DNA Replication

Flashcards covering the mechanisms, enzymes, stages, regulation, and clinical correlations of prokaryotic and eukaryotic DNA replication.

During which stage of the cell cycle does DNA replication take place?

It occurs during the S phase (Synthesis phase), prior to cell division (mitosis).

Why is DNA replication characterized as semiconservative?

Each newly formed daughter DNA molecule conserves one original parental strand and contains one newly synthesized strand built from free nucleotides.

In which direction does new DNA strand elongation proceed?

Synthesis of new DNA proceeds exclusively in the $5' \rightarrow 3'$ direction.

What chemical reaction takes place to attach an incoming nucleotide to the growing DNA chain?

The $3'$ hydroxyl group ($\text{-OH}$) of the growing strand attacks the incoming nucleoside triphosphate, releasing pyrophosphate ($PP_i$).

What is the key functional difference between polymerases and nucleases?

Polymerases synthesize nucleic acids by creating phosphodiester (PDE) bonds, whereas nucleases hydrolyze phosphodiester bonds to break nucleic acids down.

How do exonucleases differ from endonucleases?

Exonucleases cleave nucleotides from the $5'$ or $3'$ terminal ends of a nucleic acid, while endonucleases cleave internal phosphodiester bonds within the strand.

What role does DNA helicase play during DNA replication?

It unwinds the double-stranded parental DNA by cleaving hydrogen bonds between complementary base pairs, creating replication forks.

What is the function of single-stranded DNA-binding proteins (ssDNA-binding proteins / SSB)?

They bind to and stabilize the separated single-stranded template DNA to prevent them from reannealing.

What nucleotide composition typically characterizes the origin of replication (ori)?

Short consensus sequences composed predominantly of adenine-thymine ($AT$) base pairs.

Based on the provided illustration, how do the mechanisms of action of Topoisomerase I and Topoisomerase II differ?

Topoisomerase I transiently cuts a single strand of the double helix to allow relaxation and reseals it, whereas Topoisomerase II cuts both strands, passes an unbroken double helix through the break, and reseals both strands.

Which enzyme relieves positive supercoiling ahead of the replication fork in prokaryotic cells?

DNA topoisomerase II (also designated as DNA gyrase).

Why is RNA primase required to initiate DNA synthesis?

DNA polymerases cannot initiate synthesis de novo; RNA primase must synthesize a short stretch of RNA (approximately 10 nucleotides) to provide a free $3'\text{-OH}$ group for elongation.

How do RNA primer requirements differ between the leading strand and the lagging strand?

The leading strand requires only one RNA primer at the origin, whereas the lagging strand requires multiple primers for discontinuous synthesis.

What are Okazaki fragments?

Discontinuous, short segments of newly synthesized DNA linked to an RNA primer formed on the lagging strand.

Which DNA polymerases synthesize the leading and lagging strands in eukaryotic cells?

DNA polymerase $\alpha$ and DNA polymerase $\delta$ working in coordination.

What is the primary function of eukaryotic DNA polymerase $\gamma$?

Replication of mitochondrial DNA.

Which eukaryotic DNA polymerases are primarily involved in DNA repair?

DNA polymerases $\beta$ and $\epsilon$.

Which eukaryotic enzyme removes RNA primers during replication?

RNase H (exhibiting $5' \rightarrow 3'$ exonuclease activity).

Which prokaryotic enzyme removes RNA primers and fills the resulting gaps?

DNA polymerase I (employing $5' \rightarrow 3'$ exonuclease activity to remove the primer and $5' \rightarrow 3'$ polymerase activity to replace it with DNA).

What is the function of DNA ligase in DNA replication, and what energy source does it utilize?

It joins adjacent DNA fragments (such as Okazaki fragments) by catalyzing phosphodiester bond formation, a process requiring ATP cleavage.

What specific enzymatic activity is demonstrated in this diagram when an incorrect nucleotide is detected?

The $3' \rightarrow 5'$ exonuclease proofreading activity of DNA polymerase, which hydrolytically excises mismatched nucleotides.

What occurs during the termination stage of eukaryotic DNA replication?

Recoiling (double helix and supercoiling) of parental and new strands occurs, alongside reconstitution of the chromatin structure with histone and non-histone proteins.

Which antimicrobial drug classes inhibit prokaryotic DNA gyrase (topoisomerase II)?

Quinolones and fluoroquinolones (such as Levofloxacin, Ciprofloxacin, and Moxifloxacin).

Name two anticancer agents that act by inhibiting eukaryotic topoisomerase II.

Etoposide and teniposide.

According to the diagram, what structural differences exist between prokaryotic and eukaryotic chromosomes regarding replication origins and bubbles?

Eukaryotes have linear DNA with multiple origins of replication and multiple replication bubbles, while prokaryotes possess circular DNA with a single origin of replication and one replication bubble.

Why do eukaryotic cells require telomerase whereas prokaryotic cells do not?

Eukaryotes contain linear chromosomes that risk end-shortening after removal of terminal RNA primers, whereas prokaryotes possess circular DNA molecules that lack chromosome ends.

What clinical condition is associated with a defect in the mismatch repair pathway (e.g., MSH2, MLH1)?

Lynch syndrome (Hereditary non-polyposis colorectal cancer).

What disease results from a deficiency in Nucleotide Excision Repair (NER) of thymine dimers?

Xeroderma pigmentosa.

What sequential enzymatic steps perform Base Excision Repair (BER) when cytosine undergoes deamination to uracil?

1. DNA glycosylase removes the uracil base leaving an AP site; 2. AP endonuclease removes the AP site; 3. DNA polymerase synthesizes replacement DNA; 4. DNA ligase seals the strand nick.

Which metal ions serve as essential cofactors for DNA replication?

$Mg^{2+}$ and $Mn^{2+}$.