DNA Replication Foundations

100 practice Q&A flashcards covering key concepts from the DNA replication lecture notes.

What is the overall process of DNA replication as described in the notes?

A template-mediated process that yields two new DNA strands with high fidelity to maintain genome integrity.

What is the origin of replication?

The site on a chromosome where replication begins.

How many origins of replication do prokaryotic and eukaryotic chromosomes typically have?

Prokaryotes (like E. coli) have a single origin; eukaryotic chromosomes have multiple origins.

What enzyme facilitates DNA unwinding at the origin of replication?

Helicase.

What is the rate of replication for E. coli and how long does it take to replicate its genome?

Two replication forks at 500–1000 nucleotides per second, about 40 minutes to replicate the genome.

How long would replicating the human genome take with a single replication fork moving at ~50 nt/s?

Approximately 6–8 hours.

What does semiconservative DNA replication mean?

Each daughter DNA molecule contains one original strand and one newly synthesized strand.

In which direction is DNA synthesized by polymerases?

5' to 3' direction.

Why is a free 3'-OH group required for DNA synthesis?

It provides the site for nucleophilic attack by the incoming nucleotide during elongation.

Which enzyme lays down the RNA primer to start DNA synthesis?

Primase.

What are Okazaki fragments?

Short fragments of DNA synthesized on the lagging strand as the replication fork opens.

How is the leading strand synthesized relative to the replication fork?

Continuously, from 5' to 3' as the parental strand unwinds.

What are the four basic steps of DNA replication?

Separation of strands, formation of replication fork with primers and fragments, chain elongation by DNA polymerase, removal of primers and sealing of nicks by ligase.

Name the three eukaryotic DNA polymerase complexes at the fork and their roles.

α (primase and initial nucleotides), δ (replicative synthesis, highly processive, 3'-5' exonuclease), ε (replicative synthesis and error repair, 3'-5' exonuclease).

Name the three prokaryotic DNA polymerases and which one does most replication.

DNA polymerase I, II, III; Pol III carries out most replication.

What are the catalytic activities of E. coli DNA polymerase I?

5'→3' polymerase, 5'→3' exonuclease, and 3'→5' exonuclease proofreading.

How processive is DNA polymerase III in E. coli?

Very processive; synthesizes more than 500,000 nucleotides before dissociating.

How processive is DNA polymerase I in E. coli?

Not very processive; synthesizes about 3–200 nucleotides before falling off.

Do Pol I and Pol III have 3'→5' proofreading activity, and what about 5'→3' exonuclease?

Both Pol I and Pol III have 3'→5' proofreading; Pol III lacks 5'→3' exonuclease activity.

What are the three main eukaryotic polymerases at the fork and their functions?

α: primase and initial nucleotides; δ: replicative synthesis, highly processive, 3'-5' exonuclease; ε: replicative synthesis and error repair, 3'-5' exonuclease.

What are the non-replication polymerases in eukaryotes and their roles?

β: base excision repair; γ: mitochondrial DNA replication.

What is the role of primase in replication?

To synthesize an RNA primer that provides a free 3'-OH for DNA extension.

What is the role of helicase in replication?

To unwind the double helix at the replication fork.

What is the role of topoisomerase (gyrase in prokaryotes) in replication?

To relieve supercoiling ahead of the fork by introducing nicks.

What is the role of single-stranded DNA-binding proteins (SSBs)?

To prevent newly separated strands from reannealing.

What is the role of ligase in replication?

To seal nicks in the phosphodiester backbone after primers are removed.

How are the replication processes in prokaryotes and eukaryotes related?

They are functionally conserved; many proteins are analogous, though eukaryotic enyzmes are more complex.

How do origins of replication differ between eukaryotes and prokaryotes?

Eukaryotes have multiple origins per chromosome, prokaryotes generally have a single origin.

Where does DNA replication occur in eukaryotes?

In the nucleus.

What must happen to chromatin before DNA replication in eukaryotes?

Chromatin must be unpacked with histone separation.

What is S-phase in the cell cycle?

The phase in which DNA synthesis occurs.

List the phases of the eukaryotic cell cycle in order.

G1, S, G2, M.

What cell fates are possible in G1?

Senescence (G0), differentiation (G0), apoptosis, and proliferation.

What happens to DNA content during S phase?

DNA content doubles from 2N to 4N in about 6–8 hours.

What happens in G2 phase?

Preparation for M-phase; centrosomes duplicate.

What occurs during M phase?

Mitosis.

What is telomere?

The end of eukaryotic chromosomes consisting of tandem repeats of a hexanucleotide sequence.

Why are telomeres important for chromosome integrity?

They prevent loss of DNA at chromosome ends due to the end replication problem.

What is telomerase?

An RNA-dependent DNA polymerase (reverse transcriptase) that maintains telomeres by adding repeats; carries its own RNA template.

Why does telomere shortening relate to aging?

Telomere length can serve as a clock for cellular aging; shortening is associated with aging.

What is the clinical correlation of telomerase activity with cancer?

Telomerase is upregulated in many cancers, helping cells bypass senescence.

What is Dyskeratosis Congenita (DKC) and its key features?

An inherited telomere biology disorder with skin hyperpigmentation, oral leukoplakia, nail dystrophy; most deaths due to bone marrow failure.

What genetic mutations are linked to autosomal dominant DKC?

Mutations in telomerase RNA component and TERT (telomerase reverse transcriptase).

How does DKC affect stem cell proliferation and blood counts?

Reduces proliferative capacity, leading to aplastic anemia and low blood/immune cell counts.

What is the most common cause of death in DKC patients?

Bone marrow failure (aplastic anemia).

How can telomere length change across generations in families with DKC?

Telomeres shorten with each generation, causing worsening symptoms.

What is Idiopathic Pulmonary Fibrosis (IPF) and its telomere link?

A form of pulmonary fibrosis; familial IPF often has shortened telomeres or telomere-related mutations.

What are common IPF symptoms?

Chronic cough and shortness of breath due to lung fibrosis.

Which genes are associated with telomere-related mutations in IPF?

TR (telomerase RNA) and TERT.

What is the role of TR in telomere biology?

Telomerase RNA component that serves as the RNA template.

What is the role of TERT in telomere biology?

Telomerase reverse transcriptase, the catalytic subunit of telomerase.

What is the telomere length clock concept?

Telomere length serves as a clock to time cellular age.

How is telomerase activity typically different in aging vs cancer cells?

Telomerase is generally not expressed in normal somatic cells but is upregulated in many cancers.

What is the clinical picture of IPF in relation to telomere biology?

Shortest telomeres or telomere mutations found in a subset of familial IPF; mutations in related genes may be present.

What does the term end replication problem describe?

The inability of conventional DNA polymerases to fully replicate telomeres, leading to progressive shortening without telomerase.

What is the role of the alcohol of Z? (Note: intentionally avoid non-existent terms)

N/A

Which polymerase in eukaryotes is primarily involved in base excision repair outside replication forks?

β polymerase.

Which polymerase is primarily responsible for mitochondrial DNA replication in eukaryotes?

γ polymerase.

Where does replication take place in prokaryotes?

In the cytoplasm (no nucleus).

What is the significance of the chromatin unpacking step for replication?

Enables access of replication machinery to DNA and enables progression through chromatin.

What ensures high fidelity during replication at the fork in eukaryotes?

Proofreading by 3'-5' exonuclease activities of δ and ε polymerases.

What is the functional equivalence between prokaryotic gyrase and eukaryotic topoisomerase?

Both relieve DNA supercoiling ahead of the replication fork; gyrase is the prokaryotic form.

What is meant by semi-discontinuous replication?

Leading strand is synthesized continuously; lagging strand is synthesized as short Okazaki fragments.

What is the basic role of RNA primers in replication?

Provide a free 3'-OH group for DNA extension and initiation of synthesis.

Which strand is synthesized in fragments and what are those fragments called?

The lagging strand; Okazaki fragments.

What drives the initiation of replication at origins?

Helicase unwinds DNA and primase lays down RNA primers to begin synthesis.

Which step of replication is directly followed by ligation?

Removal of RNA primers and sealing of nicks by ligase.

What is the typical size of the E. coli chromosome?

Approximately 4.6 x 10^6 base pairs.

What feature characterizes the origin of replication in E. coli?

A single origin on the circular chromosome.

What feature characterizes the telomere sequence in the notes?

Tandem repeats of a hexanucleotide sequence similar to GGGGTT.

What is the consequence of telomere shortening for cellular aging?

Progressive loss of telomeric DNA contributes to aging phenotypes.

What is the role of RNA in telomerase?

Telomerase carries an RNA template used to extend telomere repeats.

Which phase is associated with histone production?

S-phase, concurrent with DNA synthesis.

Which phase immediately precedes M-phase in the cell cycle?

G2 phase.

What potential cell fate occurs when cells exit the cell cycle into G0?

Senescence or differentiation.

Why is replication fidelity important for preventing cancer?

High fidelity reduces mutations that can drive cancer development.

What is the primary function of ligase during replication?

To seal nicks after RNA primers are removed and fragments are joined.

What happens to replication timing between prokaryotes and eukaryotes?

Eukaryotic replication occurs during S-phase at defined times; prokaryotes replicate when nutrients permit, with less strict timing.

Which component directly provides the 3'-OH end for DNA synthesis on the growing strand?

The primer provides the free 3'-OH; the growing strand extends from it.

What is the directionality of primer synthesis relative to the growing DNA strand?

Primers set the 3'-OH for extension in the 5' to 3' synthesis direction.

What is the main concept behind replication forks being symmetrical?

Two replication forks move bidirectionally from origins, creating a replication bubble.

Which enzyme lays down RNA primer and initiates Okazaki fragment formation?

Primase.

Which enzyme relieves torsional strain during unwinding of DNA?

Topoisomerase (gyrase in prokaryotes).