DNA replication is ________ and __________
semi-conservative
bidirectional
Origin of replication
- sequence specific site recognized by replication proteins
- hundreds of origins per chromosome in euk (only 1 in prok)
Replication fork
where DNA helicases have unwound the double helix and the replication machinery sits and synthesizes complementary strands
Origin binding proteins
- Recognize replication origin
- Proteins bind to origin and become part of the complex, also recruits Polymerase
Helicase
enzymes that catalyze the breaking of H-bonds between base pairs and the subsequent ‘unwinding’ of the helix.
Single-Stranded Binding Proteins
bind to the melted strands of original DNA to prevent them from re-annealing or getting attacked by nucleases
Primase
Enzyme that catalyzes the addition of the RNA primer to begin replication
(RNA primer required for DNA pol)
DNA polymerase I (3 different activities)
Removes RNA primer, replace with DNA
1) 5’–>3’ exonuclease activity: removes RNA primer
2) 3’–>5’ exonuclease activity: proof-reading
3) 5’–>3’ DNA synthesis: fills gap once RNA is removed
_________ is distributive and falls on and off the DNA easily and _______ is processive
DNA polymerase I
DNA polymerase III
DNA Polymerase III (2 activities)
- Elongation of DNA from RNA primer
- uses sliding clamp (processive)
- multi-protein complex
1) 5’–>3’ DNA synthesis
2) 3’–>5’ Exonuclease activity (proofreading)
DNA polymerase III determines the correct base to input based on ________ and _________
H-bonds and geometry
DNA ligase
- enzyme responsible for sealing Okazaki fragments together once the RNA primers have been replaced by Pol I
- Uses cofactors (ATP-euk, NAD+ - prok)
Sliding clamp
- Holds on to DNA and allows DNA pol III to hold on to DNA strongly, but enables it to move
- PCNA (euk), Beta clamp (prok)
Telomere
- sequence at the ends of chromosomes, consisting of a large number of repeating segments
- gets consistently shorter with every chromosome replication
Dysteratosis congenita
telomere shortening syndrome
Topoisomerase/gyrase
- Enzyme responsible for relieving torsional strain in the DNA helix in the region ahead of the replication fork
- Does so by breaking and rejoining dsDNA
- topogyrase for prok
Exonucleases
chews from the end
Endonuclease
cuts within a DNA strand - breaks phosphodiester bond between sugar and phosphate
DNA polymerase requires that the deoxyribonucleotide to have a _________ group for phosphodiester bond synthesis
3 hydroxy group
DNA synthesis requires a ______ and proceeds in the _______ direction
RNA primer
Proceeds in the 5’–>3’ direction
Errors in replication are corrected by the __________ activity of the DNA polymerase
3’–>5’ exonuclease proofreading
7 Steps of DNA synthesis on leading strand
1) origin binding proteins bint to origin
2) DNA melted apart by helicases
3) Topoisomerase relieves tension ahead of replication fork
4) ssbinding proteins protect unwound ssDNA
5) RNA primer synthesized by RNA primase
6) Pol III elongats DNA from RNA primer, complementary to leading strand
7) New and old strands annealed
DNA synthesis on lagging strand (5 steps - after DNA unwound)
1) primase attaches RNA primer to lagging strand segment
2) Pol III elongates DNA from RNA primer back a short ways, forming an Okazaki fragment
3) RNA primer removed and replaced with DNA by Pol I
4) Fragments sealed together with DNA ligase
5) new and old strands annealed
In eukaryotes: DNA pol E synthesizes on the _________ strand and DNA pol d synthesizes on the _______ strand
leading
lagging
