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FRNSC 420 - EXAM 2-Questions with Correct Answers/ Verified/ 100% Pass
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FRNSC 420 - EXAM 2-Questions with Correct Answers/ Verified/ 100% Pass
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FRNSC 420 - EXAM 2-Questions with Correct Answers/ Verified/ 100% Passnucleosome assembly/reassembly after replication - ✔️✔️- old H3-H4 tetramers remain bound to
one of two daughter duplexes at random
- old H2A-H2B dimers dissociate and compete to associate with tetramers
- DNA sliding clamps mark replicating DNA and recruit chromosome assembly factors (CAFs) as
chaperones
- CAFs free nucleosomes and escort to newly synthesized DNA; NAP-1 escorts new/old H2A-H2B
dimers, CAF-1 escorts new H3-H4 tetramers
nucleosome imprinting - ✔️✔️old complexes recruit enzymes to add modifications to new
nucleosomes
- acetylated Lys residues on old tetramer tails will recruit bromodomains with acetyl transferase
- acetyl transferase will acetylate new tetramers adjacent to the old ones
- if left unmodified, structure and gene expression will be unregulated
substrates for DNA polymerase - ✔️✔️- primer:template junction: required for synthesis, dsDNA
next to ssDNA region
- template: portion of junction that acts as a guide
- primer: 3'-OH for nucleotides to be added on to
- Mg²⁺: promotes polymerase activity through ionic interactions
- dNTPs: added to the growing chain
replication fork - ✔️✔️Y-shaped region that directs synthesis, allows for synthesis of two strands at
once
- continuous replication of the leading strand towards the fork
- discontinuous replication of the lagging strand using Okazaki fragments away from the fork
driving force of DNA synthesis - ✔️✔️the free energy of nucleotide addition is low, but when
coupled with the hydrolysis of pyrophosphate (PPi), the reaction is favored
- initiation is an SN2 reaction with 3'-OH acting as nucleophile to attack the α-phosphate of incoming
dNTP
- extension of primer 3' end by one nucleotide and release of PPi
- PPi is hydrolyzed by pyrophosphatase to yield two inorganic phosphate (Pi) molecules
,3-D structure of DNA polymerase - ✔️✔️like a cupped right hand with palm, fingers, and thumb
- palm: a β-sheet holding active site. binds metal (Mg or Zn) to initiate synthesis and monitors base
pairing through H-bond contacts with minor groove. allows for proofreading, will release primer at a
mismatch
- fingers: α-helices that act in positioning nucleotides, pushing active site components together. Arg
and Lys residues form electrostatic interactions to ensure proper orientation; Tyr residues base stack
for stability. also can bend the backbone to expose first template base
- thumb: α-helices that maintain correct positioning of primer and active site. maintains association
between polymerase and template. works in concert w/ palm to move synthesized DNA from active
site, contributes to processivity
physical ways DNA polymerase maintains fidelity - ✔️✔️active site has discriminator site of neutral,
nonpolar, and aromatic amino acids that can differentiate between NTPs and dNTPs
- normally, AAs interact w/ ribose ring and 3'-OH of neighboring base pair attack phosphate group of
incoming nucleotide
- activity is blocked with 2'-OH group in NTPs due to steric hinderance; base will tilt and not be
recognized by neighbor 3'-OH
- dNTP binding pocket is small, most other ribonucleotides wont fit
enzymatic ways DNA polymerase maintains fidelity - ✔️✔️a 3' to 5' exonuclease in the replisome
has proofreading, will degrade unpaired DNA
- geometry of strand is altered from a mismatch, leading to decrease in polymerase activity
- primer:template junction is destabilized, creating several unpaired bases
- exonuclease has high affinity for ssDNA, will recognize and hydrolyze unpaired/mismatched bases
3'-5' vs 5'-3' exonucleases - ✔️✔️- 3' to 5' has proofreading and moves backwards on the strand to
removed mismatched bases
- 5' to 3' is not proofreading; repair for damaged DNA and removal of RNA primers (Taq polymerase)
primase - ✔️✔️a slow RNA polymerase that makes short RNA primers from ssDNA template
- synthesized RNA acts as primer for primer:template junction
- most active when associated with other proteins (ex: helicase)
- one primer for every leading strand, multiple for each Okazaki fragment in lagging strand
- primase in E. coli is DnaG w/ recognition site CTG
, helicase - ✔️✔️loads on ssDNA & separates dsDNA through breaking hydrogen bonds of base pairs
- found at replication fork on lagging strand
- hexameric (6 domains) with two tiers (C tier faces fork) that encircle ssDNA
- uses ATP hydrolysis to move (~2 bp per ATP)
helicase polarity - ✔️✔️moves in single direction along ssDNA (3'-5' or 5'-3')
- lagging strand always moves 5' to 3' towards replication fork
- can be determined through displacement of one strand of dsDNA; restriction enzyme cleaves
cccDNA, leaving two fragments
- helicase will either move 5'-3' or 3'-5' to remove fragment
single-stranded binding (SSB) proteins - ✔️✔️bind to ssDNA after displacement by helicase for
stability
- bind independent of sequence through electrostatic interactions w/ phosphate backbone (base
stacking)
- cooperative binding coats the strand with proteins to inhibit further hydrogen bonding
- ssDNA takes on an extended, straight confirmation following coating
DNA sliding clamp - ✔️✔️donut-shaped protein that associates w/ DNA polymerase and encircles
dsDNA
- β subunit in E. coli, PCNA in eukaryotes
- allows for polymerase to replicate without repeatedly dissociating from the template, increases
processivity
- leaves room for 1/2 molecules of water in between DNA, no direct contact
- loaded on rep. fork by sliding clamp loader
- loses affinity for polymerase in absence of primer:template junction; enzyme dissociates and clamp
remains on strand after replication
- removed by loader and put on next Okazaki fragment
DNA sliding clamp loaders - ✔️✔️five-subunit complex that catalyzes the opening and placement of
DNA sliding clamps; couples ATP binding and hydrolysis
- γ-complexes in E. coli, RF-C in eukaryotes
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