BIOL 4843: EXAM 2
148 Cards in this Set
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Chromatin
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Condensed DNA and protein
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Histones
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octameric protein complex with 4 different subunits (2 of each)
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Nucleosomes
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DNA wrapped around histone core. 1st level of packing in Eukaryotes.
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Core Histones
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Positive charged core consisting of H2A, H2B, H3, and H4 that DNA wraps around.
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Linker DNA
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DNA that links nucleosomes
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Structure of Nucleosome
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DNA wraps around nucleosomes 2 times in negative (left handed) supercoils.
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Histone-Fold Motif
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Each histone contains 3 alpha helices and 2 short loops.
-Contain either the H3-H4 dimer or H2A-H2B dimer that contain 3 DNA binding sites.
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DNA Binding to Histones
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DNA binds to histones nonspecifically via the PDE or minor groove.
-Since DNA has to bend around histone core, A-T tracts are favored because they promote bending.
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N-Terminal Histone Tails
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tails that protrude from the histone core that are important for nucleosome organization.
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H1 Linker Protein
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Facilitates DNA packing by affecting DNA angles coming in/out of nucleosome. 1 per nucleosome.
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30 nm Filament
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Nucleosomes compacted into a filament promoted by H1. N-terminal tails of core histones are required.
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Chromosomal Scaffold
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-Level above 30 nm filament.
-loops and coils of DNA that are attached to a protein scaffold.
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Remodeling Nucleosome Arrangement
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-chromatin remodeling complex that either reposition, eject, or replace nucleosomes.
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Modifications of Nucleosome Arrangement
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-chemically modify amino acid residues on the tails to switch closed DNA to open DNA.
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Promoter Exposure in Nucleosomes
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Use ATP to move nucleosomes to expose the promoter site.
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Histone Chaperones
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-assist the assembly of histone octamers onto DNA
-acidic proteins that bind to either H3-H4 tetramer or H2A-H2B dimer.
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Modifying Histone Tails
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-plays a role in chromosome structure by chemically modifying specific amino acids on the tails. Either by phosphorylation or methylation.
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Acetylation of Histones
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enhance accessibility to transcribe.
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HAT
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acetylate particular residues that usually "opens" the DNA to allow more transcription
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HDAC
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removes acetylate groups that "closes" the DNA to decrease transcription rate.
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Epigenetics
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study of inherited changes in phenotype or gene expression caused by mechanisms other than changes in the underlying DNA sequence. (i.e. histone tail modifications and DNA methylation).
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Fidelity
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how accurately the DNA polymerase is able to synthesize the daughter cells of DNA.
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Characteristics of Replication
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Semiconservative, bidirectional, and semi discontinuous.
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Semiconservative
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Mode of DNA replication in which the daughter complex has one intact parental strand and one newly synthesized strand.
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Semidiscontinuous
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Mode of DNA replication in which one strand, the leading strand, is replicated continuously, but the opposite strand, the lagging strand, is replicated in shorter, discontinuous segments.
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Meselson and Stahl
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Proved that DNA was semiconservative using nitrogen isotopes and CsCl.
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DNA Polymerase (DNAP)
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Enzymes that synthesize DNA in a 5'→3' direction.
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Daughter Strand
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Newly synthesized DNA template (parental DNA)
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Leading Strand
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continuously produced strand
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Lagging Strand
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discontinuously produced strand
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Okazaki Fragments
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short, discontinuous strands produced that are DNA fragments.
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RNA Primer
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build 5'→3', that creates a 3' OH to build DNA off of.
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Adding Nucleotides to DNA Strand
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dNTP adds nucleotides to DNA strand
dNTP→dNMP+PPi→Pi+Pi (Irreveserable reaction)
PPi=pyrophosphate
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Pyrophosphatase
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Splits PPi into two molecules of Pi so that the reverse reaction cannot occur.
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Exonucleases
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Enzymes that degrade DNA at the ends.
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3'→5' Exonuclease
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-Proof-reads the newly synthesized DNA.
-Releases dNMP at different active sites
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5'→3' Exonuclease
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-clean up functions on the lagging strand by removing RNA primers
-only 5'→3' exonuclease activity can occur while polymerizing because it is moving in the same direction as polymerase.
-As Pol I moves along strand, NMPs are released (creating a nick) and dNTP's are added (extending 3…
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Pol I
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Function: Okazaki fragment processing and DNA repair
# of Subunits: 1
Contains 3'→5' and 5'→3' exonuclease activity
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Pol II
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Function: Translesion synthesis
# of subunits: 1
Only contains 3'→5' exonuclease
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Pol III
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Function: Chromosome replication
# of subunits: 3
Only contains 3'→5' exonuclease
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Pol IV
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Function: Translesion synthesis
# of subunits: 1
No exonuclease activity
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Pol V
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Function: Translesion synthesis
# of subunits: 2
No exonuclease activity
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General Structure of DNAP
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Right hand model
a. Fingers: dNTP binding site
b. Thumb: Tightens grip on DNA
c. Palm: Polymerase active site
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Conformational Structures of DNAP
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-Open: dNTP binds to fingers
-Closed: moved dNTP into active position and creates an active site.
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Metal Ions in DNAP Catalysis
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2 Mg²⁺ ions attached by Asp residues
- 1 Mg²⁺ ion deprotonates the 3' OH group to form 3'-O⁻ nucleophile
- Other Mg²⁺ ion binds to the incoming dNTP and facilitates departure of the pyrophosphate leaving group.
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DNAP Processivity
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how long DNAP can "hold on" before falling off.
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Distributive Synthesis
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repositions DNAP by dissociating and rebinding to DNA
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Processive Synthesis
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repositions DNAP by sliding forward on DNA
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Pol III Core
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Heterotrimer with 3 subunits
1. α: polymerase activity
2. ε: proofreading
3. θ: stabilizes ε
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Pol III Holoenzyme
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Replicates both leading and lagging strands.
Contains the following
2 Pol III Cores
2 β sliding clamps
1 clamp loader
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β Sliding Clamp
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Homodimer in Pol III holoenzyme that clamps onto DNA.
- Increases speed and processivity of DNAP III.
- Has to be attached to DNA via clamp loader
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Clamp Loader
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-Binds β sliding clamp to DNA at the primer-template junction via ATP.
-DNA has to bend out the side of the clamp loader, therefore only ssDNA can be used.
-Clamp loader is ejected via ATP hydrolysis
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Helicase
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-Hexameric rings at replication fork that uses energy NTP hydrolysis to drive strand separation.
-DnaB makes helicase that moves 5'→3'
-Ring shape enhances grip on DNA
-Attached to Pol III via the clamp loader
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Topoisomerase
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-Untwists DNA
- Gyrase (Type II topoisomerase) is prime replicative topoisomerase.
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Primase
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-synthesizes short RNA primers specifically for initiating DNA polymerase action.
-Primer attaches to helicase and makes an RNA primer (3' OH for DNAP)
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Pol I
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-contains 5'→3' exonuclease activity that removes RNA primers and can synthesize DNA.
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RNaseH
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-another enzyme that removes RNA primers, however, it cannot synthesize DNA.
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Nick Translation Activity
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remove RNA primers at the end of an Okazaki fragment and replace it with DNA.
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DNA Ligase
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-repairs nick and makes PDE backbone bond (requires ATP).
-only acts on 5' terminus of DNA.
-won't act on 5' terminus of RNA to make sure RNA primer is removed before nick is sealed.
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SSB
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-quickly binds to ssDNA to protect it from endonucleases.
-melts hairpins to stimulate DNAP activity
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Trombone Model of Replication
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Description of DNA replication on the lagging strand, with its repeated cycles of loop growth and disassembly, by analogy with the movement of a slide on a trombone.
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Okazaki Fragment Cycle
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1. Pol III is tethered and DNA needs to loop.
2. Loop grows during Okazaki fragment synthesis
3. Loop is released to start new synthesis of Okazaki fragments
4. Loop is reformed.
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Clamp Left on DNA during Okazaki Fragment Cycle
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Why is the sliding clamp left?
- B/c ligase and Pol I associate with left behind clamp
- Directs proteins to correct location
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Recycling of Clamps
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Clamps need to be recycled because there is a limited number of them in the cell.
δ (Delta) Subunit of the clamp loader removes the clamp.
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DnaA
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Initiator protein that binds to A-T rich sites to destabilize the bonds at the origin for replication.
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DnaB
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Helicase with 6 subunits
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DnaC
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Helicase loader that works in 5'→3' direction
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Dam Methylase
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-Methylates the N⁶ position of A residues on both strands of the GATC sequence.
-Immediately after replication, new strand is not yet methylated, thus leaving the stand hemimethylated.
-Hemimethylated state is only temporary.
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SeqA Protein
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-SeqA binds to hemimethylated GATC sites and thereby sequesters the newly replicated origin, preventing DnaA from rebinding the replicated origin.
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Nucleotide-Bound State of DnaA
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DnaA-ATP is hydrolyzed to DnaA-ADP, rendering it inactive for open complex formation, thus preventing reinitiation.
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Hda Protein
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Stimulates DnaA-ATP to hydrolyze its ATP to form DnaA-ADP.
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Ter Sites
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-Opposite to the oriC to stop replication forks from colliding.
-Tus monomer binds to ter sites to stop helicases.
-Replication forks can bypass the first Tus-Ter block, but cannot bypass the second Tus-Ter block.
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End Replication Problem
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-The inability to replicate the final segment of DNA at the 3' end of the lagging strand where there is no primer to provide a free 3'-OH group.
-Results in daughter strands that will be smaller.
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Telomeres
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-"cap" for linear chromosome
-short, repeated non-coding sequences that buffer against losing coding DNA during rounds of replication.
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Telomerase
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-Enzyme that has reverse transciptase activity and a short RNA sequence that primes the addition of nucleotide repeats to the 3' ends of DNA.
-Telomerase activity ensures that no unique sequence information is lost as a result of the end replication problem.
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Point Mutation
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a change in a single base pair
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Transition Mutation
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exchange of a purine-pyrimidine base pair for the other purine-pyrimidine base pair. C-G becomes T-A
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Transversion Mutation
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replacement of a purine-pyrimidine base pair with a pyrimidine-puring base pair. C-G becomes G-C
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Silent Mutation
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produces a codon for the same amino acid
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Missense Mutation
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change results in a different amino acid
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Nonsense Mutation
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change results in a stop codon.
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Insertion Mutation
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one or more base pairs added
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Deletion Mutation
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loss of one of more base pairs
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Indels
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Collectively refers to insertions or deletions
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Triplet Expansion Disease
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-Insertion of triplet sequences
-Repeat of triplet sequences has to reach threshold before disease present.
-DNA slippage is a possible mechanism for DNA expansion.
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Hydrolysis DNA Alteration
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-Hydrolytic cleavage causes nucelotides to alter.
-Example: Deammination by water of cytosine to uracil.
-Generally a slow process.
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Hydrolytic Cleavage of N-Glycosidic Bond
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-Water clips off the base, creating an abasic site.
-Occurs at a higher rate for purines than pyrimmidines (depurination)
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Deamination by Nitrous Acid
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-NaNO₃→HNO₂
HNO₂ reacts with cytosine and adenine
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Oxidative Damage DNA Alteration
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Occurs via Reactive Oxygen Species (ROS) e.g. peroxides
-can modify bases
-break the backbone
-affect sugars
-remove bases (creating abasic sites)
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8-Oxoguanine
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-Results from ROR interacting with G
-8-Oxoguanine can pair with C and A.
-Okay to pair with C, but not A.
-Mutation if paired with A (cause cancers).
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Alkylation DNA Alteration
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-Alteration by adding an alkyl group to the base.
-Effects depend on what group is added and where it is attached to the molecule.
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Polycyclic Aromatic Hydrocarbons (PAH's)
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-When PAH's are consumed, they end up in the liver where they are hydroxylated.
-Hydroxylated PAH's become reactive expoxides that can alkylate DNA.
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Nitrogen Mustard Gas
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-Cross linking agent that links adjacent G's that causes damage to the DNA.
-Alkylation alteration
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Ames Test
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Test used to determine if a chemical is a mutagen of DNA or not.
Steps:
1. Salmonella (auxotroph) with a mutation for histidine is plated on growth medium without histidine.
2. Filter paper of chemical is added to the center of petri dish.
3. If colonies grow, it is a mutagen becaus…
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Ames Test of PAH's
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Can be used on PAH's because mutagens are hydroxylated in the liver and the Ames test includes a step in which the compound being tested is incubated in the liver extract.
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Bleomycin Chemotherapy
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Binds to DNA and generates reactive oxygen species and causes DNA breaks in the PDE backbone.
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Doxorubicin Chemotherapy
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Can be inserted between base pairs and causes frameshift mutations and blocks replication forks.
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Cisplatin Chemotherapy
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Cross-linking drug that is a alkylating agent that forms covalent adducts with the N-7 position of the two purine residues.
Stalls the replication fork and division and can kill the cell.
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Pyrimidine Dimers
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Cross links between adjacent pyrimidines on the same strand that stall DNAP. Caused by solar radiation and UV light.
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Gamma Rays
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Stronger than UV light and can directly break PDE backbones.
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Errant Cell Process
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-DNA damage can result from errant replication which can produce point mutations or small insertions or deletions.
-Errant recombination can result in large-scale chromosomal abnormalities.
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Mismatch Repair (MMR)
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Mismatches corrected to reflect parent strand.
-Mismatch repair is costly because the mismatch and the GATC cleavage site can be more than 1,000 bp away, which requires a lot of dNTPs to repair a single mismatched base.
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Mismatch Repair (MMR) Process
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1. MMR proteins identify mismatches on the demethylated strand.
2. Mismatched base pairs are recognized by the MutS protein. MutS binds to MutL protein, creating the MutS-MutL complex.
3. MutS-MutL scan DNA bidirectionally, forming a loop.
4. When MutS-MutL complex arrives at a hemimet…
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Mismatch Repair in Eukaryotes
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Eukaryotic cells lack homologs to bacterial MutH and Dam Methylase, which means they do not use methylation to distinguish between new and old strands.
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Direct Repair by DNA Photolyase
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uses the energy derived from absorbed visible light to reverse the damage of UV light.
-Energy absorbed by light in the first chromophore in the enzyme results in electron transfer from FADH⁻ to form free radical FADH⁺
-FADH⁺ donates its electron to the pyrimidine dimer, reversing the …
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Direct Repair by Methyltransferases
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1. Guanine residue of normal G-C base pair is methylated on O⁶,O⁶-methylguanine base pairs with thymine, making it highly mutagenic.
2. O⁶-methylguanine-DNA methytransferase transfers the methyl group from O⁶-methylguanine onto one of its own Cys residues.
3. The methylated Cys residu…
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Base Excision Repair (BER)
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-functions at the level of a single damaged nucleotide that distorts DNA very little.
-main pathway for single strand DNA breaks that lack a ligatable junction and therefore require "cleaning" of the 3' or 5' terminus for ligation.
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Base Excision Repair (BER) in Bacteria
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1. Recognition of damaged base is performed by DNA glycosylase.
2. DNA glycosylase cleaves the nucleotide base from pentose by hydrolyzing the N-β-glycosyl bond, leaving an apurinic or apyrimidine site.
3. AP nuclease nicks the backbone at the abasic site
4. Segment of DNA is removed …
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Base Excision Repair (BER) in Eukaryotes
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-Occurs in 2 pathways
-Insertion of correct nucleotide does not occur by re-forming the glycosyl bond with a new correct base. Instead, ssDNA is cleaved at the abasic site by AP endonuclease, creating a nick with a 3' hydroxyl and 5' deoxyribose phosphate.
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Long Patch Base Excision Repair (BER) in Eukaryotes
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-Up to 10 nucleotides
1. DNA polymerase extends the DNA strand from the 3' terminus, displacing the 5' ssDNA.
2. This is followed by cleavage by a flap endonuclease and ligation.
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Short Patch Base Excision Repair (BER) in Eukaryotes
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-Only replaces damaged nucleotide
1. Pol β inserts one nucleotide prior to ligation.
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Uracil DNA Glycosylase
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-Used to find damaged DNA deep inside the helix
-Scans the minor groove of the helix
-Damaged DNA is kinked and flipped out of the helix into the active site.
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Nucleotide Excision Repair (NER) in DNA
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-Targets large, bulky lesions and removes DNA on either side of them.
-NER does not require specific recognition of a damaged nucleotide
-Dominant repair pathway for removing pyrimidine dimers, 6-4 photoproducts, and other bulky base adducts.
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Exinuclease
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enzymatic activity that creates 2 incisions in one DNA strand, excising the lesion.
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Nucleotide Excision Repair (NER) Process in E. Coli
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1. UvrA₂UvrB complex scans DNA for damage.
2. Once bulky damaged base is located, the strands become separated to form ssDNA bubble containing the lesion and UvrA dissociates, leaving UvrB tightly bound to the damaged site.
3. UvrB recruits UvrC exinuclease to make incisions in the DNA…
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Nucleotide Excision Repair (NER) Process in Eukaryotes
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Looks similar to E. Coli with small differences...
1. Uses XPC instead of UvrB
2. Uses XPG instead of UvrC
3. RNAP is the first enzyme to find the lesion.
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Translesion DNAP (TLS DNAP) Repair
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-Uses TLS DNAP to synthesize the DNA across the lesion.
-Not specific with adding a nucelotide and can cause a mutation.
-Usually lack proofreading.
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Translesion DNAP (TLS DNAP) Repair Process
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1. When Pol III encounters a lesion, it stalls.
2. TLS Pol takes over the sliding clamp and extends the leading strand across the lesion.
3. After lesion bypass, Pol III resumes its function with the sliding clamp.
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Genetic Recombination
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exchange of genetic information with or between chromosomes.
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Recombination to Fix a Double Strand Break (forming a branched intermediate)
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1. Needs an undamaged template (copy of the damaged strands)
2. End processing of the broken DNA (3' overhand)
3. Recombinase: stand invasion
4. DNA synthesis
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Synthesis-Dependent Strand Annealing (SDSA)
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Starts with a "branched intermediate"
1. Newly synthesized DNA strand is displaced off the template
2. Displaced strand anneals to "original" strand.
3. DNA synthesis: ligation via ligase to close PDE backbone.
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Double Strand Break Repair (DSBR)
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Starts with a "branched intermediate"
1. Both stands captured.
2. DNA synthesis (stable, intertwined mixture while the strands are attached to each other).
3. Special nucleases cut the DNA strands.
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Fork Regression
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way to provide a complementary DNA strand time to allow lesion repair.
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Fork Regression Process (Lesion Repaired)
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1. Fork approaches lesion and stalls.
2. Fork regresses in the opposite direction.
If the lesion is repaired...
1. Repair machinery makes the repair.
2. The regressed DNA is digested by nuclease.
3. Replication starts again.
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Fork Regression Process (Lesion Unrepaired)
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1. Fork approaches lesion and stalls.
2. Fork regresses in the opposite direction.
Lesion remains unrepaired...
1. Replication by fork regression synthesizes enough DNA to get past the lesion by using the other newly formed strand.
2. Branch migration then carries the newly forme…
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Fork Collapse
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When a replication fork encounters a template strand break, one arm of the fork is lost and the fork collapses.
1. The 5' end at the break is processed to create a 3' single-stranded extension.
2. The 3' single-stranded end is used in strand invasion.
3. The invasion creates a branc…
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Branched Migration
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Movement of the branch point in a branched DNA formed from two DNA molecules with identical sequences.
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Gap Repair for Single Stranded Regions
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-A process for repairing gaps left when the replication fork bypasses a lesion.
-More likely on a lagging strand.
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Gap Repair Process
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1. Strand invasion at the gap site, using undamaged arm of the replication fork for repair.
2. Branch migration generates two Holliday intermediates, with the undamaged strand serving as a template.
3. Holiday intermediates are cleaved by Holliday intermediate resolvases, and the nick…
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Proteins For Recombinational Repair
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1. End Processing: RecBCD complex (generate 3' overhang)
2. Strand Invasion: recombinase (RecA) with RecBCD or RecFOR helping load RecA on ssDNA.
3. Branch Migration/Resolution: RuVAB, RuVC, SSB, polymerase, and ligase.
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Initiation of Recominational Repair
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RecBCD Complex: helicase and nuclease activity
1. Generates the 3' overhang
2. Helps load RecA onto 3' overhang.
RecBCD binds to linear DNA at free (broken) end and moves inward along the double helix.
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Chi Sequence
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sequence that alters endonuclease activity of RecC, reducing degradation of the strand with the 3' terminus and increasing degradation of the strand with the 5' terminus, creating the 3' overhang.
- 5'-GCTGGTGG-3'
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RecFOR
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-used in the gap repair process.
1. SSB associating with ssDNA blocks RecA
2. Factor X recruits RecFOR
3. RecFOR helps RecA load and displace SSB
RecFOR is a mediator: proteins that function to load other proteins on DNA.
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RecA
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Protein that is a recombinase.
-Higher affinity for ssDNA
-align ssDNA with homologous strand
-promotes strand invasion
-active form is helical filament
-up to thousands of subunits that cooperatively assemble, coding ssDNA
Goal of Rec A is strand invasion.
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Cooperativity of RecA
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Binding of first RecA is slow because it is blocked by SSB, however, once the first RecA is bound, others rapidly bind in 5'→3' direction.
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Control of RecA
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Need to control the synthesis and activity of RecA
1. Transcriptional level: only allow enough transcription as needed.
2. Activity affects formation, function, and assembly of the filament.
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RuvAB Complex
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Complex that processes Holliday intermediates for the repair of UV damage.
RuvA and RuvB: branch migration
RuvB: similar to helices, however, doesn't separate strands (more like a translocase).
RuvC: resolvase (cuts DNA strands) that results in a nick that needs to be fixed by ligase…
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Homologous Recombination in Eukaryotes
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Used to...
1. Repair DNA damage, meiosis, and mitosis.
2. Spo11 (in all eukaryotes): introduce dsDNA breaks for meiotic recombination
3. Gene Conversion: switching gene expression.
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Nonhomologous End Joining
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-Seen in all eukaryotes and some bacteria.
-How cells repair dsDNA breaks when an undamaged DNA copy is not present for homologous recombination.
-"Gluing ends together"
-"Gluing ends together"
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General Rules of Nonhomologous End Joining
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1. Ends of break are bound by proteins
2. Nuclease activity that widen the break
3. Generation of overhangs and strands anneal
4. Synthesis of DNA to fill in gaps (usually delete a small region).
5. End with ligation
In Eukaryotes, telomeric repeats protect the ends of chromosome…
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Site-Specific Recombination
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a type of genetic recombination that occurs only at specific sequences
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Recombination Site
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short, specific DNA sequence
Consists of 2 inverted repeats flanking a core sequence
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Recombinase
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Enzyme that recognizes recombination sites.
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Inversion Recombination Outcome
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-IR sites are oppositely oriented (2 IR's are facing each other)
-Recombinase recognizes IR
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Deletion Recombination Outcome
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-Core sites are in the same orientation. (IR's facing in the same direction on the same DNA strand)
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Insertion Recombination Outcome
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-Core sites on different DNA strands.
- IR's facing the same direction
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Synaptic Complex
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4 subunits of recombinase aligned with the 2 cores.
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Recombination Process
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-Multistep process with two rounds of single strand cleavage and rejoining
-Holliday intermediates generated during the process
-Tyr and Ser in active sites
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BIOL 4843: EXAM 2