Chem 153B

Draw the mechanism of elongation that occurs as a result of catalysis by DNA Polymerase. How is the mechanism of elongation different for RNA Polymerase?

The mechanism of elongation is the same for RNAP; however, each pentose (now a ribose) has an additional hydroxyl group on its 2' carbon.

What molecule is given off after each round of nucleotide addition? Draw this molecule.

Pyrophosphate

What are the three fundamental properties of DNA Polymerases?

1) Catalyze the polymerization of deoxyribonucleotides in the 5'-->3' direction: (dNMP)n+dNTP --> (dNMP)n+1 + PPi

2) Require a template (DNA, sometimes RNA = Reverse Transcriptase)

3) Require a primer: DNA or RNA

What process does reverse transcriptase catalyze?

The generation of complementary DNA from an RNA template.

What is a primer?

A short piece of duplex (double-stranded DNA or DNA/RNA hybrid duplex) required to initiate the polymerization reaction (only required by DNA Polymerases).

What are the fundamental properties of RNA Polymerase?

1) Catalyze the polymerization of ribonucleotides in the 5'-3' direction: (rNMP)n+rNTP --> (rNMP)n+1 + PPi

2) Require a template (usually DNA)

3) Do NOT require a primer

Flask #1 contains DNAP, dNTPs, and a single-stranded piece of DNA. Flask #2 contains RNAP, rNTPS, and a single-stranded piece of DNA. What happens in each flask?

Nothing happens in Flask #1 because DNAP requires a primer to begin polymerization. RNAP will synthesize a strand of RNA complementary to the DNA template strand in Flask #2 because RNAP does not require a template.

Why is DNA/RNA polymerization mostly occurring in the forward direction considering all chemical reactions are reversible?

Because the PPi produced is hydrolyzed in vivo by pyrophosphatases or hydrolyzed in the active site of the polymerase. If PPi is not hydrolyzed in the active site, it is released from the active site into the cell, where the pyrophosphatases take care of it (inactivate it via hydrolysis).

PPi could reattack the phosphodiester bond and go in reverse, but it is inactivated when it is cleaved.

In what direction does the degradation of DNA occur?

In the 3'-->5' direction (the opposite direction from synthesis - think of it as the reverse reaction occurring!)

If a reaction is going in one direction, what has typically occurred (consider that all biochemical reactions are reversible)?

Something has happened that has made one of the products of the reaction inaccessible for reacting (which prevents the reverse reaction from occurring!)

What does it mean that DNA polymerases are arranged into different families?

They have different structures/architectures/domains. The families divide them into groups based on similar structures/architectures/domains.

What does Pol stand for?

DNA polymerase

What do roman numerals mean when they are in the names of DNAPs?

DNAPs with roman numerals in their name are bacterial enzymes.

What do Greek letters mean when they are in the names of DNAPs?

DNAPs with Greek letters in their name are eukaryotic enzymes.

What does translesion mean?

Refers to polymerase enzymes that can polymerize DNA across damaged DNA (important in the process of DNA repair).

What kind of reverse transcriptase did we learn about? What process is it involved in?

Telomerase - extends the 3' end of chromosomes

What kind of structure do many DNA polymerases adopt?

A right hand-like structure.

In the right hand-like structure, what parts of the hand represent each domain?

Thumb - domain involved in binding the template primer duplex so it doesn't dissociate from the enzyme (to maintain the interaction between the DNA substrate and the enzyme)

Palm - other active site

Fingers - polymerase active site (where polymerization occurs, region where catalysis happens)

Name the two major families of RNA polymerase and their structural distinctions.

Single subunit RNAPs (one protein): Bacteriophages: T7, T3 // Mitochondria // Chloroplasts (have their own genome)

Multi-subunit RNAPs (multiple proteins): Crab Claw shape: Bacteria: α2ββω core (5 subunits, sigma -->specificity)

Eukaryotic RNAPs have similar architecture to bacterial RNAPs but have many more subunits: 3 major RNAPs, Pol I, II, III

Where is the active site of multi-subunit RNAPs?

Inside the crab claw

Are there conserved residues between eukaryotic and bacterial RNAPs?

Yes, there are conserved residues in the active site of the RNAP. Means they share a sequence of amino acids in a specific region, or the amino acids present in a specific region share similar properties (an acidic residue at the same position, an aromatic residue at the same position, etc.).

How do you determine the template strand from the primer strand when looking at the active site of DNAP?

Look for the incoming, unlinked dNTP (should see three phosphates attached to it instead of one) that will be incorporated during synthesis. Whichever strand it is below is the strand being extended by one nucleotide at a time (the primer strand). The opposite strand is the template strand. You should also see the incoming dNTP base pairing with the complementary base on the template strand (despite it not being attached yet).

Why is polymerization not happening in this crystal?

The nucleophile is absent (no 3'-hydroxyl at the 3' end of the primer). It therefore cannot attack the α-phosphorus of the incoming dNTP and add it to the growing strand, which stops polymerization from occurring.

If you want to solve the structure of an enzyme with its substrate, what do you have to do?

You have to disable it somehow (so it can bind the substrate but not react). If you want to crystallize the enzyme with the substrate, you can modify the substrate or the nucleophile/electrophile that will not react.

Can you have chemical reactions occurring in the solid state?

Yes, chemical reactions can occur even when enzymes are crystallized.

What two ions are present in the active site of DNA polymerase?

Magnesium (Mg2+) and manganese (Mn2+). Or can be two magnesium ions - the two can be used interchangeably.

What is each ion present in the active site of DNAP close to?

Both are very close to the substrate of the reaction (the dNTP). Mg2+ is very close to the terminal deoxyribose on the primer strand (very close to the nucleophile - the 3'-OH). Mn2+ is very close to the β and γ phosphates of the incoming dNTP (very close to the leaving group of the reaction). Each ion is specifically positioned within the active site.

How are the two metal ions held in place in the active site?

Two aspartate (or glutamate)residues positioned close to the two ions. Aspartate residues are negatively charged amino acids (deprotonated at cellular pH), so they make electrostatic interactions with the positively charged magnesium and manganese, thereby positioning them in the active site.

What is required to initiate the attack of the 3'-OH on the α phosphorus of the incoming dNTP?

It must be deprotonated (increases its nucleophilicity and makes it negatively charged) by a general base in the enzyme. A negatively charged oxygen in the active site is not very stable. The first Mg2+ ion (MgA) stabilizes the negatively charged oxygen (increases the nucleophilicity of the 3'-OH of the primer strand).

What is the role of MgA in the active site of DNAP/RNAP?

Lowers pKa of 3' O (previously deprotonated by a general base, stabilizes it in its negatively charged form) to activate the 3'-OH for the attack on the α-phosphate of the incoming (r/d)NTP (increases its nucleophilicity)

Can be stably associated with the enzyme or come with the incoming (r/d) NTP

Bridges the two substrates together (enzyme and (r/d)NTP, puts them in the right orientation) by interacting with the non-bridging phosphate oxygen on the α phosphate group of the incoming (r/d) NTP

In general, what does an enzyme do when it catalyzes a reaction? What ion is involved in this process with the active site of DNAP?

It lowers the transition state energy of the reaction and also orients the substrate in the right orientation for the reaction. MgA does this in the active site of DNAP by interacting with the non-bridging phosphate oxygen on the incoming (r/d) NTP).

What the role of MgB in the active site of DNAP/RNAP?

Stabilizes the negative charge that builds up on the leaving oxygen. Also binds the β and γ phosphate oxygens of the incoming (r/d)NTP (interacts mainly with the leaving group, works to stabilize its highly negative charge).

What is the function that MgA and MgB share?

They stabilize both the structure and charge of the pentavalent transition state.

What additional molecule is bound in the active site of DNAP aside from the Mg2+/Mn2+ ions and negatively charged amino acid residues? What do these molecules help with?

Water molecules, help with metal ion positioning in the active site.

Why is sugar discrimination particularly critical for DNA polymerases? What happens if sugar discrimination fails?

Because of cellular NTP concentrations. There is a much larger concentration of rNTPs than there is dNTPs (which DNAPs need to use). These rNTPS compete with the dNTP to enter the active site and get incorporated into the DNA primer being extended (need to avoid this!). Substrate abundance makes this discrimination more difficult.

Cells need to limit/avoid misincorporation of ribonucleotides into DNA. If a ribonucleotide is incorporated, this leads to strand breaks/replication blocks (distorts DNA structure, makes it more unstable due to the 2'-OH).

What is the general mechanism for discrimination for deoxyribonucleotides over ribonucleotides by DNAPs?

The aromatic/large side chain (typically tyrosine, phenylalanine) found in the DNA polymerases close to the dNTP binding side provides a STERIC GATE against rNTP entry. A 2'-OH being present would lead to a steric clash with the aromatic residue.

How is this phenylalanine side chain involved in selecting dNTPs over rNTPs?

It prevents rNTPs from entering the active site because a 2'-OH would not fit.

What is required of a pair of molecules for them to be able to interact via base stacking?

Both must be AROMATIC.

What change in the active site of RNAPs allow for the incorporation of rNTPS (without being sterically blocked by an aromatic side chain like in DNAPs)?

Instead of tyrosine/phenylalanine, RNAPs have a aspartic acid or glycine at the same position. These different amino acids with smaller side chains allow for rNTP entry and incorporation.

What is the general mechanism of sugar discrimination in RNA polymerases? Why is this process less critical than for DNAPs?

Less critical because rNTPS are typically in excess in the cell - the normal substrates for the RNAPs are in abundance.

Discrimination occurs through recognition of the 2'-OH of riboses by side chains in the active site of RNAP. Typically hydrogen bonding with the 2'-OH of the rNTP (typically donate h-bond to the oxygen). In a multi-subunit RNAP, multiple residues can interact directly with the 2'-OH to stabilize the binding of the rNTP in the active site.

Requires amino acids with smaller sidechains in the active site to allow for the entry of rNTPS.

What is fidelity?

Refers to the ability of a polymerase to incorporate the correct nucleotide into the nucleotide sequence being built (selecting the proper nucleotide in the active site).

Overall error rate of DNAPs vs RNAPs during replication (includes polymerization and proofreading activity)?

10^-8 for DNAPs (1 error every 100,000,000 base pairs)

10^-4 to 10^-5 for RNAPs (1 error every 10,000 or 100,000 base pairs)

Error rates, however, vary by family for DNA polymerases - some have higher fidelity than others (those involved in replication (A,B,C) have the highest fidelity)

Why are the A,B,C family Polymerases (involved in replication, highly accurate) shown twice on this chart?

The higher fidelity range represents when these families have editing activity (A+,B+,C+), while the lower fidelity range represents when these families have no editing activity (A-,B-,C-).

Why is it important that DNA polymerases have high fidelity?

Anytime a DNA polymerase makes a mistake, there will be a mutation in the DNA that will be copied (causing a mutation to be inherited in the newly replicated DNA). Important to minimize the amount of mutation (want a little bit of mutation to evolve the genome slowly - too much will damage important genes).

How do replicative DNA polymerases select correct nucleotides from incorrect nucleotides? What does this mechanism suggest?

Nucleotides forming Watson-Crick base pairs fit the active site (because Watson-Crick base pairs are isosteric - they take up the same space and maintain the spacing of the sugar-phosphate backbone). Nucleotides forming non Watson-Crick base pairs do not fit in the active site and are ejected.

This suggests that hydrogen bonding between bases is not a major determinant for nucleotide selection by DNA polymerase (could just be based on the shape of the base pair - of the two bases next to each other in the active site).

ONLY THE SHAPE/FIT OF THE BASE PAIR IN THE ACTIVE SITE MATTERS (NOT HYDROGEN BONDING)

What does heterocyclic mean?

Refers to a molecule containing a ring of atoms of at least two elements (one of which is generally carbon).

Artificial nucleotides can be incorporated into DNA, however:

Artificial nucleotides are not incorporated as efficiently as Watson and Crick bases.

How can mismatch base pairs occur in the active site of DNAP?

Mismatch base pairs can form in the active site of DNAP due to transient base isomerization. Bases can isomerize from the more stable keto form to the enol form. When in the enol form, a base can maintain a Watson-Crick geometry with a non Watson-Crick partner and fit into the active site of DNA polymerase (can base pair differently when in the enol form). Example: G(enol):T (has the same geometry as a G-C base pair), G:T(enol).

Can also occur via deprotonation of a ring nitrogen (another isomerization reaction). Examples: G-:T, G:T-.

Get a Watson-Crick geometry with the wrong nucleotide. They fit the active site of DNAP and result in misincorporation/mutations.

What other mismatches would you predict to occur as frequently as G-T mismatches in the active site of DNA polymerases?

A-C mismatches (imino C forms 2 h-bonds with A or imino A forms 2 h-bonds with C). To have Watson and Crick type shape/geometry, you NEED a purine and a pyrimidine.

Two pyrimidines are too far apart/too small (would not fit, leave active site basically empty). Two purines would not fit because they would be too big (too close - too many atoms!).

How do we get to an error rate of 10^-8 for DNAP?

Although base misincorporations occur with a probability of 10^-3 to 10^-5, DNAP has PROOFREADING ACTIVITY that improves its fidelity. Most DNAPs involved in replication have proofreading activity.

What can the proofreading activity of DNAP do/fix?

It can fix misincorporated nucleotides (non Watson-Crick base pairs). There is no mechanism that can fix incorporating the wrong sugar (ribose instead of deoxyribose, .)

What are the 3 enzymatic activities associated to three distinct active sites on the single polypeptide chain of DNA Pol I?

5'-->3' exonuclease active site (closer to N-terminus)
5'-->3' polymerase active site (closer to C terminus)
3'-->5' exonuclease active site (closer to C terminus)

DNA Pol I a single protein with MW of 109 kDa

What is proofreading?

The correction of a misincorporated nucleotide by replacing it with the proper Watson-Crick partner.

What occurs at the 3'-5' exonuclease active site of DNAP? What is the role of this active site?

Hydrolysis of the last phosphodiester bond on the 3' end of the DNA. A water molecule attacks the last phosphodiester bond, cleaving off the last nucleotide on the 3' end of the DNA. The water molecule is deprotonated before it attacks the phosphorus of the phosphodiester bond (increases its nucleophilicity).

Results in the production of nucleotide monophosphate.

The role of this active site/reaction if to edit newly polymerized sequences.

Why do polymerization and the 3'-->5' exonuclease reactions need to have separate active sites?

The 3'-->5' exonuclease reaction is not the reversal of the 5'-->3' polymerization because the attacking group is water rather than pyrophosphate.

What needs to happen within the DNAP to allow editing to be possible?

Editing of mistakes requires a switch between polymerization mode and editing mode within the DNAP. Need to switch the 3' end of the newly synthesized DNA into the 3'-->5' exonuclease active site (editing active site).

What structural transition mediates the switch of the primer strand into the editing site?

The mismatch induces a dissociation of the primer strand from the template, allowing more flexibility and entry into the editing site (local unwinding/dissociation of the two strands due to the mismatch).

The mismatch does not fit in the polymerase active site and results in the ejection of the primer strand from the polymerase active site and into the editing site.

What about RNA polymerases? What is their proofreading mechanism?

No 3'-->5' exonuclease domains in RNAPs, but there is still some proofreading. Proofreading by RNAPs involves backtracking and cleavage of a dinucleotide no longer base paired to the template (because of the mismatch). The catalytic mechanism uses a water molecule to hydrolyze the dinucleotide. Less accurate than DNAP's proofreading mechanism (10^-4-5 error rate).

A mismatch causes dissociation of primer and template, triggering the polymerase to stop and backtrack on the template (reverse translocation). Polymerase is unable to accept any NTPs in its active site in this conformation. Instead, it uses a water molecule in the active site to trigger hydrolysis of the phosphodiester bond of the unpaired dinucleotide (in the same active site as the polymerase active site).

Why is RNAP's higher error rate (and less efficient proofreading mechanism) not as much of a concern?

RNAPs make mRNA that will be translated into protein - but most RNAPs do not generate a nucleic acid term that will be transmitted to the next generation - in general, just makes copies of genes for expression. MISTAKES ARE NOT INHERITED.

What occurs at the 5'-->3' exonuclease active site of DNA Pol. I?

NO INVOLVEMENT IN EDITING/PROOFREADING. Allows the replacement of damaged or abnormal DNA sequences by "Nick translation." Also allows the removal of RNA sequences embedded in DNA (removal of replication primers). Uses the same mechanism of using a water molecule to attack a phosphodiester bond - but this time, it attacks the nucleotide on the 5' end of the DNA instead of the 3' end.

DOES NOT CONTRIBUTE TO FIDELITY. INVOLVED IN DNA REPAIR. The "nick" moves towards the 3' end after the 5'--->3' exonuclease reaction is finished.

USED IN DNA REPLICATION (REMOVES RNA PRIMERS) AND IN DNA REPAIR.

How does Nick translation work?

Pol. I combines 5'-->3' exonuclease and 5'-->3' polymerase activities simultaneously to remove nucleotides of one strand and replace them with newly synthesized nucleotides (activities work at the same time).

After proofreading is finished, how does the DNAP switch the DNA from the editing active site back into the polymerase active site?

Once the phosphodiester bond is hydrolyzed (thereby cleaving off the last base), you reconstruct a fully base-paired template-primer duplex, resulting in a substrate that is correct for the polymerase (causing a switch back to polymerase).

What is meant by the "speed" of a polymerase?

The overall rate of nucleotide polymerization per unit time.

Which type of polymerase has a faster speed: RNAP or DNAP? Why is this the case?

DNAP has a significantly faster speed. This is true because while RNAP only copies individual genes, DNAP has to copy the entire genome (hence why it must work faster).

What is meant by "processivity" of a polymerase?

Ability of an enzyme (polymerase) to remain attached to its substrate (template) and perform multiple rounds of catalysis (nucleotide addition) before dissociating from the template (ability to polymerize for a long distance without dissociating from the template).

What is a poorly processive enzyme called?

Distributive

Why is high processivity mechanistically problematic?

High processivity is a necessity for DNAPs involved in DNA replication but is mechanistically problematic because of the need to translocate on the template after each round of nucleotide addition, providing opportunities for dissociation (because when DNAP moves to the next nucleotide, contact with the template is temporarily lost).

Why is high processivity a necessity for DNAPs involved in DNA replication?

Need to be able to polymerize long distances without falling off of the template in order to prevent interruptions in the replication process. More important for DNAP than RNAP.

For what type of polymerases is processivity more important/problematic?

It is more problematic for DNAPs because they interact only with two nucleic acid strands vs 3 for RNAPs (only interacting with 2 strands makes it harder for DNAPs to stay attached to the DNA).


It is more important for DNAPs so that they can replicate genomic DNA without interruptions.

Processivity is determined by the way the polymerases interact with their substrates (hence processivity being more problematic for DNA, as it only interacts with 2 nucleic acid strands).

DNAPs and RNAPs do not interact the same way with the DNA used for polymerization. How so?

DNAPs do not interact with the non-template strand. RNAPs maintain contact with the non-template strand in the "transcription bubble." Therefore, RNAPs interact with 3 nucleic acid strands, while DNAPs only interact with 2. This results in RNAPs being less likely to dissociate (higher processivity as a result).

DNAPs use additional proteins to confer higher processivity.

What do DNAPs use to increase processivity?

Processivity factors, specifically sliding clamps (a type of accessory protein). ADDITIONAL ACCESSORY PROTEINS NOT PART OF THE POLYMERASE ITSELF! Prevent the polymerase from falling off the template.

Specific type of processivity factor used in bacteria.

β-clamps wrap around DNA and also interact with DNA Pol III and force the DNAP to maintain contact with the DNA template, thus enhancing processivity. DNA Pol III core is poorly processive by itself, which is problematic for replication in vivo. β-clamps are dimers (two polypeptides interacting).

Type of processivity factor used in eukaryotes.

PCNA - a trimer sliding clamp. Overall structure the same as β-clamp, just made of 3 proteins instead of 2. Operates exactly the same way to increase processivity. Also can help load the polymerase onto the DNA.

What does PCNA stand for?

Proliferating Cells Nuclear Antigen because it is found very abundantly in cells that are replicating actively.

Some DNA polymerases can be highly processive without clamps. Name one that is, and why it is processive alone.

Eukaryotic Pol ε is highly processive without PCNA. Processivity is due to the presence of an additional protein domain, the P-domain, which encircles the DNA (forces the protein to remain bound to the DNA template). In less processive "normal" DNAPs like Pol δ, polymerase does not surround the DNA and only covers one side of the DNA, which is a recipe for dissociation.

POL EPSILON - INVOLVED IN DNA REPLICATION IN EUKARYOTES

Pol δ also involved in eukaryotic DNA replication (DELTA), needs processivity factor

What is chromatin? What problems does this cause for replication in eukaryotes?

DNA wrapped around nucleosomes (this forms the chromatin structure) - packages the DNA. Need to disrupt this structure to replicate the DNA (and reassemble it after replication is complete).

CHROMATIN NOT FOUND IN PROKARYOTES

Makes DNA harder to access for replication in eukaryotes.

DNA replication by DNAPs: key principles?

It is copying the genetic material to prepare for cell division.

Replication is semi-conservative - one parental strand is transmitted into each daughter DNA molecule.

Replication is bi-directional from the origin of replication (1 origin of replication in eubacterial chromosomes, several origins in eukaryotes).

The origin of replication is where replication is initiated/begins (middle of the replication bubble).

What is the bidirectionality problem?

If DNA is synthesized in both directions from each origin of replication, on each replication fork there is one strand (the leading strand) that can be synthesized continuously (5'-->3' heading towards the fork). However, synthesis of DNA on the lagging strand requires continuous synthesis of primers to be able to polymerize in that same direction (because it is 3'->5' in this direction, opposite the direction that DNA is polymerized). Still polymerizes in the 5'-->3' direction, but is moving towards the replication fork with each new Okazaki fragment.

What are the fragments that are synthesized discontinuously on the lagging strand called?

Okazaki fragments (not linked to each other), require primers.

Why are the primers on the lagging strand made of RNA?

Primers are made of RNA since RNAPs do not require primers.

How was the identity of the primers determined (how was it determined that they were made of RNA)?

Alkaline hydrolysis of Okazaki fragments (high temperature, high pH). dNTPs labeled with a single radioactive group on the α-phosphorus. rNTPs unlabeled. Allow replication to occur with these species. High temp/high pH activates the 2'-OH of RNA for hydrolysis of the phosphodiester bonds of RNA - cleaves off individual RNA molecules.

Saw a ribonucleotiede with a 3' radioactively labeled α phosphorus, indicating a transfer of radiolabel from DNA--->ribonucleotide, indicating a 5'RNA-->3'DNA junction (so the primers must be made out of RNA!).

Mechanism of RNA degradation during alkanaline hydrolysis?

2'OH gets deprotonated and attacks the phosphorus of the phosphodiester bond.

Key parts of the replisome of E. Coli (bacteria)?

1) Helicases (DnaB): unwind DNA at the replication fork in a reaction coupled to ATP hydrolysis

2) Single-stranded DNA binding proteins (SSB): bind and stabilize the DNA in a single-stranded conformation after the melting by helicases (strands want to rehybridize to each other due to base complementarity, SSB stops them from doing this)

3) Primase enzyme: synthesizes RNA primers of the lagging strand

4) DNA Pol III: the replicase which synthesizes most of the DNA during replication

5) DNA topoisomerase II: relaxes supercoiled DNA that forms ahead of the replication fork (acts downstream of fork - unwinding DNA generates tension downstream of fork, topoisomerase helps relieve this tension)

6) Rnase H: removes RNA primers (hydrolyzes RNA primers)

7) DNA Pol I: removes RNA primers, replaces RNA primers with DNA via Nick translation (5'-->3' exonuclease activity on DNA Pol. I can act on RNA as well as DNA, fills in gaps between Okazaki fragments)

8) DNA Ligase: joins the Okazaki fragments (need to ligate the extended piece to the following Okazaki fragment).

What does it mean to be "upstream?" What does it mean to be "downstream?"

Upstream: toward the 5' end of the DNA/RNA molecule

Downstream: toward the 3' end of the DNA/RNA molecule

Why is it necessary to remove the RNA primers from the Okazaki fragments?

Because the presence of RNA embedded in DNA would cause problems when this strand is replicated next, as DNAPs won't use a template strand with RNA.

Because the presence of RNA embedded in DNA might lead to strand breaks (if RNA present in double-stranded regions). Makes double-helix more fragile, single-stranded breaks will be INHERITED by the daughter cell.

Proteins that interact with DNA (interact with B-form DNA to recognize gene sequences) will not be able to interact with a RNA-DNA duplex because RNA is A-type (A-type helix for both strands because RNA is stuck in A-type).

Where do the SSBs bind in bacteria?

The single-stranded DNA on the lagging strand.

What can DNA ligase ligate?

DNA to DNA, DNA to RNA, RNA to RNA

What makes this model inaccurate? How is this possible?

DNA Pol III does not move in different directions on the leading and lagging strands. Replication is coupled by the 2 DNA Pol III enzymes that move in the same direction.

This is possible because the DNA on the lagging strand is looped out, allowing the polymerase on the lagging strand to move in the same direction as the polymerase on the leading strand.

How many subunits does DNA Pol III have and what are their names? What is each subunit's activity/purpose?

3 subunits; α, ε, θ.
α = catalytic site for polymerization (no 5'->3 or 3'->5' exonuclease activity) (largest subunit)(no other activities)
ε = 3'->5' exonuclease (corrects mistakes of the α subunit via editing, exactly the same as Pol I except this activity is in a separate protein instead of one protein with 2 active sites)
θ = structural role, small
(3 proteins interacting together)

Each DNA Pol III interacts with? How is this element loaded onto the DNA?

β-clamp (homodimer of 2 x 41 kDa): ATP-dependent processivity factor (associates with the core of Pol and maintains the polymerase in contact with the duplex DNA to make it more processive)

Clamp loader Complex (CLC): ATP-dependent loading of the β-clamp on to DNA and unloading (3 subunits: δ, δ', τ)

What are the purposes of the different subunits of the CLC?

τ is the dimerization factor: holds the Pol III cores together (so they stay together during polymerization)
δ, δ' are involved in the ATP-dependent loading of the β-clamp on to DNA and unloading

polymerization on the lagging strand requires the constant loading of β-clamps onto the DNA

How many subunits does DnaB helicase have?

6 (shaped like a donut)

Why is there no 5'-->3' exonuclease activity for Pol III

Because it is not involved in processing Okazaki fragments (does not remove the RNA primers, Pol I does this along with Rnase H)

Because it is not involved in DNA repair

No need for a 5'-->3' exonuclease activity!

Explain the cycle of molecular events during lagging strand synthesis.

1) The primase synthesizes a new RNA primer upstream in the lagging strand; the two polymerase replicate DNA

2) A sliding clamp is assembled around the new RNA primer (CLC helps); primase dissociates

3) The lagging strand polymerase detaches (after reaching the 5' end of the previous Okazaki fragment) and associates with the newly deposited sliding clamp

4) The lagging strand polymerase starts to synthesize the next Okazaki fragment; primase will reinitiate synthesis of the next RNA primer

Note: the lagging strand loop is released after each Okazaki fragment and reassembled for each new fragment

BACK TO STEP ONE; CYCLE REPEATS

How many DNA Pol III cores are associated with the replication fork? What is the advantage of 3 polymerase replisomes vs 2 polymerases?

THREE! Two polymerases copy the leading/lagging strands and their polymerase activity is coupled to each other. A third Pol III core associates with the τ subunit of the CLC.

Advantage of this: the third Pol III could take over if the 2nd Pol III falls off the lagging strand. A TriPolIII replisome synthesizes longer DNA strands (MORE PROCESSIVE) and leaves less gaps to be filled in by Pol I in the lagging strand (DNA replication is overall more efficient with the third Pol III present - processing of Okazaki fragments is very efficient).

Gaps would be present because RNA would have been removed but not replaced with DNA (improperly processed Okazaki fragments) or the DNA fragments have not been ligated with each other.

The movement of the Pol III on the leading strand mediates ______

the detachment of the polymerase on the lagging strand

What happens when DNAPs and RNAPs (interference between replication and transcription) collide (speed of DNAPs >>>>> speed of RNAPs)?

In bacteria, replication and transcription occur at the same time. In eukaryotes this is different. DNAP chases RNAP and will catch up and bump into it. Collision between DNAP/RNAP results in both polymerases (Pol III and RNAP) dissociating from the DNA (DNAP ejected from the β-clamp)
and the use of the RNA synthesized by RNAP as a primer for replication. DNAP can resume replication immediately after the collision.

After both dissociate, a new β-clamp is assembled onto the RNA-DNA hybrid, Pol III binds to the newly assembled β-clamp, and Pol III extends the mRNA (the prior β-clamp remains attached to the DNA duplex DNAP was working on).

DNA Pol I can remove the RNA and replace it with DNA via Nick translation afterwards.

This evolved to avoid replication blocks.

Are DNAP/RNAP collisions problematic?

Yes because they would lead to replication blocks. The process stops upon collision, stopping replication (signals cell that there is something wrong). Truncated RNA (from premature transcription termination) not much of a problem, as there are mechanisms to degrade truncated proteins/RNA.

Features of eukaryotic DNA replication linked to the size and organization of eukaryotic genomes:

Large size of eukaryotic chromosomes, limited time for DNA synthesis requires multiple origins of replication

Replication machinery needs to deal with nucleosome packaging of eukaryotic DNA (tightly wrapped DNA around histones)

Problem of linear chromosomes (causes problems with replicating chromosome ends)

Architecture of the eukaryotic replisome?

1) Pol ε (fully processive, does not need a processivity factor, but is associated with one in the case of the replisome - this processivity factor is PCNA): replicates leading strand

2) Pol δ: replicates lagging strand (synthesizes most of the length of the Okazaki fragments)

3) MCM: hexameric ATP-dependent DNA helicase (separates the two strands from each other at the replication fork)

4) PCNA (proliferating cells nuclear antigen): trimeric sliding clamp (both Pols attached to this)

5) Pol α and primase complex: complex contains both activities, primase (RNA primer synthesis), and DNA polymerase α (shortly extends RNA primers)

6) RPA (replication protein A) - the single-stranded binding proteins that stabilize the single stranded regions on the lagging strand (prevents reassociation)

7) FEN1: nuclease that removes RNA primers

8) RFC Clamp Loader: loads PCNA onto the DNA strand/primers

Also loops the lagging strand so the two polymerases can synthesize DNA in the same direction (coupling their activity, they move together).