CMMB 411 - 3 DNA Replication

properties of DNA polymerase

synthesizes the new DNA strand only from 5’ to 3’, travels reads the template DNA strand in the 3’ to 5’ direction

cannot synthesize a new DNA strand de novo, requires an RNA primer with a 3’OH to add the first dNTP

DNA polymerase active site

single site that can bind to four different substrates and catalyze four different reactions (incorporation of dATP, dCTP, dGTP, dTTP)

E. coli DNA pol

five types - I, II, III, IV, V

5’ to 3’

DNA pol synthesizes in the _____ direction

3’ to 5’

DNA pol reads the template strand from the ______ direction

DNA pol II

translesion synthesis, has 3’ to 5’ exonuclease activity only

DNA pol IV and V

translesion synthesis, has no exonuclease activity at all

DNA pol I

for okazaki fragment processing and DNA repair, has both 3’ to 5’ and 5’ to 3’ exonuclease processing

structure of polymerases

resembles a right hand, palm, thumb and finger domain

palm domain

polymerases, contains the catalytic site where the correct dNTP is incorporated into the growing daughter DNA strand

finger domain

polymerases, binds the incoming dNTP and brings it close to the Mg2+ ions in the catalytic site, fingers close to achieve this

thumb domain

polymerases, not involved in catalysis but positions the primer and terminal 3’ OH in the correct orientation in the active site

DNA polymerase reaction

to incorporate deoxynucleotides, active site contains insertion and post-insertion site

insertion site - location of base pairing between template nucleotide and incoming dNTP

post-insertion site - contains the 3’ terminal base pair of primer or daughter DNA strand

after incorporation of the dNTP, the DNA pol translocates along the template DNA strand so that the insertion site is ready to accept the next dNTP

oxygen of terminal ribose 3’OH is the nucleophile that attacks the a-phosphorus of the incoming dNTP, a-phosphate is incorporated as a phosphodiester bond in the daughter DNA strand

pyrophosphate (PPi) is rapidly hydrolyzed by pyrophosphate to two phosphate groups providing the energy for DNA synthesis

exonucelase activity of DNA pol I

has two distinct exonuclease sites for both 3’ to 5’ and 5’ to 3’

3’ to 5’ exonuclease (proofreading activity)

removes incorrect base at the 3’ terminus of the DNA daughter strand due to incorporation of the wrong dNMP during DNA replication

synthesizing in 5’ to 3’ → can just backtrack the other way if makes a mistake

5’ to 3’ exonuclease (nick translation)

removes dNMPs/NMPs from the 5’ end of a nick and fills in the gap (polymerization) with correct dNTPs

used to remove RNA primers during DNA replication and repair DNA lesions

endonuclease activity

cuts internally, e.g. MNase (cuts linker DNA into fragments)

exonuclease activity

cuts the end of a strand of DNA, can be 3’ to 5’ or 5’ to 3’

metal ions in DNA pol

two Mg2+ ions coordinated by conserved aspartate residues in active site of DNA polymerase

one deprotonates the terminal 3’OH to form the O- nucleophile that attacks the a-phosphorus of incoming dNTP

other neutralizes the negative charge of the B and Y phosphates of the dNTP and stabilizes pyrophosphate facilitating its release

similar process in 3’ to 5’ nucleases except Mg 2+ acts to remove the incorrect dNMP

accuracy of DNA pol

must be highly accurate to prevent mutations

• creates a specific shaped active site for catalysis with conformation change

• AT and GC base pairs form a similar shape that properly fits into the active site, thereby allowing rapid dNTP incorporation (induced fit)

• base mispairing produces wrong geometry for active site → stalls so proofreading can replace

• incorporate a wrong nt 1/10,000 incorporations but proofreading reduces errors by 100-fold

processivity

efficiency of DNA pol in DNA synthesis, the average number of dNTPs incorporated before the enzyme falls off the template DNA

determines the speed of DNA synthesis

distributive synthesis

DNA pol detaches from the template DNA after incorporating each dNTP and then reattaches to template DNA to resume synthesis of daughter strand (low processivity)

processivity synthesis

DNA pol remains tightly bound to dsDNA and slides along the template DNA throughout DNA synthesis with help of the sliding clamp (high processivity)

DNA pol III

1000x more processivity than DNA pol I, a replicative polymerase

full function requires holoenzyme (not just core) for max processivity (1 kb/s)

DNA pol III core

heterotrimer of alpha (DNA pol activity), epsilon (3’ to 5’ exonuclease proofreading) and theta (nonessential subunits)

DNA pol III holoenzyme

includes three pol III cores, three B sliding clamps, and a clamp loader complex (tau)

1 core for leading strand, 2 cores for lagging strand

clamp loader

five subunit complex containing three tau subunits with a flexible C-terminal linker that binds to the Pol III core and binds DnaB helicase and has ATPase activity

delta subunit opens the sliding clamp and the delta’ subunit modulates the delta subunit function

two small subunits (psi and chi) bind the clamp loader to stabilize the complex

beta sliding clamp

increases the efficiency of DNA pol

a homodimer of six repeated domains that forms a ring around the duplex of new synthesized DNA

contacts the DNA pol and helps it to not dissociate from DNA during replication, greatly increases processivity and speeds up polymerization

clamp loader is required to position it onto DNA

also involved in post-replication DNA repair

clamp loader process

1. ATP binding to the tau subunits causes a conformation change allowing the clamp loader to bind and open the sliding clamp (the clamp loader without the C-terminal is called gamma Y)

2. clamp loader-sliding clamp complex is brought to a primer-template junction on the DNA (RNA-ssDNA junction) and the opened sliding clamp is positioned around the primed DNA at the junction

3. ATP hydrolysis by the clamp loader causes it to detach from the sliding clamp and DNA

4. once released from the clamp loader, the sliding clamp closes around the primed DNA

5. release of ADP and Pi and binding to a new ATP allows the clamp loader to initiate a new cycle of loading

helicase (DnaB)

separates template DNA, unwinds double stranded nucleic acids (dsDNA, hybrids, dsRNA) using ATP hydrolysis

pulls strand through hole, a ring-shaped hexamer that encircles one of the two single DNA strands at the replication fork near the ssDNA-dsDNA junction

unwinding occurs ahead of the DNA polymerase during replication

depending on which single DNA strand the DNA helicase has encircled, it can travel either 5’ to 3’ or 3’ to 5’ (e.g. travels 5’ to 3’ on the lagging strand template of the replication fork)

speed of unwinding is faster when tethered to the DNA pol III holoenzyme through the tau subunits

topoisomerases

remove supercoils, mainly gyrase which requires ATP - relieves positive supercoiling tension caused by DNA unwinding by introducing negative supercoils

dsDNA in front of the replication fork needs to be relaxed by reducing the Lk since helicase cannot untwist the DNA strands

topoisomerases untwist the DNA by cutting one or both strands of DNA to unwind it before resealing the phosphodiester bonds

primase (DnaG)

synthesizes RNA primers (11-13 rnt) de novo, provides a 3’ OH so that DNA pol can incorporate the first dNTP

leading strand - starts with activation of the E. coli DnaG primase when it binds to DnaB helicase, only one RNA primer

lagging strand - needs 1000s of RNA primers to synthesize Okazaki fragments

removing RNA primers

DNA pol I removes the primers with its 5’ to 3’ exonuclease activity or RNase H digests most of the RNA primer in an RNA-DNA hybrid (and DNA pol I removes the last rNMP attached to DNA that RNase H cannot)

DNA pol I binds to the primer-template junction and repairs the gap by filling it with the complementary sequence of dNMPs (nick translation)

ligase seals the nick in the phosphodiester backbone using ATP (seals between two DNA fragments)

ligase

seals the nick in the phosphodiester backbone using ATP (seals between two DNA fragments)

RNase H

digests most of the RNA primer in an RNA-DNA hybrid (and DNA pol I removes the last rNMP attached to DNA that RNase H cannot)

single strand binding protein (SSB)

unwound ssDNA at the replication fork is not naked, coated by protein

quickly binds to ssDNA generated by helicase to stabilize and protect it from endonucleases in the cell

prevents ssDNA from reannealing and forming secondary structures like hairpins that impede DNA pol

binding to DNA is cooperative because each SSB binds to another and increases rate of coating ssDNA

issues of simultaneous synthesis of leading and lagging strands

• lagging strand is slower (in fragments)

• more ssDNA would need to accumulate on lagging strand to protect it

• all cores are in the same holoenzyme - 1 for leading, 2 for lagging, need to stay together to not tear apart enzyme (they’re attached by the tau complex clamp loader)

E. coli replisome

contains the DNA pol III holoenzyme, DnaB helicase, DnaG primase, at the replication fork and coordinates priming, leading, and lagging strand synthesis

helicase unwinds DNA and leading strand template is exposed and bound by a pol III core and sliding clamp immediately (few SSB required)

lagging strand - delay in binding of DNA pol III cores, so ssDNA is spooled out in a loop and entirely coated with SSB (loops => trombone model)

DnaG primase transiently associates with the DnaB helicase to synthesize the primer on the lagging strand template then the clamp loader assembles the clamp and DNA pol III core at the primer-template junction