Define semiconservative with regards to DNA.
Each new strand is half parent strand half daughter strand.
DNA replication: Unidirectional or Bidirectional?
Bidirectional(Think about Okazaki fragments)
What is an Okazaki fragment?
Small stretches of DNA synthesized during replication in the 5’ to 3’ direction on the lagging strand.
What is the origin of replication?
A specific sequence in DNA that binding proteins recognize. There are 100’s per chromosome. Many contain multiple short repeats, including AT rich zones.
What is the replication fork?
The two areas where DNA helicases have unwound the double helix.Replication machinery sits in the forks and synthesizes complimentary strands.
What is an origin binding protein?
A protein that binds to the origin of replication at AT rich sequences.
What is helicase?
A protein that unwinds (and separates strands of) DNA by breaking the H bonds between base pairs.
What is a single strand binding protein?
A protein that binds to the single strand of DNA to prevent it from re-annealing.
What is primase?
A protein that catalyzes the synthesis of an RNA primer by copying the template strand.
What does ye know about DNA Pol I?
DNA Pol I replaces RNA primers with DNA in 5’ - 3’ direction.Proofreads DNA in 3’ - 5’ direction.No sliding clamp = low processivity (3-200)Slow (16-20 nucleotides/sec)
What does ye know about DNA Pol III?
Proofreads DNA in 3’ - 5’ direction.Has sliding clamp - high processivity (>500k)Fast (250-1000 nucleotides/sec)
What is a processivity/sliding/DNA clamp?
A protein that binds DNA polymerase to the template strand.Pol III has, Pol I does not.
What does DNA ligase do?
It binds the Okazaki fragments together once the RNA primer has been replaced with DNA by DNA Pol I.
What does telomerase do?
A protein that restores telomeres to their full length.This is not normally expressed in humans cells. Cancer, however, does express telomerase, cells never die, and then tumors show up.
What does topoisomerase do?
It relieves torsional strain in the DNA helix in the region ahead of the replication fork.It clips the phosphodiester backbone and puts it together after relieving the strain.
What does reverse transcriptase do?
Catalyzes the synthesis of DNA from an RNA template.
How does DNA Polymerase create the phosphodiester bond?
It breaks off a diphosphate group from the dNTP and uses the energy liberated from that rxn to bind the remaining phosphate group to the hydroxyl group of the previous nucleotide.
What does DNA polymerase require in order to start replicating DNA?
What direction does DNA synthesis occur in?
5’ to 3’. Duh.
List the steps occurring during replication of the leading strand.
- Origin binding proteins bind2. Helicase unwinds and separates DNA.3. Primase adds and RNA primer to DNA4. Pol III elongates DNA while topoisomerases relieve tension ahead of replication fork.5. The two strands anneal
List the steps occurring during replication of the lagging strand.
- Origin binding proteins bind2. Helicase unwinds and separates DNA3. Primase attaches and makes RNA primer4. Pol III elongates DNA from RNA primer forming Okazaki fragment.5. Pol I replaces RNA primer with DNA6. Fragments bound together by DNA ligase.7. Strands anneal
List a few causes of DNA mutation.
UV light, reactive oxygen species, chemicals
What three things need to happen in order for cancer to occur?
- Mutation in gene of self proliferation.2. Repair mechanisms misses mutation or overwhelmed by amount of mutations3. Self destruction pathways cannot be activated.
What are some examples of human DNA repair diseases?
Cockayne’s syndrome - impaired nervous system, photosensitivity, premature aging.Xeroderma Pigmentosum - damage by sunlight cannot be fixed
Thymine dimers:Cause? How repaired? Consequences of repair failure?
UV lightNucleotide excision repairMalformed helix causes Pol III to fall off, Pol II takes over = preponderance of errors
Bulky chemical adducts:Cause? How repaired? Consequences of repair failure?
Chemotoxic binding of large molecules to basesNucleotide excision repairChanges base pairing
Double strand breaks:Cause? How repaired? Consequences of repair failure?
UnknownNon homologous end joiningCan lose up to 1/2 of a chromosome
Uracil in DNACauses? How repaired? Consequences of repair failure?
Cytosine is deaminatedBase excision repairCan cause problems with replication/ transcribing DNA + recognition sites of transcription enzymes
What is DNA mismatch?
When the new base does not match appropriately with the template base.
Describe the basic steps of mismatch repair.
MLH proteins recognize mismatch. New strand identified b/c not methylated. Backbone is cleaved by endonuclease. Exonuclease chews away new strand. DNA polymerase repairs single strand gap. Ligase seals the deal.
Describe base excision repair (BER).
Glycosylase recognizes specific altered bases. Flips damaged base from helix. Breaks bond attaching it to sugar producing apurinic/apyrimidimic site. Exonuclease removes segment creating a gap. Sealed by ligase.
What lesions does base excision repair fix?
DNA lesions that are missed by nucleotide excision repair, but that do not distort DNA structure.Spontaneous base loss, spontaneous deamination, base alkylation, base oxidation
What is nucleotide excision repair (NER)?
A process that removes DNA lesions that distort DNA structure and result in blocking polymerase movement.
What are the basic steps of NER?
Recognized by enzyme that also has endonuclease. Cleaves backbone on both sides of lesion. Helicase unwinds to release oligonucleotide. Chewed away by exonuclcease. Polymerase, then ligase.
What is recombinatorial repair?
Genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA.Use to repair harmful breaks that occur on both strand of DNA.
What are the basic steps of recombinational repair for double break in DNA?
Exonuclease chews up the end of each single strand. Nearby double helix donates part of its single strand. Now each double helix is missing one portion. DNA Pol elongates remaining missing portions. Ligase seals together. Phosphodiester link is cleaved. Whoa.
What is non homologous end joining?
Repair mechanism for broken strands that ensures no homology between broken ends. Thus insertions or deletions of excessive nucleotides is the norm. Fail intelligent design.
What allows replication to continue in the face of DNA lesions that other repair pathways fail to remove?
Damage bypass or tolerance. Sliding clamp jumps over mutation. Leads to genomic instability.