DNA Replication

• 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