tgt w/ DNA ligase

DNA Polymerase III Clamp Loader

• clamp is loaded onto the DNA-RNA hybrid by a clamp loader in ATP dependent rxn

  • prevents DNA polymerase from falling off

  • uses power of ATP hydrolysis to provide mechanical force so that it’s able to open up the ring structure and latch it down

• clamp loading requires ATP binding and hydrolysis to add the clamp to the substrate

• latches over RNA/DNA hybrid structure formed after primase adds RNA primer

  • going over a double-stranded region

  • clamps onto the back of polymerase and follow along behind polymerase

DNA Polymerase III Clamp Loader FIGURE

Replication is Semi-Discontinuous: Continuous Hypothesis

Not true

• missing a polymerase that could add free nucleotides to the 5’ end and grow strand in opposite direction

Replication is Semi-Discontinuous: Semi-Continuous Hypothesis

True

• one strand primed at one end and then goes all the way until end of linear chromosome

  • other strand is synthesized in pieces

  • enough single-stranded region has to be pulled out fork and for primase

Processivity

• how many nucleotides is DNA polymerase able to add before falling off

Leading Strand Synthesis

• helicase (DnaB) unwinds DNA at the replication fork

• primase adds a primer to generate a free 3’OH

• clam loader loads on a clamp to ensure synthesis is pocessive

• DNA polymerase synthesizes the leading strand is synthesized in one piece in 5’→3’ directions

  • follows helicase until end of sequence

Lagging Strand Synthesis

• synthesized in Okazaki fragments

• primase repeatedly adds primers to generate new 3’OH groups

• clamp loader repeatedly loads sliding clamps onto new RNA-DNA primer template hybrids

• DNA polymerase extends each fragment until it reaches the primer of the previous Okazaki fragment

• cycle repeats until lagging strand synthesis is complete

Lagging Strand Synthesis (Slide notes)

• trombone mechanism

• lagging strand synthesis required loop formation

• single strand at fork comes out far enough for primase to bind and add RNA primer (w/ free 3’OH)

• happens in opposite direction of fork

• once no more single-stranded synthesis → falls off

• process is repeated → generates fragments as replication process occurs

RNA and Gaps Problem

• synthesis of short fragments of DNA generates two new problems

  • RNA problem → polymerase can’t fill the gap (needs 3’OH)

  • once the last nucleotide is added, can’t generate the last phosphodiester bonds (done by DNA ligase)

• generating Okazaki fragments involves short regions of RNA-DNA hybrid complexes in the lagging strand

  • lagging strand contains many RNA primers, leading strand = 1 RNA fragment

    • we don’t want RNA in our genome; has to be removed

• DNA pol I removes the RNA and replaces it w/ DNA thru the specialized exonuclease activity (nick translation)

• DNA ligase seals the remaining nick

RNA and Gaps Problem Figure

DNA Polymerase I: 5’→3’ Exonuclease Activity

• distinct from 3’→5’ proofreading exonuclease

• the 5’→3’ domain is in front of the enzyme and performs nick translation

  • removes RNA in the 5’→3’ direction

• mild protease treatment separates this domain from the remainder of the enzyme (the large fragment or Klenow fragment which is responsible for the nick translation function of DNA pol I)

Nick Translation

• occurs in the 5’→3’ exonuclease domain

  • RNA removed one at a a time, and dNTPs are added as RNA is removed (same active site)

• nick translation = a break or nick in the DNA is moved along with the enzyme

• important in: DNA repair, and removal of RNA primers during replication

Proteins acting at the Replication Fork

Proteins that are required to replicate genome during s-phase; DNA polymerase cannot replicate the entire genome, several other proteins are required

• SSB: binds and stabilizes single-stranded DNA generated by helicase

• helicase: DNA unwinding (polymerase only works w/ single-stranded template

• primase: RNA primer synthesis

• DNA pol III: new strand elongation (E. coli only has this)

• DNA pol I (pol ⍺): filling of gaps; excision of primers

• DNA ligase: ligation

• DNA gyrase (topoisomerase II): supercoiling; reduces strain

  • pushing polymerase thru a genome (especially circular) results in lots of (+) supercoiling → strain/stress

More than on DNA polymerase (Paula DeLucia)

• DeLucia and Cairns isolated a mutant E.coli w/ no DNA pol I activity but could still survive

• the polymerase responsible for replicating E.coli chromosome in vivo is DNA pol III (discovered by Kornberg)

• DNA pol I is the prototype for all DNA polymerases (enzymatically and structurally)

  • the catalytic site and overall fold (hand structure) is conserved, editing capacity varies depending on the polymerase

E. coli has at least 5 DNA polymerases

• DNA polymerase I is abundant, but insufficient for replication for the E. coli chromosome

  • rate (600 nucleotides/min) is slower than observed for replication fork movement

  • low processivity

• the primary function of DNA pol I is cleanup during replication, recombination and repair

DNA Pol II, III, IV. and V

• DNA pol I: involved in RNA removal post-replication and DNA repair

• DNA pol II: involved in DNA repair

• DNA pol III: the principle replication enzyme in E. coli

• IV and V: involved in an unusual form of DNA repair

  • translesion polymerases that repair damage from UV radiation