DNA Alexandra Kathryn Vedeler We will cover… qProkaryotes vs Eukaryotes qDNA structure qNucleosides and qTypes of DNA qDNA replication qProkaryotes qEukaryotes qDNA repair PROKARYOTES EUKARYOTES

• Circular DNA • Linear DNA – nucleus • NO nucleus, mitochondria or • Circular DNA - mitochondria other organelles • Plasmids • Non-chromosomal DNA • Can lead to antibiotic-resistance* Let’s brake it down

Circular DNA Linear DNA • Double-stranded DNA (dsDNA) • Double-stranded DNA (dsDNA) • Supercoils • Histones Acetylation = reduces positive charge, decreases bonding Histones Deacetylation = increases bonding between histones & DNA • Classes: • H1, H2A, H2B, H3, H4 • H1 = linker DNA • Octamer = 2x(H1, H2A, H2B, H3, H4) • Positive charge at physiological pH • Pos. charged aa residues à tight bonding of histones to DNA Nucleosome = 2x(H1, H2A, H2B, H3, H4) DNA structure

• Deoxyribose • Nucleobase • Phosphate group • Negative charge PURINES PYRIMIDINES Nucleobases Adenine Thymine

Cytosine

Guanine

Uracil (RNA)

“TUC-TUC around the pyramids” nucleoSide/ + =

+ =

nucleoTide Bonds

Beta-N-Glycosidic bond

Ester bond

The Double Helix

• Deoxyribose-phosphate backbone • Hydrophilic � • Van der Waals interactions • Base pairs • Hydrophobic � • H-bonds The Double Helix

Major & minor grooves • Provide access for binding of regulatory proteins

Dactinomycin (anticancer drug) acts on the minor groove à inhibits DNA replication Chagaff rule • The amount of A = T • The amount of G = C • The amount of purines = pyrimidines Types of DNA

DNA B-type DNA A-type DNA Z-type High humidity DNA Dehydrated B-DNA Helical rotation Right-handed Right-handed Left-handed Denaturation of DNA = Base pairs per 10bp 11bp 12bp loss of helical structure 360o Helical diameter 20Å = 2.0 nm 26Å = 2.6 nm 18Å = 1.8 nm Absorption at 260nm

Characteristics Chromosomal DNA DNA-RNA hybrids or Influences gene double stranded RNA expression and regulation Quiz time PURINES PYRIMIDINES

Nucleobases Adenine Thymine

Cytosine

Guanine

Uracil (RNA)

“TUC-TUC around the pyramids” nucleoSide/nucleoTide + =

+ = Types of DNA Most predominant DNA helix

DNA B-type DNA A-type DNA Z-type High humidity DNA Dehydrated B-DNA Helical rotation Right-handed Right-handed Left-handed

Base pairs per 360o 10bp 11bp 12bp

Helical diameter 20Å = 2.0 nm 26Å = 2.6 nm 18Å = 1.8 nm

Characteristics Chromosomal DNA DNA-RNA hybrids or Influences gene double stranded expression and RNA regulation REPLICATION

Requirements

TEMPLATE PRIMER PRECURSORS ENZYMES Single strand of original Short strand of RNA 5´deoxynucleotiSide DNA DNA = provides free triphosphates (dNTP’s) Sliding clamps = provides sequence 3´hydroxyl groups = provides energy information Nucleases Ligases Single-strand binding proteins (SSB’s) PROKARIOTIC REPLIKATION Steps

1. Separation 2. Replication fork formation 3. Supercoils 4. Replication 5. Chain elongation 6. Excision of RNA primers 7. Termination 1. Separation

• = Ori • AT-rich segment

• E.coli: OriC 2. Replication Fork Formation

DnaA protein DNA Single-stranded DNA- (DnaB) binding protein (SSB) Binds to DnaA boxes Binds to ssDNA Binds to ssDNA

Unwinds dsDNA by AT-rich segment melts breaking H-bonds Provides ss-template between base pairs for polymerases

+ dsDNA à ssDNA Supercoils Protects DNA from *Requires nucleases

ATP IS NOT AN ENZYME Supercoil

SSB

dsDNA Helicase 3. Supercoils Type 1 DNA • Cleaves 1 of the DNA strands Positive supercoils • Works for negative supercoils - DNA region ahead of the replication fork Negative supercoils - DNA region behind the replication fork Type 2 DNA topoisomerase • Cleaves both strands • Works on negative and positive supercoils • Required ATP 4. Replication

DNA RNA primer Provides deoxiribonucleoTides for new DNA strand Provides short single strand RNA sequence (DnaG) • 3´à 5´: reading direction DNA dependent RNA polymerase • 5´à 3´: synthesizing direction • Synthesize the RNA primer • Leading strand • Hybrid duplexes: A – U, C – G • • Lagging strand Leading strand: only one • • Lagging strand: constantly synthesized Leading strand

RNA primers DNA polymerase

Topoisomerase

SSB

Helicase

Primase

Lagging strand 5. Chain Elongation DNA Polymerase III

• Holoenzyme = Active enzyme • � - subunit = DNA sliding clamps • Highly possessive • 5´-deoxiribonucleoSide triphosphates (dNTP’s) Proofreading • 3´ à 5´ exonuclease • Removes error in opposite direction • 5´à 3´polymerase • Replaces wrong nucleotides with correct ones Leading strand

RNA primers DNA polymerase

Topoisomerase

SSB

Helicase

Primase

DNA Pol III

Lagging strand 6. Excision of RNA Primers

DNA Polymerase I • Removes RNA primer • 5´à 3´exonuclease activity • Synthesize new DNA • 5´à 3´polymerase activity • Proofreading

• 3´à 5´exonuclease activity DNA Pol I 7. Termination

DNA ligase 2- • Joins Okasaki fragments PO3 - • Final phosphodiester linkage: 3´à 5´ • Requires ATP

TUS Terminus utilization substance + Ter Replication terminator

STOP

Quiz time Requirements

TEMPLATE PRIMER PRECURSORS ENZYMES Single strand of original Short strand of RNA 5´deoxynucleotiSide DNA Polymerases DNA = provides free triphosphates (dNTP’s) Sliding clamps = provides sequence 3´hydroxyl groups = provides energy Helicases information Primases Nucleases Ligases Topoisomerases Single-strand binding proteins (SSB’s) Do you remember the steps?

1. Separation 2. Replication fork formation 3. Supercoils 4. Replication 5. Chain elongation 6. Excision of RNA primers 7. Termination What enzymes remove supercoils?

Topoisomerase I & II DNA polymerase I DNA polymerase III

Exonuclease à à activity 5´ 3´ - removes RNA primer 3´ 5´ - proofreading 3´à 5´ - proofreading

Polymerase Fills gap after removal of RNA Replication, proofreading activity primer, DNA repair and editing Eukaryotic DNA synthesis PROKAROTIC EUKARYOTIC

Origin Single Multiple

Primer excision DNA pol I RNase H -1 (FEN-1) DNA polymerases Numbered Greek letters I, II, III, IV, V �, �, �, �, � DNA polymerases Polymerase Function Proofreading α Initiates DNA synthesis alpha Primase; RNA primer No 5´à 3´polymerase β DNA repair No beta Gap filling δ Complete DNA synthesis Yes delta on lagging strand 3´à 5´exonuclease ε Complete DNA synthesis Yes epsilon of leading strand 3´à 5´exonuclease γ Replicates mitochondrial Yes gamma DNA 3´à 5´exonuclease

= complexes of non-coding DNA + proteins located at the end of linear • Prevent attack from nucleases • Noncoding hexametric sequences; AGGGTT • shortening acts as a reverse transcriptase

= RNA dependent DNA polymerase • Found in germ cells, stem cells and cancer cells • Maintain telomere length in cell • DNA pol α Reverse transcriptase is involved in replication of HIV DNA Repair DNA Repair: The triple R’s

• Recognize • Remove • Replace Methyl-directed mismatch repair excision repair • Mut proteins • Glycosylases • Parental strand fully methylated = • AP-endonucleases + deoxyribose correct phosphate lyase • Endonuclease activity • Apyrimidinic / Apurinic = AP-site • Polymerase activity • DNA polymerase + DNA ligase Nucleotide Excision Repair = Repair of damaged DNA due to UV-radiation

UV light à covalent bonding of two pyrimidines (thymine's), dimer Xeroderma pigmentosum DNA polymerase activity • Pyrimidine dimers form in skin • Rare, genetic disorder • uvrABC excinuclease • Cannot repair DNA (endonuclease) • Mutations & skin cancer • DNA polymerase & DNA ligase Quiz time DNA polymerases Polymerase Function Proofreading α Initiates DNA synthesis alpha Primase; RNA primer No 5´à 3´polymerase β DNA repair No beta Gap filling δ Complete DNA synthesis Yes delta on lagging strand 3´à 5´exonuclease ε Complete DNA synthesis Yes epsilon of leading strand 3´à 5´exonuclease γ Replicates mitochondrial Yes gamma DNA 3´à 5´exonuclease What causes xeroderma pigmentosum? a. Accumulation of purine dimers b. Accumulation of pyrimidine dimers c. Methylation of parental DNA strand d. Accumulation of AP-sites GOOD LUCK!