DNA Organization, Replication, and Repair (Lecture 1; Lecture generated)

Chemical Composition and Primary Structure of DNA

• Monomeric units of DNA are nucleotides, each consisting of three fundamental components:

  • A nitrogenous base (purine or pyrimidine).

  • A pentose sugar (deoxyribose).

  • A phosphate group (derived from phosphoric acid).

• Nitrogenous Bases:

  • Purines: Consist of a double-ring structure.

  • Adenine (A).

  • Guanine (G).

  • Pyrimidines: Consist of a single-ring structure.

  • Cytosine (C).

  • Thymine (T).

• Pentose Sugar Structure:

  • Deoxyribose is a five-carbon sugar lacking a hydroxyl group ($\text{-OH}$) at the $2'$ carbon position, having a hydrogen atom ($\text{-H}$) instead.

• Phosphodiester Backbone and Polynucleotide Formation:

  • Phosphodiester bonds link adjacent nucleotides together into single-stranded polynucleotides.

  • A covalent phosphodiester bond forms between the $3'\text{-OH}$ group of one deoxyribose sugar and the $5'\text{-OH}$ group of the next deoxyribose sugar via a phosphate bridging group.

  • Nucleotides exist in monophosphate (NMP), diphosphate (NDP), or triphosphate (NTP) forms depending on the number of attached phosphate groups.

Double Helical Secondary Structure of DNA

• Double Helix Architecture:

  • DNA is composed of two linear polynucleotide chains wound around a central imaginary helical axis.

  • The two strands are antiparallel, running in opposite directions: one strand runs $5' \rightarrow 3'$ while the complementary strand runs $3' \rightarrow 5'$.

  • The sugar-phosphate backbones form the outer structural frame, whereas the nitrogenous bases extend inward perpendicular to the helical axis.

  • The coiling of the strands creates alternating major grooves and minor grooves along the outer surface of the helix.

• Complementary Base Pairing:

  • Specific hydrogen bonding occurs between paired nitrogenous bases across the opposing strands:

  • Adenine (A) pairs with Thymine (T) via two hydrogen bonds ($11.1\text{ Å}$ span).

  • Guanine (G) pairs with Cytosine (C) via three hydrogen bonds ($10.8\text{ Å}$ span).

Physical Properties of DNA: Denaturation, Renaturation, and Hybridization

• DNA Denaturation (Melting):

  • High temperatures or alkaline reagents break the hydrogen bonds joining complementary base pairs, causing the double-stranded DNA molecule to separate into two single strands.

  • Phosphodiester bonds within the sugar-phosphate backbone are resistant to heat and alkali treatment and remain intact during denaturation.

• Melting Temperature ($T_m$):

  • $T_m$ is defined as the exact temperature at which $50\%$ of a DNA sample becomes single-stranded.

  • $T_m$ is directly dependent on base composition: DNA containing a higher GC content has a higher $T_m$ because G-C pairs are linked by three hydrogen bonds compared to the two hydrogen bonds of A-T pairs.

• Renaturation and Hybridization:

  • Denaturation is reversible: restoring physiological temperature or neutral pH allows separated complementary strands to reassociate, a process termed renaturation or reannealing.

  • Hybridization is the sequence-specific binding of a single strand of DNA to a complementary sequence of single-stranded DNA or RNA.

Genomic Organization: Mitochondrial vs. Nuclear DNA

• Overview of Genetic Storage:

  • Genomic DNA stores all cellular genetic information, which is inherited through replication and expressed through transcription and translation.

• Mitochondrial DNA (mtDNA):

  • Architecture: Circular, double-stranded DNA molecules located in the mitochondrial matrix (supporting the endosymbiotic theory of mitochondrial origin).

  • Abundance: Constitutes approximately $1\%$ of total cellular DNA.

  • Composition: Completely lacks histone proteins and is rich in GC content.

  • Gene Content: Contains 37 genes:

  • 13 genes encoding polypeptides involved in mitochondrial energy production (oxidative phosphorylation).

  • 24 genes encoding non-translated structural RNAs ($22\text{ tRNAs}$ and $2\text{ rRNAs}$).

  • Inheritance: Transmitted exclusively through maternal lineage; does not undergo homologous recombination, making it a key marker for tracing human ancestry.

• Nuclear DNA (nDNA):

  • Consists primarily of non-coding sequences ($98\%$ non-coding).

  • Linear and extremely long (approximately 6 feet / 2 meters per diploid cell), encoding roughly 22,000 functional genes.

Chromosome and Chromatin Structure

• Chromosome Components:

  • Centromere: The primary constricted region of a chromosome that serves as the attachment site for spindle microtubules; mandatory for precise mitotic and meiotic chromosome segregation.

  • Telomeres: Specialized repeated sequence caps at both physical ends of linear chromosomes consisting of tandem $5'\text{-TTAGGG-}3'$ repeats (repeated up to 1000 times); protect terminal regions and shorten with successive cell divisions.

  • Chromosomes are categorized by centromere position into metacentric, submetacentric, and acrocentric structures (acrocentric chromosomes contain satellite bodies).

• Comparison of Euchromatin and Heterochromatin:

  • Chromatin condensation:

  • Euchromatin: Less condensed.

  • Heterochromatin: Highly condensed.

  • Anatomical location:

  • Euchromatin: Located along chromosome arms.

  • Heterochromatin: Concentrated at centromeres, telomeres, and specific structural regions.

  • Sequence types:

  • Euchromatin: Unique, single-copy sequences.

  • Heterochromatin: Highly repeated sequences.

  • Gene presence:

  • Euchromatin: Contains many active genes.

  • Heterochromatin: Contains few genes.

  • Transcriptional activity:

  • Euchromatin: Frequently transcribed.

  • Heterochromatin: Infrequently transcribed or transcriptionally silent.

• Hierarchical Levels of DNA Packaging:

  1. Naked double-stranded DNA helix ($2\text{ nm}$ width).

  2. Nucleosome ("beads-on-a-string" level, $11\text{ nm}$ width): The fundamental structural unit of chromatin. Consists of a histone octamer core (two molecules each of core histones H2A, H2B, H3, and H4) wrapped 1.65 times ($\sim 2$  turns) by double-stranded DNA.

  3. Linker DNA and Histone H1: Linker DNA connects adjacent nucleosomes. Linker histone H1 binds to the outside of the nucleosome core to stabilize higher-order packaging.

  4. Solenoid / Chromatin Fiber ($30\text{ nm}$ width): Nucleosomes fold and supercoil into compact tubular coils.

  5. Looped Domains ($300\text{ nm}$ loops): The $30\text{ nm}$ fiber forms loops averaging $300\text{ nm}$ in length anchored to a nuclear protein scaffold.

  6. Compressed Fiber ($250\text{ nm}$ wide fiber, compressed to $700\text{ nm}$ chromatids).

  7. Mitotic Chromosome ($1400\text{ nm}$ width): Fully condensed chromosome state achieved during cell division.

DNA Replication: Overview and Cell Cycle Context

• Cell Cycle Timing:

  • Proliferating cells undergo an ordered cycle of growth and division.

  • S Phase (Synthesis phase): Dedicated period during interphase where total nuclear DNA genome replication occurs (lasts approximately 9 hours in human cells).

  • $G_2$ Phase: Period dedicated to cell growth, proofreading, and DNA repair prior to mitosis.

• Key Features of DNA Synthesis:

  • Semiconservative: Each new daughter double-stranded DNA molecule retains one original parental template strand and receives one newly synthesized daughter strand.

  • Bidirectional: Synthesis proceeds simultaneously in both directions away from origins of replication.

  • Origins of Replication (ori):

  • Eukaryotes contain multiple origins per chromosome ($\sim 100$ origins/chromosome) to permit duplication of large genomes within S phase.

  • Prokaryotes contain a single origin of replication per circular chromosome.

Components of the Replication Machinery

• Replication Fork:

  • The Y-shaped dynamic region created where parental DNA strands are separated and active nucleotide addition occurs.

• Enzymatic Roles in Replication:

  • Helicase: Unwinds the parental DNA double helix by breaking hydrogen bonds between complementary base pairs.

  • Topoisomerase (DNA Topoisomerase): Relaxes torsional strain and removes positive supercoils ahead of the advancing fork by transiently cleaving and resealing the sugar-phosphate backbone.

  • Single-Stranded Binding Proteins (SSBP): Bind to single-stranded template DNA to prevent reannealing of complementary strands and protect against nucleolytic cleavage.

  • Primase (Subunit of DNA Polymerase $\alpha$): Synthesizes short RNA primers ($\sim 15$  nucleotides long) complementary to template strands, providing a free $3'\text{-OH}$ group required for initiation of DNA synthesis.

  • DNA Polymerase:

  • Adds complementary deoxyribonucleoside triphosphates (dNTPs) to the free $3'\text{-OH}$ group of the growing strand, forming a new phosphodiester bond and releasing inorganic pyrophosphate ($PP_i$).

  • Always reads parental template strands in the $3' \rightarrow 5'$ direction.

  • Always synthesizes new daughter strands in the $5' \rightarrow 3'$ direction.

  • Contains proofreading activity via $3' \rightarrow 5'$ exonuclease function to excise mismatched terminal nucleotides.

  • DNA Ligase: Catalyzes phosphodiester bond formation between a free $3'\text{-OH}$ group and an adjacent free $5'\text{-phosphate}$ group to seal nicks in the backbone.

• Roles of Eukaryotic DNA Polymerases:

  • DNA Polymerase $\alpha$ (alpha): Contains primase activity; initiates DNA synthesis by laying down short RNA:DNA primers. Proofreading: No.

  • DNA Polymerase $\beta$ (beta): Functions specifically in nuclear DNA repair mechanisms. Proofreading: No.

  • DNA Polymerase $\delta$ (delta): Elongates Okazaki fragments on the lagging strand. Proofreading: Yes ($3' \rightarrow 5'$ exonuclease).

  • DNA Polymerase $\epsilon$ (epsilon): Elongates the leading strand continuously. Proofreading: Yes ($3' \rightarrow 5'$ exonuclease).

  • DNA Polymerase $\gamma$ (gamma): Replicates and repairs mitochondrial DNA. Proofreading: Yes ($3' \rightarrow 5'$ exonuclease).

Mechanism of DNA Strand Synthesis

• Leading Strand Synthesis:

  • The leading strand template runs $3' \rightarrow 5'$ toward the replication fork.

  • Synthesized continuously in the $5' \rightarrow 3'$ direction moving toward the advancing replication fork.

  • Initiated by a single RNA primer and extended continuously by DNA polymerase $\epsilon$ .

• Lagging Strand Synthesis:

  • The lagging strand template runs $3' \rightarrow 5'$ away from the replication fork.

  • Synthesized discontinuously in the $5' \rightarrow 3'$ direction moving away from the advancing replication fork.

  • Produces short segment intermediates called Okazaki fragments ($\sim 200$  nucleotides in eukaryotic cells).

  • Requires multiple RNA primers synthesized periodically by DNA polymerase $\alpha$/primase.

  • DNA polymerase $\delta$ extends Okazaki fragments until reaching the previously synthesized fragment.

  • RNA primers are excised via $5' \rightarrow 3'$ exonuclease activity, gaps are filled with dNTPs by DNA polymerase, and remaining backbone nicks are covalently joined by DNA ligase.

The End-Replication Problem and Telomerase

• End-Replication Problem Mechanics:

  • Removal of the RNA primer at the extreme $3'$ end of the lagging strand template leaves a terminal gap that cannot be filled by DNA polymerase due to the absence of a upstream free $3'\text{-OH}$ primer.

  • Successive cycles of replication lead to progressive loss of terminal genetic material and telomere shortening in dividing cells.

• Telomerase Enzyme Structure and Mechanism:

  • Telomerase is a specialized ribonucleoprotein complex containing two essential components:

  • Telomerase Reverse Transcriptase subunit (encoded by the $TERT$ gene).

  • Telomerase RNA template subunit (encoded by the $TERC$ gene, carrying sequence $3'\text{-AAUCCC-}5'$).

  • Mechanism: Telomerase binds the $3'$ single-stranded DNA overhang and synthesizes repetitive $5'\text{-TTAGGG-}3'$ hexanucleotide sequences ($3\text{--}20\text{ kb}$ total repeat length) via reverse transcription.

  • Once extended, DNA polymerase $\alpha$ (with primase) synthesizes a complementary strand on the newly extended overhang.

  • Somatic cells do not express telomerase, leading to progressive chromosomal shortening, senescence, and cell aging.

  • Telomerase is persistently expressed in germline cells, stem cells, and cancer cells, conferring cellular immortality.

DNA Damage, Mutagens, and Cellular Stability

• Sources and Types of DNA Lesions:

  • Replication Errors: Base misincorporations escaping polymerase proofreading occur at a rate of approximately 1 error in every $10^9\text{--}10^{10}$ base pairs.

  • Spontaneous Endogenous Damage:

  • Depurination: Cleavage of the $N$ -glycosidic bond releasing a purine base (A or G) leaving an apurinic site ($>1000$  events/cell/day).

  • Deamination: Loss of amine groups; cytosine deaminates to uracil ($>100$  events/cell/day). Deamination of 5-methylcytosine converts it to thymine, which escapes detection as a standard base.

  • Spontaneous alkylation.

  • Mutagen-Induced Damage:

  • Endogenous Mutagens: Reactive oxygen species (ROS), free radicals.

  • Exogenous Mutagens: Alkylating agents, radiation (ionizing radiation, X-rays, UV radiation), chemical pollutants, pesticides, tobacco smoke (e.g., benzo[a]pyrene), plant/microbial toxins, chemotherapeutics.

  • Carcinogens: Mutagens specifically capable of transforming normal cells into malignant neoplastic cells.

• Consequences of Unrepaired DNA Damage:

  • Failure to repair DNA damage causes permanent base changes (mutations).

  • Defective repair disrupts the balance between genomic stability and instability, leading to hereditary diseases, carcinogenesis, and genetic divergence.

Pathways of DNA Repair

• Classification of DNA Repair Mechanisms:

  • Single-Strand Damage Pathways: Direct Damage Reversal, Base Excision Repair (BER), Mismatch Repair (MMR), Nucleotide Excision Repair (NER).

  • Double-Strand Break Pathways: Homologous Recombination (HR), Non-Homologous End Joining (NHEJ).

• Direct Damage Reversal:

  • Simplest repair mechanism; corrects specific chemical alterations without cutting the phosphodiester backbone.

  • Primary application: Removal of drug-induced alkylation at guanine bases ($O^6$ -methylguanine).

  • Enzyme: $O^6$ -methylguanine-DNA methyltransferase transfers the offending methyl group from guanine directly to a cysteine residue ($Cys\text{-SH} \rightarrow Cys\text{-S-CH}_3$) on the enzyme itself.

  • Stoichiometric / Wasteful Process: The transfer permanently inactivates the methyltransferase enzyme; exactly one enzyme molecule is consumed for every single repaired alkylation site.

• Generic DNA Excision Repair Pathway:

  • Universal sequential steps across excision pathways:

  1. Detection: Recognition of DNA damage or helical distortion.

  2. Excision: Cleavage and removal of the damaged base or nucleotide sequence by endo- and exonucleases.

  3. Resynthesis: Replacement of missing nucleotides by repair DNA polymerase using the intact strand as template.

  4. Ligation: Sealing of remaining backbone nicks by DNA ligase.

• Base Excision Repair (BER):

  • Corrects small non-helix-distorting base damage caused by spontaneous depurination, cytosine deamination to uracil, or oxidative/alkylation damage.

  • Mechanism: Specific DNA glycosylases recognize damaged bases and cleave the $N$ -glycosidic bond; the phosphodiester sugar-phosphate backbone remains intact prior to enzymatic cleavage.

• Mismatch Repair (MMR):

  • Corrects non-damaged mispaired bases and small insertions or deletions (indels) introduced during DNA replication that escape polymerase proofreading.

  • Strand Discrimination: MMR enzymes detect structural distortions and selectively excise nucleotides on the newly synthesized strand, which is recognized by the presence of unsealed nicks.

  • Clinical Correlation: Inherited mutations in MMR genes cause Lynch Syndrome (Hereditary Non-Polyposis Colorectal Cancer, HNPCC), an autosomal dominant disease responsible for $15\%$  of all colorectal cancers.

• Nucleotide Excision Repair (NER):

  • Detects and removes bulky, helix-distorting lesions that cause replication blocks.

  • Major Causes of Lesions:

  • Benzo[a]pyrene-guanine adducts formed by carcinogens in tobacco smoke.

  • Pyrimidine dimers (e.g., thymine-thymine dimers, 6-4 photoproducts) induced by UV radiation exposure.

  • Clinical Correlation: Failure to repair UV-induced thymine dimers leads to skin cancer. Inherited mutations in NER genes cause Xeroderma Pigmentosum (XP), an autosomal recessive condition characterized by severe UV light sensitivity ("children of the night") and a high incidence of early-onset skin cancers.

!Benzo[a]pyrene oxidation and adduct formation with guanine in DNA

• Double-Strand Break (DSB) Repair Pathways:

  • Causes of DSBs: Ionizing radiation, reactive oxygen species, topoisomerase failures, replication across a nick, mechanical stress, or physiological breaks ($V(D)J$  recombination and class switching in lymphocytes).

  • Homologous Recombination (HR):

  • Uses genetic information from an intact sister chromatid as a template to accurately repair double-strand breaks.

  • Highly accurate and error-free mechanism.

  • Restricted primarily to late S and $G_2$  phases of the cell cycle when sister chromatids are available.

  • Involves proteins such as RAD51, BRCA1, BRCA2, and the MRN complex (RAD50, MRE11, NBS1).

  • Non-Homologous End Joining (NHEJ):

  • Primary DSB repair pathway in mammalian cells.

  • Directly ligates broken DNA ends together without requiring sequence homology.

  • Active throughout the entire cell cycle (predominant in $G_1$  phase).

  • Error-Prone: Almost always results in nucleotide deletions or insertions at the cleavage junction; the repaired DNA sequence is never identical to the original sequence.

  • Involves proteins Ku70/86, DNA-PKcs, Artemis, DNA polymerase $\mu$ /$\lambda$ , XRCC4, and DNA ligase IV.