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.

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.

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.

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.