Theme 1C Flashcards
DNA Replication & Repair
Semiconservative replication
each daughter cell remains paired with its complementary parental strand
Conservative replication
after replication, both daughter strands pair up
Dispersive replication
daughter strands will have a mixture of parental and newly-synthesized DNA
Semiconservative DNA Replication: Meselson and Stahl (3rd Classical Experiment)
track parental and newly-synthesized DNA strands over several generations with nitrogen isotopes
nitrogen isotopes are incorporated into DNA molecules via nitrogenous bases
Concluded that DNA replication is semiconservative
DNA synthesis reaction
nucleotides can only be added to the new strand at the 3’-OH end
hydrolysis of pyrophosphate provides energy for the formation of new phosphodiester bond
DNA synthesis occurs in the
what direction?
5’-3’ direction
DNA polymerases
synthesizes new strand only in the 5’-3’ direction
travels and reads the template strand.
cannot synthesize a new strand de novo - requires RNA primer with a 3’-OH for synthesis
has single active site that can catalyze four different reactions (incorporation of dATP, dCTP, dGTP, dTTP)
Replisome
molecular machine of enzymes that replicate DNA
- helicase
- primase
- single-strand binding protein
- DNA topoisomerase/gyrase
- DNA polymerase III
- DNA polymerase I
- sliding clamp
- DNA ligase
Helicase
unwinds the double helix by breaking hydrogen bonds
Primase
synthesizes RNA primers for DNA polymerase
Single-strand binding protein
stabilizes ssDNA before replication by preventing reannealing so that the strands can serve as template
DNA topoisomerase/gyrase
removes super coils that form ahead of the replication form, relives torque of mainly circular DNA
DNA polymerase I
removes RNA primer and fills gaps with DNA
DNA polymerase III
synthesizes DNA by adding nucleotides to the new DNA strand
Sliding clamp
attaches to DNA Pol III to DNA template, makes replication more efficient
DNA ligase
joins the ends of the DNA segments by forming phosphodiester bonds
Replication forks
synthesis of two new strands from the template strand occurs concurrently at the forks
Because DNA polymerase can only synthesize in the 5’-3’ direction:
- one new strand is synthesized continuously (leading strand)
- other new strand is synthesized discontinuously (lagging strand) in small fragments
Okazaki fragments
small DNA fragments of lagging strand
How does DNA replication work?
- DNA helicase unwinds double helix
- RNA primase lays down RNA primer
- Topoisomerase prevents twisting ahead of replication fork during unwinding
- as helicase unwinds, DNA Pol III synthesizes leading and lagging strand
- DNA Pol I removes RNA primer of Okazaki fragments and fills in gaps with dNTPs - but it can’t fill in all nicks
- DNA ligase seals the nicks by reforming the phosphodiester bond
What direction is the template strand read by DNA polymerase during DNA replication
3’-5’
Initiation
unwinding and separation of two template DNA strands at the origin of replication site (oriC)
Elongation
simultaneous synthesis of the two new DNA strands from the template by DNA polymerase
Termination
DNA replication stops when it reaches a termination site (circular DNA) or at the end of a chromosome (linear DNA)
Review slide 10
Polymerase chain reaction
slide 11
End replication problem
requirement for RNA primer to initiate all new DNA synthesis presents problem, as no DNA polymerase can fill the gaps at the chromosomal ends
- therefore, there will be additive loss at the chromosomal ends for every round of DNA replication/cell division
Solution to end replication problem
telomeres
- non-coding single-stranded DNA are added to the 3’ end of the chromosomes by telomeres
- usually repeats of 5-8 G’s and T’s
- telomeres can be worn away after each DNA replication as they do not have genes in them
- exist to ensure ends of linear chromosomes are fully replicated
- when all telomere region is done, the cell stops dividing
– leading cause of natural death
– telomeres in older individuals are shorter
Telomerase
enzyme that restores shortened telomeres
- not present in most eukayotic cells
- present in gametes and stem cells (which keep dividing and dividing)
Human telomerase (hTERT) mutations can be used as a biomarker in cancer because many cancers aquire _______________ to negate the limitations of rapid cell division
mutations that activate the telomere gene
How do cancer cells keep dividing?
they have telomeres and cells somehow reactivate telomerase genes that allows for rapid division
- vaccine would attack telomere genes
High fidelity of DNA replication
DNA must fully replicate devoid of all errors
- telomeres exist to ensure ends of linear chromosomes are fully replicated
- failure to maintain this causes defected genomes possibly resulting in disease (cancer) and death of organism
- DNA repair mechanisms mediated by enzyme complexes ensure that replication error rate is low
Proofreading activities of DNA polymerase
- first way to catch and fix mistake
- DNA Pol III synthesizes new strand
(optimum conformation of active site and incoming nucleotide allows catalysis of the correct base pair but mistakes can still happen)
- DNA pol III detects mistake and uses 3’-5’ exonuclease activitiy to remove most recent mismatched nucleotides
- it replaces correct nucleotide and resumes synthesis of the new DNA strand
DNA mismatch repair (MMR)
MMR covers for replication errors not corrected by proofreading
- recognition of mismatch damage by DNA binding protein MutS and MutL
MutH
endonuclease daughter strand several nucleotides away from mismatch
Exo1
5’-3’ exonuclease excises region of daughter strand surrounding mismatch
DNA Pol III (MMR)
fills gaps and repairs mismatch
The nick left after gap is sealed in MMR is sealed by
DNA ligase
In DNA replication, DNA polymerase catalyzes new synthesis in the ____ direction
5’-3’
The leading strand is synthesized continuously in the _____ direction
5’-3’
Telomerase enzyme is needed because
RNA primers are removed (After replication)
DNA pol can only add nucleotides to an existing 3’ end of a nucleic acid
Linear chromosomes are shortened with each cell division
