DNA Replication

Cards (42)

  • fundamental rule of DNA replication

    -semi-conservative (1 old 1 new strand)
    - begins at an origin (1 bacteria many for humans)
    - proceeds bi-directionally
    -proceeds in 5' to 3' direction
    -semi-discontinuous
  • What was the Meselson Stahl experiment?

    -grew bacteria with a heavy 14N containing isotope then replicated it in another with a light 15N containing isotope
    -DNA was then separated by density centrifugation
  • What did Meselson Stahl prove?

    -proved that DNA replicated through a semiconservative model
  • DNA Polymerase reaction

    (new strand) +dNTP- (DNA Pol)-> new strand + PPi
  • What reaction do DNA Polymerases catalyze?

    -phosphoryl group transfer
    -by catalyzing the phosphodiester link formation between adjacent nucleotides
    -pyrophosphate generated is hydrolyzed by pyrophosphatase.
  • What are the primary requirements for DNA polymerases to work?

    -single unpaired DNA strand
    -primer (pre-existing short strand of DNA or RNA with a free 3' OH where a new nucleotide can be added).
  • catalytic mechanism for DNA polymerases
    -needs two Mg2+ ions at the active site
    -One Mg2+ helps deprotonate the 3' OH group, making it a more effective nucleophile
    -The 3' OH attacks the alpha phosphate on the incoming dNTP
    -The other Mg2+ binds to the incoming dNTP and stabilizes its negative charge.
  • What is Processivity?
    the average number of nucleotides added before the polymerase dissociates from the template strand
  • What is the error rate for replication? How do cells achieve this remarkably low rate?
    -mistake is only made once every 10^9 to 10^10 meaning that an error occurs once per 1,000 to 10,000 replications
    -due to proofreading activity (3' to 5' exonuclease activity) which corrects mistakes and improves the accuracy of polymerization 100 to 1000 fold.
  • What is proofreading?

    DNA pol identifies mismatches and mistakes and repairs them in the DNA
  • How does the 3' to 5' exonuclease activity of DNA Polymerase fix errors?

    -aka proofreading activity
    -digests nucleotides with the 3' OH from the 3' to 5' direction
    -when a mistake is identified it is excised out and the correct nucleotide is added
  • components of bacterial DNA polymerase III
    -Two core domains containing ɑ, ε, and θ subunits (linked by clamp loader)
    -Core domains interact with a dimer of β subunits that increase processivity.
    -Core, clamp loader, and β sliding clamp all complete the holoenzyme
  • core function in DNA pol
    polymerase and proofreading
  • clamp loader function in DNA pol
    helps load the β clamp onto the core
  • β clamp function in DNA pol
    increases processivity
  • What proteins are involved in the Initiation phase of replication in bacteria?
    - 20 molecules of DNaA protein
    -DNaB-DNaC complex
    -DNaC
    -SSB, helicase, primase , fork, pol III complex (not proteins, but still important)
  • 20 molecules of DNaA protein function

    binds to oriC (at 9bp repeat) to unwind the double helix (at 13bp)
  • oriC
    contains three repeats of a conserved 13bp sequence and 4 repeat 9bp sequence
  • DNaB-DNaC complex function in initiation

    -binds to the unwound DNA
    -DnaC loads DnaB helicase protein at the forks and dissociates
  • SSBs function in initiation
    bind to the single-stranded region that was dissociated by DnaC and prevents duplexes from forming
  • helicase in initiation
    primase that is associated with DnaB
  • primase function in initiation

    -binds to DnaB
    -synthesizes RNA primers on the leading strand
  • fork function in initiation

    -expands causing movement that makes DnaA proteins to dissociate
  • Pol III complex in initiation
    binds to the fork making the transition from the initiation complex to the replicative complex (synthesizing new DNA using the leading and lagging templates)
  • initiation is a tightly regulated so that replication occurs only once in each cell cycle how?
    -Once dissociated from OriC, DnaA takes 20 minutes to be activated before it can bind to OriC again
    -or regulated by DNA methylation
  • DNA methylation steps

    -OriC methylated by Dam methylase at the N6 position of adenine within a palindromic sequence GATC
    -Only the parent strand is methylated, but DNA replication generates two hemimethylated daughter strands
    -the protein SeqA binds hemimethylated OriC and sequesters it to the plasma membrane
    - OriC is released from the plasma membrane after some time at which point Dam methylase methylates the daughter strand
    -Both strands must be fully methylated before DNaA can bind and initiate a new round of replication
  • What is Replisome?
    an entire complex with enzymes and protein factors needed in addition to DNA polymerase for elongation and promotes rapid DNA synthesis
  • What are the main enzymes/proteins of the Replisome?

    helicase, topoisomerase/gyrase, SSBs, DNA pol III, DNA pol I, DNA ligase
  • helicase function in the replisome

    unwinds DNA duplex by using ATP; also creates topological stress
  • Topoisomerase/Gyrase function in the replisome
    relaxes supercoils generated upstream of the replication fork
  • Single-stranded binding proteins(SSBs) function in the replisome
    stabilize the separated strands preventing premature re-annealing
  • primase function in the replisome

    generates RNA primers (one for leading and multiple for lagging)
  • DNA Pol III function in the replisome
    replicates genome
  • DNA Pol I function in the replisome
    removes RNA primers, fill in gaps left by primers
  • DNA ligase function in the replisome
    seals nicks in the backbone
  • How is the leading strand elongated?

    -unwound by helicase resulting in topological stress that is relieved by gyrase
    -SSBs bind to separate strands
    -primase (DnaG protein) synthesizes a short RNA primer at the origin
    -Deoxynucleotides are added to the primer by DNA polymerase III
    -proceeds continuously keeping pace with the unwinding DNA at the fork.
  • How is the lagging strand elongated?

    -DNA synthesis is continued until the fragments extend as far as the primer of the previously added Okazaki fragment
    -a new primer is synthesized near the replication fork to begin the process again
  • How does one replisome synthesize both leading and lagging strands?

    -DNA replication has to occur in the 5' to 3' direction so the leading and lagging strand synthesis coordinate simultaneously in opposite directions by a single DNA POL III complex
    -This is accomplished because the lagging strand loops around
    -One set of DNA polymerase III core is used for leading strand synthesis
    -The other set of core subunits cycles from one Okazaki fragment to the next on the looped lagging strand.
  • What is the 5' to 3' exonuclease (Nick Translation) activity of DNA Pol I? What role does it play in DNA replication?
    -DNA or RNA strand are paired to a template are simultaneously degraded by the 5' to 3' exonuclease activity and replaced by the polymerase activity of the same enzyme
    -A Nick is a broken phosphodiester bound with a free 3' OH and free phosphate group
    -Nick translation has a role in DNA repair and removal of RNA primers during DNA replication.
  • What role does DNA ligase play in replication?

    -seals nicks in backbones
    -joins okazaki fragments