3.3 - DNA replication Flashcards
DNA replication
- synthesis of new DNA
- occurs during S-phase of the cell cycle
- semi-conservative
- 2 strands of parental double helix unwind, and each specifies a new daughter strand by base-pairing rules
- 3 Steps: Initiation, elongation (of the new strand), termination
Initiation in DNA Replication
- Begins at an origin of replication (OriC)
- involves: initiator proteins, helicases, primase, polymerase III
- at the end of initiation: helicases are ready to unwind, RNA primers are in place, polymerases are ready for elongation
Initiation Components: Initiator proteins
-separate the strands on OriC
Initiation Components: Helicases
- loaded only
- 1 at each fork
Initiation Components: Primase
-synthesizes 2 RNA Primers
Initiation Components: Primer
- synthesized by primase
- short (4-6 nt) RNA
- complementary to DNA template
- antiparallel
- (attracts DNA polymerase to build)
Initiation Components: Polymerase III
- loaded only
- 2 at each fork
Elongation in DNA Replication
- Helicases unwind duplex bidirectionally
- DNA polymerase adds DNA nucleotides to the 3′ end
- involves: helicases, DNA polymerase III dimers, dNTPs, primases (RNA primers), DNA polymerae I, DNA Ligase
Elongation Components: Helicases
- unwind duplex bidirectionally
- 2 forks move away from the origin
Elongation Components: DNA polymerase III
- adds single nucleotides to 3’ OH of pre-existing polynucleotide basepaired to a DNA template
- 5’ > 3’ polymerase activity
- will also “proofread” 3’ - 5’
Elongation Components: dNTPs
deoxynucleoside triphosphate precursors
- dATP – deoxyadenosine triphosphate
- dGTP – deoxyguanosine triphosphate
- dCTP – deoxycytidine triphosphate
- dTTP – deoxythymidine triphosphate
Lagging strand
- lagging strand synthesis is discontinuous (okazaki fragments)
- Parent template is 5’ > 3’
- Contain primase that synthesizes RNA primer
Elongation Components: DNA polymerase I
- Primer removal and gap filling in the completion of an Okazaki fragment
- at 3’OH of one Okazaki fragment (so can only go 5’ to 3’ direction)
1) breaks phosphodiester “forward” (5’ > 3’)
2) adds deoxynucleotide to 3’OH (5’ > 3’) - these two activities alternate until primer is removed
- will also “proofread” 3’ - 5’
Elongation Components: DNA Ligase
- nicks still exist between okazaki fragment, therefore…..
- forms the final phosphodiester linkage on lagging strand between 3’OH of one okazaki fragment and 5’PO4 of next okazaki fragment
Mismatched base-pairs (proof-reading)
- mismatched bps create distortion in diameter detected by trailing portion of polymerase
- polymerizing activity stops
- 3’ to 5’ exonuclease activity breaks 3-4 phospodiester linkages including mismatched nucleotides
- resumes 5’ to 3’ polymerase activity
Termination in DNA Replication
- stops DNA replication by addition of telomere sequence at end
- includes in circular chromosome: topoisomerase II
- includes in linear chromosome: telomerase, DNA polymerase I, DNA ligase
Telomere Synthesis
-does not occur after chromosome replication in all cells
1) after telomerase extension of 3’ end
2) primase-primed DNA polymerase synthesis of other strand
3) ligase forms final phosphodiester linkage
4) repair polymerase removes primer
Leading strand parent template
- parent runs runs 3’ > 5’
- therefore, leading strand synthesizes 5’ > 3’ w/o problems
