Biochem - DNA Replication Flashcards
Central Dogma
DNA —> RNA –> Protein
Central dogma for all organisms except…
some viruses
Significance of DNA replication
transmit genetic info to offspring, be performed with high fidelity, defects –> genetic instability –> mutations and disorders
Semiconservative DNA replication
parental strands are permanently separated
each forms a duplex molecule with the new complementary daughter strand
AT, GC H bonds
2 H bonds for A-T
3 H bonds for G-C
Substrates for DNA polymerases?
Deoxyribonucleoside triphosphates (dNTPs)
What actually gets added to the growing DNA chain?
dNMPs - processive process
How a dNTP –> dNMP and adds to the DNA chain
Incoming dNTP forms bp with complementary NT on parental strand
phosphodiester bond is formed
Pyrophosphate is released (PPi)
generates large energy change –> drives DNA synthesis
Type of bond made between incoming NT to the DNA chain
phosphodiester bond made between 5’ phosphate of incoming dNTP and free 3’ OH of the end of the chain
Bond is a 3’-5’ phosphodiester bond
Hydrolysis of PPi
PPi –> 2Pi generates a large energy change
DNA replication occurs in …
S phase of cell cycle in eukaryotic cells
oriC
origin of replication in E. coli
contains short AT rich sequences
Primosome
Protein complex that initiates DNA synthesis
Initiate unwinding of the duplex DNA
stabilize the DNA template single strands
initiate the synthesis of the RNA primers required for DNA synthesis
Primosome proteins?
DnaA DnaC DnaB (Helicase) SSB DnaG (DNA primase)
DnaA
tetrameric DNA binding protein with specificity for oriC
Involved in directing other primosome proteins to the origin
ATP required for initial melting of AT region
DnaC
Helicase inhibitor that is required for loading DnaB onto the strand
DnaB
DNA helicase
further separates the duplex DNA
ATP is hydrolyzed
SSB
Single stranded DNA binding protein
Stabilized ssDNA
DnaG
DNA Primase
generates short RNA primers for DNA synthesis
Primosome binding process
DnaA binds to the replication origin DnaB binds to the DNA DnaB opens the helix and binds DnaG to form primosome RNA primer synthesis DNA polymerase can start first DNA chain
Semidiscontinuous manner
DNA has anti-parallel structure
DNA synthesis can only occur in 5–>3’ direction
Replication fork moves in 5–>3 for one strand but 3–>5 for the lagging strand
Leading strand
Synthesized continuously in 5–>3’ direction
Moves in same direction as fork
Lagging strand
Synthesized in opposite direction
Discontinuous DNA fragments must be joined together by DNA ligase
Okazaki fragments
discontinuous DNA fragments on the lagging strand
Replisome
elongation of DNA requires additional proteins
Replisome proteins
DNA polymerase
DNA ligase - ATP hydrolysis
Topoisomerases
Sliding clamp
Sliding clamp
Made up of 2 halves
Clamp loader places clamp on DNA in ATP dependent process
Clamp keeps DNA pol to be processive
DNA ligase
seals nicks between DNA fragments
Seals in 3’–>5’ fashion
ATP hydryolysis
E. coli is NAD-dependent
E. coli DNA pol I
Monomeric 5-->3' elongation 3-->5' exonuclease proofreading 5-->3' exonuclease Remove RNA primers Replace RNA primers with DNA Slow, low processivity
E. coli DNA pol III
5-->3' elongation 3-->5' exonuclease proofreading No 5-->3' exonuclease Recognizes RNA primers Fast, high processivity Major replicative polymerase
Termination of replication
replication forks meet in region with multiple copies of ter sequence
Proteins bind and trap/arrest the fork by inhibiting DnaB
Why PPi –> 2Pi
If you keep taking out the product, then you can keep pushing the reaction to go forward
Why is eukaryotic DNA replication more complicated?
Size and organization of euk DNA
Multiple origins of replication, many forks
Primer removal (5–>3’ exonuclease) is done by RNaseH
ORC in eukaryotes
Origin recognition complex - 6 proteins
Analogous to DnaA
Phosphorylation of ORC allows binding of proteins like primosome
Onset of S phase - DNA pol alpha-primase binds to make it an active complex
DNA pol alpha
Nuclear
Initiation, primase activity, synthesize 20nt
5–>3’ polymerase
No 3–>5’ exonuclease
DNA pol beta
Nuclear
DNA repair
5–>3’ polymerase
3–>5’ exonuclease
DNA pol delta
Nuclear
Elongation - chromosomal DNA replication
5–>3’ polymerase (highly processsive)
3–>5’ exonuclease
DNA pol epsilon
Nuclear
Elongation - chromosomal DNA replication
5–>3’ polymerase (highly processive)
3–>5’ exonuclease
DNA pol gamma
Mitochondrial
Replication of mitochondrial genome
5–>3’ polymerase
3–>5’ exonuclease
Nucleosomes
H1, H2A, H2B, H3, H4 Positively charged (ionic bond with DNA) Core histones = 2A, 2B, 3, 4 + Linker H1 Duplicated during DNA synthesis Synthesized mostly in S phase
Telomere
non-coding NT sequences at ends of linear chromosomes
Maintain the structural integrity of eukaryotic chromosomes
Protozoa - GGGGTT
Euk - AGGGTT
Telomere sequences
Protozoa - GGGGTT
Euk - AGGGTT
Shelterin
Protein in telomeres that functions to protect ends from being recognized as DNA breaks
Problem with telomeres?
RNA primer on the end of lagging strand at 5’ is shorter with each DNA replication
Removal of the primer leads to a shorter strand
Telomerase
Ribonucleoprotein
Contains RNA molecule that serves as template for elongation of G rich strand of telomeric DNA
Has a protein with polymerase activity
Only found in - germ cells, early embryonic cells, cells that divide continuously
Telomerase process
Telomerase extends the 3’ end of DNA
RNA primer is synthesized by primase at end of 3’ end
RNA primer serves as primer for DNA polymerase
Polymerase extends in 5’–>3’
RNA primer is removed
Hutchinson-Guilford Progeria
Disease of very premature aging
Due to mutations in nuclear proteins - lamins
Dramatically shortened telomeres at early age
Why do SSB proteins exist?
Don’t let ssDNA base pair, get degraded, form a duplex
Cell thinks ssDNA is a virus
