1. Figure 12-12. A model of eukaryotic DNA replication: in vitro replication of SV40 viral DNA . Replication of eukaryotic DNA viruses has served as a model for studying eukaryotic DNA replication. This is because DNA viruses have a well-defined origin of replication and generally use the same proteins that cells use to replicate their DNA. Below is a step-by-step outline of replication of the SV-40 eukaryotic DNA virus:
A. The viral protein called T-antigen (Tag) binds to the origin and melts the DNA. T-antigen hexamer is a helicase. T-antigen helicase serves the same function as DnaA and DnaB proteins in E. coli, except that Tag helicase moves toward the 5' end of the DNA strand, opposite of the direction of movement of the E. coli DnaB helicase.
B. Replication protein A (RPA) is a single strand DNA binding protein that stabilizes melted ssDNA. It functions like SSB in E. coli.
C. Eukaryotic primase binds to the ssDNA together with DNA polymerase-alpha. Like E. coli primase, the eukaryotic primase makes a short RNA primer which is then extended by DNA polymerase-alpha. RFC, replication factor C binds to and stimulates Pol-alpha activity.
D. The protein proliferating cell nuclear antigen (PCNA) replaces DNA polymerase-alpha with the highly processive DNA polymerase-delta. DNA polymerase-delta is similar to DNA polymerase III in E. coli. Remember that processive means that this polymerase can copy long stretches of template without falling off.
E. Lagging strand synthesis is carried out discontinuously by a complex of pol-alpha, primase and RFC.
In summary, the enzymology of DNA replication in prokaryotes and eukaryotes is similar. In both cases a battery of proteins is required, partly due to the fact that DNA polymerase is limited in its ability to manipulate the DNA molecule.
2. Figure 6-40 (Alberts): This is a diagram comparing addition of a nucleoside triphosphate to the 3' end of a growing DNA chain with addition to the 5' end, which has never been found for DNA replication. It doesn't explain why 5' addition did not evolve and we will probably not know the answer to that question, but it does show why different enzymes are needed to carry out the two reactions. It has to do mainly with the energetics of the process of phosphodiester bond formation. For polymerization in the 5'-to-3' direction, which occurs in DNA replication, each incoming deoxyribonucleoside triphosphate has the energy needed to add itself to the DNA chain stored in its triphosphate groups. For a polymerase working in the opposite direction, i.e. 3'-to-5', the end of the growing DNA chain carries the stored energy needed to add the next deoxyribonucleoside triphosphate in the triphosphate group on the end of the chain. This is called head-growth polymerization, and it is used to link amino acids together during translation to make polypeptide chains, but for whatever reason, it did not evolve as the successful way of polymerizing DNA chains.
3. Figure 12-13. Mechanism of action of telomerase. An RNP called telomerase elongates the 3' telomeric end of the lagging-strand DNA template. The telomerase shown here adds TTTTGGGG nucleotide repeats by a reverse transcription mechanism. The telomerase contains its own template, a strand of RNA that base-pairs with the DNA template. The telomerase then adds deoxyribonucleotides complementary to the RNA template until position 35 is reached. The newly synthesized DNA is then thought to slip back and self-hybridize to expose more RNA template. The telomerase then goes back to work synthesizing more complementary DNA, slippage of the newly made DNA occurs again, re-exposing the RNA template. This process can repeat many times allowing telomerase to produce long stretches of repeated sequence at the end of the chromosome. Note the use of non-Watson-Crick base pairs, which are not as stable as the standard pairs.
Media Connection: Telomerase Replication (Animation 12.4)
1. Figure 5.11 (Cooper text). Model of the E. coli replication fork in which the lagging strand template is folded such that both polymerase III cores move in the same direction as overall movement of the replication fork. We have seen how DNA can twist into supercoils due to helicase activity during template unwinding and how these must be handled during replication in both eukaryotes and procaryotes. Now we will consider in more detail the two classes of topoisomerases and their roles in DNA replication.
2. Figure 12-14: This figure shows the mechanism of action of E. coli topo I. The key step is the cutting of a phosphodiester bond in one of the DNA strands and immediate transfer of this bond to a tyrosine residue in the topoisomerase itself, to produce a DNA-to-protein phosphodiester bond. The cut 3' end of the DNA is then passed under the other strand and resealed to form again a phosphodiester bond, and this passing under removes a negative supercoil. No energy source outside of the DNA and enzyme is required since equivalent bonds are being broken and reformed. What is the definition of a type I topoisomerase? These are enzymes that cleave only one strand of a DNA double helix and then they reseal it to reform the same chemical bonds (phosphodiester bonds) as the starting DNA. The E. coli type I enzyme can remove only negative supercoils, so it does not play a major role in growing fork progression, since this unwinding process generates positive supercoils. E. coli topo I is an essential enzyme for viability since inactivation of the gene encoding this enzyme causes cell death. Its functions are to: i) help maintain the optimum superhelical density of the chromosome by removing negative supercoils, one at a time; ii) may regulate transcription; and iii) may function in DNA repair. In E. coli, the negative supercoils accumulate due to the action of topo II, or DNA gyrase, during replication.
3. Figure 12-16: Type II topoisomerases change DNA topology by breaking and rejoining double-stranded DNA. Topo II enzymes are defined as those that cut both strands on one side of a double helix, pass another portion of the helix through the cut, and reseal the cut on the opposite side with energy provided by ATP hydrolysis. These activities of the E. coli topo II (also called DNA gyrase) have the effect of either changing a positive supercoil into a negative one, or increasing the number of negative supercoils by two. Both prokaryotic and eukaryotic type II topoisomerases catalyze catenation and decatenation, that is the linking and unlinking of two different DNA duplexes. This will become important at the very end of replication of a circular DNA molecule, for example.
4. Figure 12-17: Molecular model for the catalytic activity of E. coli
topoisomerase II (DNA gyrase). This enzyme introduces negative supercoils
at or near the origin of replication in the DNA template. DnaA proteins
bind here, and only initiate replication on a negatively supercoiled template.
A second function of DNA gyrase is to remove positive supercoils that form
in front of the moving replication fork during elongation of the growing
stands.
Here the mechanism is shown. G is a segment of DNA double helix to which
the gyrase binds and this binding drives a major conformational change in
gyrase. The asterisks mark the ATP binding site. Following ATP binding,
another DNA segment is bound (T segment). Then, the G segment is cut, the
T segment is passed through the break in the G segment, and the G segment
is resealed. Then the T segment is release by another major conformational
change in gyrase. ATP hydrolysis is then used to energize a conformational
change back to form 2. From here another cycle of removal of two more supercoils
can occur, or alternatively, the G segment can be released from the enzyme.
5. The story in yeast, the best studied eukaryotic cell for topoisomerase activity, is a little different. In yeast, both topo I and II can relax both positive and negative supercoils, and function in the movement of replication forks. Deletion of the yeast topo I gene is not lethal but does slow growth. Topo II apparently is enough to keep replication going. However, deletions in the topo II gene are lethal, which makes sense because this enzyme is required to separate the two progeny DNA helices during cell division.
1. E. coli Pol III makes about one wrong base pairing per 10,000 nucleotide additions to DNA. Without correction, this would cause a potentially harmful mutation in one out of every ten genes per replication. The measured mutation rate is about one in a billion nucleotide additions (five orders of magnitude fewer). The increased accuracy or fidelity of replication is mainly due to the proofreading function of DNA polymerase.
2. Figure 12-20: Outline of an experiment showing that the 3'-exonuclease activity of E. coli DNA polymerase I can remove a mismatched base (i.e. one that does not follow Chargaff's rules) at the 3' end of a DNA strand. Note the tritium (3H) labeled thymidines correctly paired to adenosine residues and the phosphorous (32P) labeled cytidine that is mispaired. The radioactive emissions of tritium can be distinguished from those of phosphorous 32. When E. coli pol I and thymidine triphosphate are added, the radioactive cytidine is removed from the chain but not the tritiated thymidine, showing that the polymerase can selectively remove mismatched nucleotides.
3. Figure 12-21: A schematic model of the proofreading functions of DNA polymerases. The diagram resembles a partially open right hand. The fingers are the domains of the polymerase that bind the single-stranded template DNA. The catalytic site, where nucleotides are added, is located where the fingers attach to the palm. If the polymerase inserts the wrong nucleotide, this causes a melting of the double-stranded DNA, allowing the 3'-end of the growing stranding to be transferred to the 3' exonuclease site (Exo). Here the mispaired nucleotide is removed. Then the DNA strand flips back into the polymerase site and elongation resumes.
4. Proofreading is carried out by almost all bacterial DNA polymerases and the delta and epsilon DNA polymerases of animal cells, but not the alpha polymerase of animal cells. For E. coli, where we understand the replication process in greatest detail, there are four processes that contribute to high fidelity DNA replication:
A. Correct pairing of the incoming nucleotide with its complementary base according to Chargaff's rules in the template strand.
B. A shape change (conformational change) by the polymerase, which takes time to carry out, allows time for an incorrectly paired nucleotide to dissociate before it is covalently attached to the growing DNA strand by phospodiester bond formation.
C. 3' Exonuclease activity that can remove a mismatched nucleotide at the 3' terminus of the growing DNA chain.
D. Removal of a mispaired nucleotide deeper in the growing DNA chain, i.e. many nucleotide-additions after the wrong one was originally added, by the mismatch repair system.
