DNA Replication

What does it mean that DNA replication is semi-conservative? Explain it in your own words, indicating how the two daughter DNA molecules are formed.

At what point in the cell cycle is DNA replicated and why is it essential for replication to occur before cell division begins?

Before the famous Meselson and Stahl experiment, there were three theoretical models to explain DNA replication: semiconservative, conservative, and dispersive. Imagine that you perform this experiment by culturing E. coli bacteria for many generations in a medium with heavy nitrogen ($^{15}\text{N}$) until all their DNA is labelled, and then you transfer them to a medium with light nitrogen ($^{14}\text{N}$).

1. a) Deduce what types of DNA molecules (heavy $^{15}\text{N}\text{-}^{15}\text{N}$, hybrid $^{15}\text{N}\text{-}^{14}\text{N}$, or light $^{14}\text{N}\text{-}^{14}\text{N}$) and in what percentages would appear in the density gradient after one and after two generations if replication were:

1. Strictly conservative.

2. Strictly dispersive.

1. b) Reason in a justified manner which of these two alternative models was ruled out already with the analysis of the first-generation DNA and which obligatorily required waiting for the second generation to be refuted.

Name the main enzyme that synthesises the new strand of DNA during replication and explain its basic function and in which direction (5'→3' or 3'→5') it adds nucleotides.

Name the enzyme that opens the DNA double helix at the replication fork and explain what mechanical problem appears in the molecule ahead of the fork (supercoiling) and what type of enzyme solves it.

Explain the role of the RNA primer at the start of replication and reason why DNA polymerase cannot initiate a new strand from scratch.

At a replication fork, one of the new strands (A) is synthesized continuously, moving toward the fork, while the other (B) is synthesized in fragments, moving away from it. Indicate which of the new strands (A or B) is the leading strand and which is the lagging strand, and explain why one is synthesized continuously and the other in Okazaki fragments, referring to the direction in which DNA polymerase operates.

Place the following stages of replication in order and justify the chosen order: joining of fragments by DNA ligase, unwinding of the double helix by helicase, synthesis of nucleotides by DNA polymerase, removal of primers, formation of Okazaki fragments.

Name the enzyme that synthesises the RNA primers during replication and explain why each Okazaki fragment needs its own primer while the leading strand only needs one at the start.

Once the RNA primers of the lagging strand have been removed, what are the gaps filled with and which enzyme then seals the chain? Explain why the lagging strand takes longer to be completed than the leading strand.

To analyze the synthesis of new DNA strands during replication, a pulse-chase assay is performed in a bacterial culture: cells are exposed to radioactive thymidine ($[^3\text{H}]\text{-thymidine}$) during an ultrashort pulse of $5$ seconds and then transferred to a medium with an excess of non-radioactive thymidine (chase) for $5$ minutes. Following alkaline lysis and denaturation, the DNA strands are separated by density gradient centrifugation. a) Predict and justify what size pattern of radioactive fragments (small ones of about $1000$-$2000$ nucleotides or high-molecular-weight ones) will be observed at the end of the pulse ($5$ s) and how it will change after the chase ($5$ min). b) If the same assay is performed in a mutant strain with thermally blocked DNA ligase, predict the result after the $5$-minute chase and justify your answer. c) Reason why this experiment refutes the hypothesis that both strands are synthesized continuously.

During replication, which nucleotide will be inserted opposite an adenine of the template strand? And opposite a guanine? Explain which property of the DNA structure makes it possible for the copy to be faithful.

The nucleotides incorporated into the new strand arrive as triphosphates (dATP, dGTP, dCTP, dTTP). Explain where the cell obtains the energy needed to form the bonds of the growing chain.

Chemical analysis of the DNA of a species shows that $30\ \%$ of its bases are adenine. State, with reasoning, the percentages of thymine, guanine and cytosine, and explain what consequence this complementarity relationship has for DNA replication.

In a laboratory, a mutant strain of Escherichia coli is obtained that has an alteration in DNA polymerase III: its polymerization rate in the $5'\to 3'$ direction remains intact, but it has completely lost its $3'\to 5'$ exonuclease activity. a) Predict and justify what effect this mutation will have on copying fidelity and on the spontaneous mutation rate per cell division. b) If this strain is cultured in the presence of 2-aminopurine (a base analog that can mispair with cytosine), reason whether the mutation fixation rate will be higher, lower, or equal to that of the wild-type strain. c) Justify at the molecular level why the proofreading activity must remove the mismatched nucleotide in the $3'\to 5'$ direction before continuing with the addition of the next nucleotide.

DNA replication achieves extraordinary fidelity thanks to three successive barriers: the pairing specificity of DNA polymerase (an error of $1$ in every $10^5$ nucleotides), $3'\to 5'$ exonuclease proofreading (final error of $1$ in every $10^7$), and the post-replicative mismatch repair system (final error of $1$ in every $10^9$). a) A cell line from hereditary colorectal cancer (Lynch syndrome) exhibits a mutation rate $100$ times higher than normal, even though its DNA polymerases have intact polymerase and exonuclease activities. Deduce and justify what mechanism is failing in these cells. b) To correct an error after the passage of the fork, the repair system must distinguish the template (old) strand from the newly synthesized one. Reason how the cellular machinery discriminates between both strands to avoid replacing the base on the original strand. c) If a mismatch error (for example, a guanine incorrectly paired with a thymine) escapes all correction systems and the cell goes through two complete rounds of cell division, deduce what proportion of the final daughter DNA molecules will carry the permanently fixed mutation.

How many replication forks are formed from a single origin of replication and in which direction do they advance? Explain the advantage of eukaryotic chromosomes having many origins of replication.

State two differences between the replication of a bacterial chromosome and that of a human chromosome, considering the shape of the DNA and the number of origins of replication.

The DNA polymerase of E. coli adds about $1000$ nucleotides per second at each fork. Calculate how long it will take, at a minimum, to replicate its circular chromosome of $4.6$ million base pairs if it is replicated from a single origin bidirectionally, and comment on the result by comparing it with the generation time of the bacterium (about $20$ minutes).

After replication, each chromosome is made up of two sister chromatids. Explain what relationship the DNA sequences of both chromatids bear to each other, why they remain joined for a time and at what moment of cell division they separate.

Two human cell cultures are compared in the laboratory: normal dermal fibroblasts and cancer cells from a lung carcinoma cell line. Average telomere length is monitored over the course of $60$ population doublings. The fibroblasts enter replicative senescence at doubling $40$ with progressive telomeric shortening, whereas the cancer cells proliferate continuously and maintain their telomere length intact. a) Justify at the molecular level why conventional replication machinery cannot complete the $5'$ end of the lagging strand after the removal of the last RNA primer (the "end replication problem"). b) A therapy is designed that introduces into the tumor cells a synthetic oligonucleotide complementary to the template RNA of human telomerase ribonucleoprotein (hTR). Predict and justify what effect this treatment will have on telomerase activity, telomere length, and the long-term viability of the tumor cells.

Eukaryotic DNA polymerase adds about $50$ nucleotides per second and an average human chromosome can have $150$ million base pairs. a) Calculate how long it would take to replicate that chromosome if there were only one origin of replication at its center. b) Explain how the eukaryotic cell solves this speed problem. c) Reason what advantages and what risks the strategy of using many origins of replication has.

A thermosensitive yeast mutant for the gene encoding DNA ligase I is isolated. At $25\text{ }^\circ\text{C}$ the yeast divides normally, but when cultured at $37\text{ }^\circ\text{C}$ the enzyme is inactivated and the cell arrests its cell cycle before entering mitosis. a) A student claims: "At $37\text{ }^\circ\text{C}$, only the lagging strand will have breaks because Okazaki fragments are formed on it; the leading strand will be a single, perfectly continuous chain from one end of the chromosome to the other." Refute this statement, taking into account the presence of multiple replication origins (replicons) per linear chromosome. b) Specify the exact type of chemical bond that cannot be sealed in the absence of ligase (indicating the terminal $3'$ and $5'$ chemical groups) and reason why DNA polymerases cannot carry out this sealing reaction on their own. c) Justify why the presence of unsealed breaks in the double helix activates a cell cycle checkpoint, preventing mitosis.

To verify whether replication is continuous or discontinuous, researchers carry out pulse labelling with tritiated thymidine for $5$, $15$, and $60$ seconds in bacteria. After denaturing the DNA, they analyse the size of the radioactive strands by centrifugation. At $5$ seconds, they only detect short fragments ($1000$–$2000$ nucleotides), but as the labelling time increases, the radioactivity progressively shifts toward high-molecular-weight fragments. a) Reason why this result supports that replication occurs in a semi-discontinuous manner rather than continuously on both strands. b) Predict and justify what result would have been obtained in the three measurements if both strands were synthesised continuously from the replication origin. c) Explain the unavoidable molecular cause (structural and enzymatic) why the cell is forced to synthesise one of the two strands based on small fragments.