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 labeled, 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.

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

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.

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.

Name the main enzyme that synthesizes 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 explain 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 synthesizes 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.

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.

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.

A temperature-sensitive 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 labeling with tritiated thymidine for $5$, $15$, and $60$ seconds in bacteria. After denaturing the DNA, they analyze the size of the radioactive strands by centrifugation. At $5$ seconds, they only detect short fragments ($1000$–$2000$ nucleotides), but as the labeling 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 synthesized continuously from the replication origin. c) Explain the unavoidable molecular cause (structural and enzymatic) why the cell is forced to synthesize one of the two strands based on small fragments.

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.

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.

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.

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).