DNA Organization and Nucleosomes

DNA Organization

• Eukaryotic DNA is structured by extreme length and abundance of noncoding sequences.
• Must be compacted to fit in the nucleus but remain accessible for selective genetic expression.
• Achieving compaction requires DNA supercoiling around histones to create nucleosomes.
• More than 98% of a haploid genome is noncoding sequences.
• Noncoding sequences act as introns (intervening regions in coding regions/exons).
• Noncoding sequences have a regulatory function for genes.
• Remaining noncoding DNA exists in pseudogenes and repetitive DNA forms.

Nucleosomes

• Basic structural unit of chromatin.
• DNA supercoils twice around an octet of histones to form nucleosomes.
• Chromatin has one nucleosome per 200 DNA bases.
• DNA wraps around histone octet and includes 30 base pairs as a linker to the next nucleosome
• Histone H1 associates with the linker region, acting as a fifth histone.
• Histones in the octet include H2A, H2B, H3, and H4.
• Amino acid sequence is conserved across species for each histone.
• Nucleosome formation does not depend on the DNA base sequence.
• DNA is further compacted by coiling into a solenoid structure.
• Nucleosomes assemble in 30-nm fibers to create this solenoid structure.
• Histone H1 stabilizes the packing of nucleosomes within these fibers.
• Chromatin fibers form heterochromatin or euchromatin when attached to nuclear scaffold proteins.
• Heterochromatin is genetically inactive due to its highly compacted state.
• Euchromatin has a more open structure.

Pseudogenes

• Single-copy DNA, contain the intact sequence for a functional polypeptide.
• Cannot be expressed due to the lack of a promoter for RNA synthesis initiation.
• Created by retroviruses making DNA copies of mRNA.
• A pseudogene DNA sequence has no promoter or introns but includes a polyA tail.
• Described as a DNA sequence representing mature mRNA.
• Retroviruses insert pseudogenes into the DNA helix, incorporating copies of their retroviral chromosome.
• Pseudogenes are permanently incorporated, remain dormant, inert, and unexpressed.

Repetitive DNA and Transposons

• Repetitive DNA consists of tandem, repeated sequences (2 to several thousand base pairs) constituting ~30% of the genome.
• Many are in centromeres/telomeres, but also exist throughout the genome.
• Repetitive DNA (satellite DNA) bands are small satellite bands differentiated during density gradient centrifugation.
• Centrifugation isolates/analyzes DNA using cesium chloride, forming a density gradient >200,000g.
• DNA migrates to match its density; satellite DNA is denser, with higher guanine-cytosine.
• Repetitive DNA forms by random, unequal crossover events, causing deletions on one chromosome and duplications on another.
• Duplications create grouped tandem sequences expanded by tandem arrangement doubling via recombination.
• Transposons (jumping genes) are produced by retroviruses.
• Unlike pseudogenes, transposons are the retrovirus and are trapped inside cells because they cannot make viral coat proteins.
• Can leave and re-enter a chromosome at a different site.
• They can take flanking sequences, creating deletion/insertion mutations.
• The two major classes of transposons make up ~10% of the genome.
• Short interspersed nuclear elements (SINEs) have 100-500 base pairs in length.
• The Alu sequence (280 base pair) is a well-known SINE and it has about 1 million copies dispersed throughout the genome (including introns).
• The Alu sequence contains the Alu restriction enzyme recognition sequence.
• The Alu sequence is derived from 7 S RNA found in signal recognition particles.
• Long interspersed nuclear elements (LINEs) are 6000-7000 base pairs long
• LINEs have the reverse transcriptase gene, meaning they are derived from retroviruses
• The L1 family of LINEs constitutes ~5% of the genome.

Repetitive DNA and Disease

• Some genetic diseases associate to increase in repetitive DNA sequences.
• The repeat sequence CpGpG is associated with fragile X syndrome.
• Huntington chorea (CAG), myotonic dystrophy (CTG), and spinobulbar muscular dystrophy (CAG) are other examples that indicate that the location of the repeat is significant.

Heterochromatin vs Euchromatin

DNA Organization - Key Points

• Eukaryotic DNA coils around histones forming nucleosomes and higher-order structures in the nucleus.
• Most DNA is transcriptionally inactive due to compaction, high methylation, and DNase resistance.
• About 2% of the eukaryotic genome codes for polypeptides (exons), with the remainder as noncoding DNA.
• Noncoding DNA functions as regulatory or intervening (intron) sequences, or as pseudogenes or repetitive DNA.

DNA synthesis

• Cellular DNA synthesis happens in response to cell division signals or repair signals for damaged DNA.
• DNA within chromatin must be made physically available to replication or repair enzymes in either case.
• DNA replication and repair occur at certain times during the cell cycle.

Cell Cycle

• The cell cycle has timed events during itnerphase and mitosis (M)
• Interphase: G1 (gap) phase, the S (synthesis) phase, and G2 phase
• The G phases have checkpoints to control movement into replication (G1) or mitosis (G2).
• The G1 and G2 phases include RNA and protein synthesis (not DNA).
• S phase: DNA replication
• M phase: chromosome separation during cell division
• Cell cycle progression involves cyclins (concentrations rise/fall through the cycle).
• Cyclins activate cyclin-dependent protein kinases (Cdks) by binding.
• Activated cyclin-Cdk phosphorylates target proteins important for the cell cycle progression.
• Checkpoints blocks the formation of daughter cells when there is DNA damage by inhibiting activated cyclin-Cdks.
• Nuclear phosphoprotein p53 exists in the G1/G2 checkpoints that activate transcription that increases the expression of genes for growth arrest, DNA repair, or apoptosis (leads to cell death).
• p53 halts the cell cycle to reduce chances for dangerous mutations.
• Tp53 is known as a tumor suppressor gene and the antimutagenic activity.
• Many human cancers associate to mutations in the Tp53 gene.

Formation of the Replication Fork

• S phase begins DNA replication: DNA strands must separate as templates (semiconservative replication).
• Chromatin's higher-order packing is reduced for replication enzymes access.
• Semiconservative replication creates 1 parent (original) strand and one daughter (new) strand in each double helix.
• DNA strands: antiparallel -> each direction contains a template strand.
• DNA synthesis: bidirectional from replication origins for eukaryotic/prokaryotic DNA.
• Eukaryotic DNA synthesis has many origins to reduce chromosome replication time.
• Action of DNA gyrase (topoisomerase II) relaxes supercoiling for polymerization enzymes (induces negative supercoils in DNA).
• Supercoiled DNA releases helicase to bind and continue helix unwinding via energy.
• Helicase separates DNA strands.
• Topoisomerase I releases strain by breaking and rejoining one helix strand (relaxes either positive/negative supercoils).
• Helix-destabilizing proteins bind to single-stranded DNA to prevent reannealing.
• Separation point: replication fork.

Mitosis (M)

• G2 checkpoint -> M checkpoint = detects improper spindle formation to prevent mis-segregation of chromatids to daughter cells.
• Once M checkpoint has begun, cells enter mitosis, progress through metaphase (chromosomes line up on metaphase plate), then anaphase (chromosomes separate pulled to opposite spindle poles).

Apoptosis

• Programmed cell death where cells cause suicide.
• Neutrophils form blebs on surface at day one that are digested by phagocytic cells, along with DNA degradation.
• Stepwise cellular apoptosis includes mitochondrial degradation.

Deoxyribonucleotide Polymerization

• Precursors for DNA synthesis utilize 5'-deoxyribonucleotide triphosphates.
• DNA polymerase creates a phosphodiester bond --> pyrophosphate cleavage that attaches it to the free 3'-hydroxy group on the growing polypeptide.
• DNA synthesis direction is always 5' to 3' because of polymerase nature; nucleotide sequences represented with 5' end at left and 3' end at right.
• DNA strand with 3' end advancing in same direction as replication fork: leading strand.
• Leading strand synthesis: continuous/ highly processive (catalyzed by DNA polymerase III (prokaryotes) that circles DNA helix and moves along template strand to add new nucleotides to growing daughter strand).
• Opposite strand direction creates replication dilemma.
• Synthesis initiates and extends away from replication fork direction: lagging strand synthesis that uses discontinuous process.
• Lagging strand has the sequential action of many enzymes to initiate, elongate, and join short DNA pieces (Okazaki fragments, ~1000 nucleotides long).
• Errors in DNA synthesis are dangerous and must be corrected to prevent genome mutations.
• DNA polymerase needs one helix turn to attach nucleotides (9-10 nucleotides); an RNA primer first synthesizes this initial helix.
• Primer removal and DNA sequence replacement allows high-fidelity base pairing, reducing potential mutation damage.
• Each new Okazaki fragment primer is synthesized (5' to 3' direction) by primase (a DNA-dependent RNA polymerase).
• Primosome: Helicase and DNA proteins + primase
• Each primer starts at/near replication fork and is extended in opposite direction; primosome keeps lagging strand synthesis with leading strand synthesis at replication fork.
• DNA polymerase III extends new exonuclease activity (polymerization of deoxynucleotides continues, until reaching 3' hydroxyl of earlier Okazaki fragment).
• RNA primer is removed one base at length using DNA polymerase I that is 5' to 3' exonuclease activity.
• Wrong errors related to RNA primer are corrected while ribonucleotide is replaced with deoxyribonucleotide.
• The last dexyribonucleotide uses DNA ligase to join the Okazaki fragment into the growing lagging strand.

Relaxation of Supercoiling

• Gyrase during DNA synthesis releases positive supercoiling strain while causing negative supercoils.
• Fluoroquinolone blocks relaxation that inhibits bacterial growth.
• Ciprofloxacin example: treat urinary tract and other bacterial infections.

Comparison Between Enzymes for Prokaryotic and Eukaryotic DNA Polymerization

• DNAP, DNA polymerase.*

Proofreading

• Mispairing during synthesis from keto-enol/amino-imino tautomeric causes mismatching (base pairing process).
• Mismatches become mutations if they are incorrectly corrected; use proofreading for detection.
• DNA polymerases I and III can proofread for mismatches in base pairs by correcting the helix shape; anything other than adenosine-thymine (AT) and guanine-cytosine (GC) creates the irregularity.
• While the helix synthesizes, helix runs a channel within DNA polymerase for accurate fit if this is irregular, activation occurs on 3' to 5' exonuclease activity within two DNA polymerase enzymes.
• DNA polymerase backs up (3' to 5' direction), removes wrong nucleotide/reinserts right nucleotide.

Telomerase and Telomeres

• Replication fork approaches chromosomes' end: initiation problems for last Okazaki fragment.
• Primosome/primase (initiates last Okazaki fragment) separates from DNA during final separation of strands + replication fork doesn't exist anymore.
• Leading strand is synthesized toward molecule's end, but lagging strand shortens by one Okazaki fragment long.
• Some cells are short until the loss of the critical sequence, which leads to cell death.
• Active proliferating cells have telomerase, solving the problems associated with lagging strand synthesis at the chromosome ends.
• Telomerase includes an RNA sequence as protein group acting as the template that is tandemly repeating.
• Synthesis of six-base sequences extends the chromosomes well beyond the genomic DNA sequence (replication fork stays intact)
• Intact primosome initiates of the terminal Okazaki fragments.
• DNA repeats are called telomeres at the chromosomes.
• Active cells preserve their telomeres, which stops terminal erosion.

Reverse Transcriptase

• Specific DNA polymerase is found among retroviruses with RNA chromosome.
• Name implies genetic information has switched from classic central dogma.
• Reverse transcriptase (RNA-dependent DNA polymerase) utilizes RNA template -> directs the synthesis of DNA molecule.
• Makes hybrid by utilizing DNA-RNA with using RNA chromosome.
• RNAase H is used as retroviral enzyme, degrades the RNA strand + it exchanges the RNA with DNA, which then forms DNA helix.
• DNA gets placed into new chromosome used as DNA in cells as template for RNA.
• Reverse transcriptase: high mutation rate due to lack of 3' to 5' exonuclease for proofreading.
• DNA polymerase has highest errors + causes genetic adaptability of retroviruses (HIV).

DNA Synthesis - Key Points

• DNA synthesis happens during repair + chromosome replication, the replication itself happens during the S phase of that cycle (requires cyclin-Cdk to pass the G1 checkpoint)
• Repair happens cycle if damage is detected.
• Unwinding helix with the monomers requires an enzyme for polymerization.
• Helicase opens the DNA + unwinds it. Creates DNA fork in several segments of DNA within synthesis.
• 5' to 3' is the major path with the help of 5' triphosphates, uses parent DNA for DNA pairs = happens with leading parts.
• In opposite paths, use fragments linked via DNA ligase use template- continuous leading/opposing lagging strand.
• DNA can restore a strand + restore mismatches. Transcriptions uses RNA, so it all skips over proofreading.
• Telomeres are constructed of repeating DNA, allows building lagging, the telomerase + end synthesis, is available within + removed in cells.

Nucleoside Reverse Transcriptase Inhibitors

• Within DNA inhibition, 3'-deoxy drugs stop growth.
• No 3' hydroxyl groups causes bonds + chain terminators.
• It wants to convert form for main DNA forms + treat HIV.
• The acids convert into DNA; causes hepatomegaly --> prevents DNA. Has impact within cells.

Transpositional Recombination

• Transposons have DNA segments + reverse transcriptase: lead to insertion --> double strand segments to RNA.
• They merge with recombinations --> leads to germ cells.
• They are able to revert genes (jumping) and can alter cells.
• Insertion blocks if DNA expression occurs --> causes a reaction.

DNA MUTATION AND REPAIR

• Mutation that escapes detection becomes "locked in" genome + becomes permanent at the following cell division.
• Errors fixed via DNA repairs; significant examples occur for fixes; mismatch, base excision, nucleotide, & repair.

Mismatch Repair

• Finds distortions from mismatch when inserting during the syntheses.
• The added info shows the strand to pinpoint a error.
• This occurs after synthesis, when adenine gets a methylation → guanine + cytonine within GATC segments.
• Repairs occurs before the segments forms.
• Uses GATC endonucleases (single-strand) → detects the wrong strand + the incorrect location.
• Damages segments → filled within repair segments → segments merged with new ligase.

Base Excision Repair

• Uracil is manufactured via cytosine, but base pairs with thymine + switches from GC --> AT.
• Thymine is identified as a mistake in DNA via glycoside, + is cut using glycosidase → Creates AP with a phosphate backbone → Phosphates is taken out → Phosphates repairs the cytosine.
• Ligase seals segment → Phosphate repairs the cytosine.