Lecture 4 Flashcards
Base-pairing enables
DNA replication
- DNA synthesis begins at replication origins
- 2 replication forks form at each replication origin
Semi-conservative replication
When DNA replicates, molecule serves as a template for its own replication
DNA polymerase synthesizes DNA using a
Parental strand as a template
- The replication fork is asymmetrical
- DNA polymerase is self-correcting
Short lengths of RNA act as primers for
DNA synthesis
Proteins at a replication fork cooperate to form a
Replication machine
Telomerase replicates the ends of eukaryotic chromosomes
telomere length varies by cell type and with age
Recall
Conserved sequences that all chromosomes have are CENTROMERES, TELOMERES< and REPLICATION ORIGIN
Glycosidic bond holes
Carbohydrates
Phosphoanhydride bonds holds
Triphosphates
H-bonds hold
DNA b/w chains
Phosphodiester bonds hold
DNA backbone
DNA is damaged
ALL the time
DNA acts as a template for
Its own replication
AT rich becuase it is much easier to
break
When this sequence is recognized, the helix will be opened at this stie to create a “bubble”
2 replication forks for each origin
Synthesis goes BIDIRECTIONALLY (away from each other)
S phase is ~8 hrs in humans = we MUST have several replication origins
A DNA double helix is opened at
Replication origins
Opened with the aid of initiator proteins => single stranded DNA templates ready for DNA synthesis
DNA synthesis occurs at
Y-shaped junctions called replication forks
The 2 replication forks formed at a replication origin move AWAY in
Opposite directions
Polymer
The chain of subunits (DNA, RNA are polymers)
Polymerase
Enzyme that makes a polymer (i.e. DNA polymerase makes DNA)
A new DNA strand is synthesized in the
5’ to 3’ direction
DNA polymerase MUST add to 3’ hydroxyl, so DNA synthesis can ONLY occur in one direction (5’ to 3’)
MUCH energy contained in phosphoanhydride bond; energy comes from the incoming triphosphate
DNA polymerase adds a deoxynucleotide to the
3’ end of a growing DNA strand
At a replication fork, the 2 newly synthesized DNA strands are of
Opposite polarities
2 different types of strands
Leading strand (5’ to 3’) Synthesized CONTINUOUSLY
Lagging strand (3' to 5') Synthesized DISCONTINUOUSLY
Mutation rate is LOW because enzyme can fix it
Removes damaged base IMMEDIATELY
At each replication fork, the lagging DNA strand is synthesized in
Pieces
During DNA synthesis, DNA polymerase proofreads its own
work
DNA polymerase contains SEPARATE sites for DNA synthesis and proofreading
Editing site is where damaged bases are fixed
RNA primers are synthesized by an RNA polymerase called primase, which uses a
DNA strand as a template
RNA primers used for DNA synthesis
Primase = making a primer
For DNA replication: RNA ptimer
RNA does ____ need a primer
NOT
Multiple enzymes are required to synthesize the
Lagging DNA strand
DNA ligase
“Ligates” or fuses DNA
DNA ligase joins together
Okazaki fragments on the lagging strand during DNA synthesis
DNA polymerase
Catalyzes the addition of nucleotides to the 3’ end of a growing strand of DNA using a parental DNA strand as a template
DNA helicase
Uses the energy of ATP hydrolysis to unwind the DNA double helix ahead of the replication fork
Single-strand DNA-binding protein
Binds to single-stranded Dna exposed by DNA helicase, preventing base pairs from re-forming before the laging strand can be replicated
DNA topoisomerase
Produces transient nicks in the DNA bafckbone to relieve the tension built up by the unwinding of DNA ahead of the DNA helicase
Sliding clamp
Keeps DNA polymerase attached to the template, allowing the enzyme to move along without falling off as it synthesizes new DNA
Clamp loader
Uses the enrgy of ATP hydrolysis to lock the sliding clamp onto DNA
Primase
Synthesizes RNA primers along the lagging-strand template
DNA ligase
Uses the energy of ATP hydrolysis to join Okazaki fragments made on the lagging-strand template
DNA synthesis is carried out by a group of proteinas that act together as a
Replication machine
DNA topoisomerases relieve the tension that builds up in front of a
Replication fork
Clips DNA, let unwind to relieve pressure repeat
Without a special mechanism to replicate the ends of linear chromosomes, DNA
Would be lost during each round of replication
Linear DNA has this problem, NOT circular DNA
Okazaki fragments start with RNA primer, remove RNA primer, DNA fills in, left with primer on one end, DNA veiws as damage and eats back until its gone
This is BAD, could eventually reach somehting important
Solution: Add telomeres to ends using complementary base-pairing
Telomeres are hundresds - thousands of sequences long
ONly EUKARYOTES need telomeres, prokaryotes do NOT
Telomeres and telomerase prevent
Linear eukaryotic chromosomes from shortening with each cell division
if you mutate this RNA, it can’t bind well to DNA, and it will get shroter (could cause cancer or some other damage)
3 different types of DNA damage:
Mistakes
Mutigens/Chemical
Double-stranded breaks (Most severe)
DNA damage occurs
Continually in cells
Cells possess a variety of mechanisms for repairing DNA
A DNA mismatch repair system removes replication errors that escape proofreading
Double-strand DNA breaks require a different strategy for repair
Failure to repair DNA damage can have
Severe consequences for a cell or organism
Depurination and deamination
The most frequent chemical reactions known to create DNA damage in cells
Depurination
Removing a purine (A or G) through spntaneous biochemical reaction, hydrolysis removes base
Without repair, causes a frameshift due to missing purine
Deamination
Occurs with cytosine: C is hydrolyzed, amine removed, C becomes U (shouldn’t be present in DNA)
Without repair, One strand will have a mutated sequence
UV Radiation
In sunlight can cause the formation of thymine dimers
Removes bonds between thymines, bringing them closer together and resulting in a kink in the DNA structure
Proteins that need to bind to DNA can’t because form isn’t right
The basic mechanism of DNA repair involves 3 steps
1) Damaged segment is excised
2) Repair DNA Polymerase fills in missing nucleotide in top strand using bottom strand as template
3) DNA ligase seals nick
Direct and Excision Repair
Mostly for damaged nucleotides
MMR
Repairs replication errors (if DNA polymerase doesn’t correct errors)
DBS
Repairs double-stranded breaks (can be done by nonhomologous end joining or homologous recombination)
General overview of DNA repair pathways
Look for mistakes, cut them out, DNA polymerase will come in and replace, DNA ligase gets rid of gaps
NO need for a ____ in DNA repair
Primer
Proteins scan DNA, look for change in DNA structure (shape is altered by errors); mistake found =
Cut out mistake (might cut just mistake or cut out big segment)
Errors made during DNA replication must be corrected to avoid
Mutations
Cells can repair double-stranded breaks in one of two ways
Nonhomologous end joining:
- Break
- Processing of DNA end by nuclease
- Bigger gap
- End joining by DNA ligase
- Deletion of DNA sequence as a result
- Very error prone
Homologous recombination
- Break
- Processing of broken ends by recombination-specific nuclease
- Double strand break ACCURATELY repaired by using undamaged DNA as template
- ALL organisms use this; may use chromosome 10 from dad to heal chromosome 10 from mom that was broken (don’t need to be identical)
- Crossing over is homoologous recombination
A single nucleotide change causes the disease
Sickel cell anemia
