L4&5 DNA Replication, DNA Mutations, and DNA Repair Systems

Comprehensive practice Q&A flashcards covering Eukaryotic DNA Replication, DNA Damage & Mutations, and DNA Repair Systems based on Module 3 Lectures 4&5.

What is the ploidy and chromosome composition of normal human somatic cells?

Human cells are diploid ($2n$), containing two copies of each chromosome: 22 pairs of autosomal chromosomes plus sex-specific chromosomes (XX or XY).

How does the DNA content change across the different phases of the eukaryotic cell cycle?

Cells have $2C$ DNA content ($2n$) in $G_1$ phase, replicate their DNA from $2C$ to $4C$ ($2n \rightarrow 4n$) during $S$ phase, maintain $4C$ content in $G_2$ phase, and divide into two daughter cells with $2C$ content each during $M$ phase.

What does it mean that eukaryotic DNA replication is semi-conservative?

During replication, the parental DNA strands melt apart and each parental strand serves as a physical template for the synthesis of a new complementary daughter strand.

What is the Origin Recognition Complex (ORC) and what is its role during replication initiation?

ORC is a 6-subunit protein complex bound to origins of replication (ORIs) throughout the cell cycle that serves as a platform for recruiting replication proteins and initiating DNA replication.

Which enzyme performs the local opening and unwinding of the DNA double helix at origins of replication?

MCM DNA helicase, which is recruited by ORC in $G_1$ phase to form the pre-replicative complex and becomes activated during $S$ phase to unwind DNA bidirectionally.

What are the three fundamental properties of all DNA Polymerases during DNA synthesis?

1) Strict unidirectionality of DNA synthesis in the $5'$ to $3'$ direction; 2) Requirement of an RNA/DNA primer to initiate synthesis; 3) Proofreading capability via $3'$ to $5'$ exonuclease activity.

How does DNA synthesis differ between the leading strand and the lagging strand?

The leading strand is oriented $3'$ to $5'$ relative to fork progression and undergoes continuous synthesis toward the fork; the lagging strand is oriented $5'$ to $3'$ and undergoes discontinuous synthesis away from the fork, forming short Okazaki fragments.

What is the function of Single-Strand DNA-binding (SSB) proteins at the replication fork?

SSB proteins bind to single-stranded DNA to protect it, prevent re-annealing and secondary structure formation, and are dislodged by DNA polymerase during synthesis.

What is the key functional difference between DNA Topoisomerase I and DNA Topoisomerase II?

Both induce nicks to release torsional tension in DNA upstream of the replication fork, but DNA Topoisomerase I is ATP-independent while DNA Topoisomerase II is ATP-dependent.

What causes the end-replication problem in linear eukaryotic chromosomes?

The removal of the terminal RNA primer on the lagging strand leaves an unfillable gap (approx. $100\text{ bp}$), resulting in progressive telomere shortening with each cell division until senescence or cell death occurs after 50 to 70 divisions.

How does Telomerase maintain telomere length in immortal cell types?

Telomerase reverse transcribes its own internal RNA template to extend the $3'$ end of telomere DNA repeats ($(\text{AGGGTTA})_n$), allowing DNA primase and DNA polymerase to complete lagging strand replication.

Which clinical agents inhibit DNA replication by targeting precursor (dNTP) synthesis?

Methotrexate and 5-fluorouracil.

What is the baseline human mutation rate per nucleotide per cell division?

$1 \times 10^{-8}$ per nucleotide per cell division (a 1 in 100 million probability per nucleotide per generation).

How is a DNA mutation distinguished from a DNA polymorphism in a population?

A DNA mutation is a rare permanent sequence change occurring at less than $1\text{\%}$ frequency in a population, whereas a polymorphism is frequent, occurring at greater than $1\text{\%}$ frequency.

What specific point mutation causes Sickle Cell Anemia?

A missense mutation in the beta-globin gene that converts Glutamate to Valine ($\text{GLU} \rightarrow \text{VAL}$), causing deoxyHemoglobin S (HbS) to polymerize under hypoxia and distort red blood cells into a sickled shape.

What chromosomal abnormality causes Chronic Myeloid Leukemia (CML) in 95% of cases?

The Philadelphia Chromosome, formed by a reciprocal translocation between chromosome 9 and chromosome 22 ($t(9;22)$) that creates a $BCR::ABL$ fusion gene driving uncontrolled cell proliferation.

What genetic mechanism causes Charcot-Marie-Tooth disease on chromosome 17?

A gene duplication of $PMP22$ on chromosome 17, which leads to myelin destabilization and peripheral neuropathy characterized by loss of muscle tissue and touch sensation.

What DNA repair pathway corrects DNA polymerase proofreading errors, and what cancer syndrome is linked to its defect?

DNA Mismatch Repair (MMR), carried out by MSH2, MSH6, MLH1, and PMS2; congenital defects cause Lynch Syndrome (hereditary non-polyposis colorectal cancer).

What types of DNA lesions are repaired by Base Excision Repair (BER), and what enzyme initiates base removal?

BER repairs small, non-helix-distorting lesions (depurination/abasic sites, Cytosine to Uracil deamination, 8-oxo-Guanine oxidation, and single-strand breaks); DNA Glycosylase initiates repair by removing the damaged base.

What mechanism causes synthetic lethality when PARP inhibitors are used in BRCA1/BRCA2-deficient tumors?

PARP inhibitors block BER and trap PARP, converting single-strand lesions into double-strand breaks during replication; because BRCA1/BRCA2-deficient cells lack functional Homologous Recombination, they cannot repair DSBs and undergo selective cell death.

Which DNA repair system removes bulky, helix-distorting DNA lesions like UV-induced thymine dimers, and what disease results from its loss?

Nucleotide Excision Repair (NER); congenital defects in NER proteins (such as XPA, XPB, XPC, XPD, XPE, POLH) cause Xeroderma Pigmentosum (XP), characterized by extreme sunlight sensitivity and high skin cancer incidence.

How do Homologous Recombination (HR) and Non-Homologous End-Joining (NHEJ) differ in fidelity and cell cycle timing?

Homologous Recombination is high-fidelity (no DNA loss), uses a sister chromatid template, and occurs mainly in $S$ and $G_2$ phases; Non-Homologous End-Joining directly ligates DSB ends with DNA loss (error-prone) and is active throughout the entire cell cycle ($G_1, S, G_2, M$).

What are the specific functions of BRCA1 and BRCA2 in Double-Strand Break Repair via Homologous Recombination?

BRCA1 senses double-strand breaks, promotes end resection, and recruits repair proteins; BRCA2 mediates homologous pairing and strand invasion using the sister chromatid.

Which main proteins carry out Non-Homologous End-Joining (NHEJ)?

Ku70/80 heterodimer, DNA-PKcs, and DNA Ligase IV (LIG4).

What are the chemical polarity features of the antiparallel DNA double helix backbone?

The sugar-phosphate backbone runs antiparallel with a free $5'$ phosphate group ($5'\text{P}$) at one terminal and a free $3'$ hydroxyl group ($3'\text{OH}$) at the opposite terminal.

Which clinical therapeutic agents act directly by inhibiting DNA polymerases?

Acyclovir (antiviral), Cytarabine (chemotherapy), and Zidovudine (antiviral).

Which antibiotic and chemotherapeutic agents inhibit DNA Topoisomerases?

Quinolones (antibiotics targeting bacterial topoisomerase/gyrase), Irinotecan, and Topotecan (chemotherapeutic agents).

Which chemotherapeutic agents function as intercalating agents that stall replication forks?

Cisplatin and Bleomycin.

How do ionizing and non-ionizing radiation differ in the specific DNA damage they inflict?

Ionizing radiation (X-rays, gamma rays) causes single- and double-strand breaks and reactive oxygen species (ROS) oxidative damage, whereas non-ionizing radiation (UV light) induces bulky lesions such as thymine-thymine dimers.

How do silent, missense, and nonsense point mutations differ in their effects on protein translation?

Silent mutations do not change the amino acid; missense mutations substitute one amino acid for another; nonsense mutations introduce a premature STOP codon, yielding a truncated protein.

Under what structural rule do insertion-deletion (indel) mutations cause a frameshift?

Indels whose nucleotide count is not divisible by $3$ shift the translation reading frame, whereas indels in multiples of $3$ gain or lose whole amino acids without altering the reading frame.

What three major pre-mRNA structural defects result from splicing mutations?

Exon skipping, intron retention, and activation of cryptic splice sites.

How do mutations located within gene promoter or enhancer regions alter gene function?

They disrupt the binding of RNA Polymerase II and general transcription factors, leading to abnormally increased or decreased gene transcription without altering the protein's coding sequence.

What gene is mutated in Cystic Fibrosis, and what type of mutation is involved?

The $CFTR$ gene, which undergoes small-scale mutations or indels that disrupt epithelial chloride channel function.

What are the four main types of large-scale chromosomal rearrangements?

Micro/macro deletions, duplications, translocations, and inversions.

What are the four primary functional consequences of large-scale chromosomal rearrangements on genes?

Gene disruption, gene amplification, gene fusion (e.g., $BCR::ABL$), and position effects.

What is the fundamental role of DNA Primase during DNA replication?

DNA Primase synthesizes short RNA primers required to supply a free $3'\text{OH}$ group for DNA Polymerase initiation on both leading and lagging strands.

What is the function of DNA Ligase during lagging strand maturation?

DNA Ligase catalyzes phosphodiester bond formation to seal nicks between adjacent Okazaki fragments after RNA primers are removed and replaced with DNA.

What is the repetitive nucleotide sequence characteristic of human telomeres?

$(\text{TTAGGG})_n$ or $(\text{AGGGTTA})_n$ repeated at the ends of linear chromosomes.

What role does PARP1 play in the Base Excision Repair (BER) pathway?

PARP1 recognizes single-strand DNA nicks and damaged bases, binding to the site to recruit downstream enzymes necessary to complete Base Excision Repair.