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.

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.

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.

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.

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.

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.

In early embryos of the insect Drosophila melanogaster, the S phase lasts only $5$ minutes, whereas in adult somatic cells it lasts for about $8$ hours. However, the replication fork progression rate is identical at both stages: about $40$ nucleotides per second. a) If the haploid genome of Drosophila has $1,8 \cdot 10^8$ base pairs, calculate the maximum average distance (in base pairs) that can exist between two adjacent active replication origins in the embryo to complete replication in $5$ minutes, assuming bidirectional replication. b) Interpret what accounts for the difference in S-phase duration between the embryo and the adult if the polymerization rate does not change. c) Reason what genomic risk it would pose to a eukaryotic cell if the same replication origin were activated ("fired") twice within the same S phase, and how the cell prevents this event.