Replication Flashcards
Meselson and Stahl Experiment
Demonstrated the semi conservative nature of DNA replication
- used centrifugation to distinguish between heavy and light nitrogen
- grew cells in heavy nitrogen and moved to a medium of of light nitrogen
Replication Forks
- origin of replication
- bidirectional replication
DNA Polymerase
- catalyse replication
- uses ssDNA as a template to add dNTPs to free 3’ OH group of a base paired nucleotide to synthesize a complementary strand
- incoming nucleotides selected by Watson Crick base pairing
- driven by energy of release of pyrophosphate and its subsequent hydrolysis to inorganic phosphate
Polymerase III
5’ - 3’ polymerase activity
3’ - 5’ exonuclease activity
Polymerase I
5’ - 3’ polymerase activity
5’ - 3’ exonuclease activity
- binds to dsDNA at ss nicks and cleaves beyond the nick to excise the DNA
- nick translation: replace nucleotides on 5’ side of ss nick, translating nick to 3’ end without changing molecule
3’ - 5’ exonuclease activity
- 3-5 activity activated by unpaired 3’ terminal nucleotide with free OH (nucleotide excised)
DNA helicase
- unwinds duplex DNA
- ATP hydrolysis used to unwind short sections of AT rich DNA: less energy needed
- binds ssDNA and moves along the duplex prising DNA apart
Single Stranded Binding Proteins
- prevent reannealing of DNA
- cooperative protein binding straightens region of chain
- stripped off for replication to occur
- keeps strands apart
- stop secondary structure formation
- aligns strands
- interacts with replication proteins
- stimulates polymerases
Topoisomerase
- supercoiling of DNA occurs ahead of helicase
- tightening of helix will create intolerable strain and energy needed to unwind would be too great
- nick in one of the strands phosphodiester bonds could allow swivel and relaxation
2 classes: type I makes ss breaks while type II makes staggered ds breaks
Type I topoisomerase
- tyrosine in active site forms covalent phosphodiester link with top ss molecule of DNA
- allows nick so rotation = relaxation
- original phosphodiester bond energy conserved = spontaneous reversible reaction
Primase
- synthesizes short RNA primer at replication origin so that DNAP can begin replication
- forms primosome with helicase
Model of DNA replication
- leading strand synthesized continously in 5-3’ direction
- lagging strand synthesized discontinously in 3-5’ direction as a series of Okazaki fragments joined to make long nascent DNA chains
- elongation of lagging strand in opposite direction to the direction of replication fork advance
E. Coli Replication Initiation
- OriC origin of bidirectional replication fork
- OriC bound by initiator protein DnaA that opens up a segment into single strands
- DnaC binding permits helicase (DnaB) binding
- primase activated by helicase
DNAP in E. Coli
- versatile activity
- 5 - 3 polymerase
- 5- 3 exonuclease (removal of primers)
- 3 - 5 exonuclease (proofreading)
Polymerase I
- 3 -5 polymerase/exonuclease
- 5 - 3 exonuclease
- lower processivity and polymerisation rate
Polymerase III
- holoenzyme required for survival
- can proofread (3-5 exonuclease) but not repair nicks (5-3 nick translation)
Structure of DNAP III
- DnaB helicase
- core (a, E, w subunits)
- B clamp + clamp loader
- y complex: DNA dependent ATPase
B sliding clamp
- clamp is a protein binding DNA pol and prevents enzyme dissociation from DNA (stabilizes molecule)
- increases processivity (promoting factor in replication)
- if ds region encountered the clamp releases it
- clamp loader loads onto the DNA and releases it
Accuracy of Polymerase
- reliance on Watson Crick base pairing alone is not sufficient for perfect accuracy
Method 1:
- correct nucleotide has higher affinity for polymerase as it correctly base pairs with template
- phosphodiester bond formation involves conformational change in DNAP so that incorrectly bound nucleotide doesn’t fit in active site
Method 2:
- 3-5 activity of DNAP
- takes place immediately after incorrect addition to growing chain
- polymerase can’t extend such a chain as 3-OH is needed
- clips off mismatches at separate catalytic site
- newly synthesized DNA transiently unpairs and polymerase has a conformational change to move this editing site into place
Replisome
- lagging strand template loops around bringing 2 DNA polymerases into a complex
- brings 3’ end of a completed okazaki fragment close to the start site for the next fragment
- trombone model
Steps of Replication
- leading strand synthesised simultaneously and laggin strand begins later (unwinding is RDS)
- as DNA unwinds, 3’OH is continually replicated
- unwinding and synthesis is concommitant
- after enough ssDNA is made, lagging strand synthesiis occurs
- fragment synthesis nears completion
- primase binds to DnaB and synthesizes a new primer then dissociates
- new B clmap loaded onto new primer by clamp loader
- synthesis of new fragment completed on lag strand
- lag strand core subunits transferred to the new template primer and its B clamp - old clamp discarded
- next B clamp readied as fragment synthesis initiated
RNA primer removal
- DNAP III elongates chain and falls off
- DNAP I binds and uses 5-3 exonuclease activity to remove RNA and replace it with DNA using 5-3 polymerase activity
- DNA ligase links fragments
Ligase
- energy input from ATP
- 3-OH and 5-P joined and catalysed by ligase
Uses of Replication Enzymes
- recombinant gene technology
- PCR
- Sanger method of sequencing
