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DNA

Telomerase

  • telomerase generates telomeres

DNA basics

  • DNA = double helix of deoxyribonucleic acid
  • composed of elements : COHNP

  • each nucleotide unit includes:

  • nitrogen base

  • pentose sugar
  • phosphate group

Nitrogen bases

  • purines (2 rings): A (adenine), G (guanine)
  • pyrimidines (1 ring): C (cytosine), U (uracil), T (thymine)

DNA vs RNA sugar difference

  • RNA: \(2'\) carbon has OH
  • DNA: \(2'\) carbon has H

Nucleotide structure and bonds

Key attachments

  • phosphate group (O) attached to \(C5'\) via ester bond
  • base (N) attached to \(C1'\) via glycosidic bond

Naming

  • base + sugar = nucleoside
  • base + sugar + phosphate = nucleoside monophosphate (nucleotide)

Phosphodiester bond formation

  • phosphodiester bond: phosphate group (O) attached to \(C3'\)
  • facilitated by polymerase via condensation reaction

Directionality :

  • \(5'\) end = phosphate end (beginning)
  • \(3'\) end = can add more nucleotides
  • mechanism: electrons on the \(3'\) OH attack the phosphate group on the incoming dXTP

Triphosphate chemistry

  • three phosphates: \(\alpha\), \(\beta\), \(\gamma\) (from the \(C5'\) outward)

  • after connecting to the DNA strand (polymerase-mediated):

  • \(\alpha\) separates from \(\beta\) and \(\gamma\)

  • pyrophosphate is released
  • \(\beta\) and \(\gamma\) are cut by pyrophosphatase

Convention :

  • assume DNA sequence is written \(5' \to 3'\) from left to right

Base pairing and helix stabilization

Base pairing rules

  • A–T/U: 2 H-bonds
  • C–G: 3 H-bonds

Forces

  • hydrogen bonds: inter-strand force

  • base stacking: intra-strand force

  • bases are hydrophobic, phosphates are hydrophilic

  • drives formation of the double helix

Denaturation and re-annealing

  • dsDNA denatures at high temperature and/or basic conditions
  • strands can re-anneal
  • denaturation increases absorbance at 260 nm (bases more exposed)

Antiparallel orientation

  • strands are antiparallel to line up bases

DNA helix forms and geometry

  • DNA is a right-handed helix with:

  • major groove

  • minor groove
  • one of each groove per 360°
  • each 360° rotation includes ~10 bp
  • major groove enables protein activities

DNA forms :

  • A-DNA: during transcription, water is scarce; ~11 bp per 360°
  • B-DNA: most common
  • Z-DNA: left-handed, zig-zag; bases can be inside/outside; certain genes’ transcription can be in Z form
  • a single DNA molecule may not be all in one form at a given time

Cell cycle overview

  • G1 (gap/growth 1): protein synthesis (DNA replication proteins), some organelle duplication
  • S (synthesis): replication should be quick because DNA is exposed and mutation risk increases
  • G2 (gap/growth 2): spindle fibers, organelles
  • mitosis
  • cytokinesis
  • cell may or may not leave the cycle (G0 or G1)

S phase: DNA replication machinery and steps

Origins and initiation

  • replication starts from origins of replication spaced across the genome
  • origins bind origin of replication complex (ORC), an assembly platform formed in G1
  • mini chromosome maintenance (MCM) complex has helicase activity

Helicase structure

  • hexameric helicase activity
  • 2× ATP binding, 2× catalytic activity, 2× empty spots
  • DNA passes through the middle

Replication bubbles and forks

  • forming replication bubbles → two replication forks

Topology management: Topoisomerase I

  • relieves torsional strain
  • transient break of one of the two strands
  • binds broken strand, unwinds, then re-ligates

ssDNA protection

  • ssDNA-binding proteins coat ssDNA to:

  • prevent re-annealing

  • protect
  • signal

Priming and polymerases

Primase

  • RNA primase forms primer of ~10–30 bp

DNA polymerase alpha

  • starts synthesis with low processivity
  • ~20–100 bp per binding
  • noted as “leading end”

Polymerase switching: delta and epsilon

  • DNA pol delta (more lagging; Okazaki fragments)
  • DNA pol epsilon (more leading)
  • higher processivity, aided by PCNA (sliding clamp)

Core polymerase functions

  • polymerization (adds nucleotides)
  • proofreading: \(3'\to5'\) exonuclease activity

Polymerase structure

  • palm: catalytic site, binds template
  • fingers: incoming nucleotide binds; closes/locks if correct
  • thumb: binds polymerase to template
  • switches to editing site if error detected

Preventing re-replication

  • geminin binds to newly synthesized material and prevents recopying

Primer removal and ligation

  • FEN1 (flap endonuclease 1) and RNase H remove RNA primers
  • gaps filled by DNA polymerase
  • DNA ligase seals DNA

Chromatin reassembly

  • histones bind, package, and condense into chromosomes

Replication directionality and principles

  • polymerase reads template \(3'\to5'\) and synthesizes \(5'\to3'\)
  • replication is semi-conservative
  • fidelity depends on \(3'\to5'\) exonuclease proofreading (delta and epsilon)

Mismatch correction mechanism :

  • mismatch kink pauses polymerase
  • daughter strand reorients to correction site
  • nucleotides removed
  • daughter strand reorients to elongation site

Antiviral nucleoside inhibitors (example)

  • AZT: thymine analog where the \(3'\) OH is replaced by N\(_3\)