DNA Replication and Repair

Question 1

Define semiconservative with regards to DNA.

Answer 1

Each new strand is half parent strand half daughter strand.

Question 2

DNA replication: Unidirectional or Bidirectional?

Answer 2

Bidirectional(Think about Okazaki fragments)

Question 3

What is an Okazaki fragment?

Answer 3

Small stretches of DNA synthesized during replication in the 5’ to 3’ direction on the lagging strand.

Question 4

What is the origin of replication?

Answer 4

A specific sequence in DNA that binding proteins recognize. There are 100’s per chromosome. Many contain multiple short repeats, including AT rich zones.

Question 5

What is the replication fork?

Answer 5

The two areas where DNA helicases have unwound the double helix.Replication machinery sits in the forks and synthesizes complimentary strands.

Question 6

What is an origin binding protein?

Answer 6

A protein that binds to the origin of replication at AT rich sequences.

Question 7

What is helicase?

Answer 7

A protein that unwinds (and separates strands of) DNA by breaking the H bonds between base pairs.

Question 8

What is a single strand binding protein?

Answer 8

A protein that binds to the single strand of DNA to prevent it from re-annealing.

Question 9

What is primase?

Answer 9

A protein that catalyzes the synthesis of an RNA primer by copying the template strand.

Question 10

What does ye know about DNA Pol I?

Answer 10

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)

Question 11

What does ye know about DNA Pol III?

Answer 11

Proofreads DNA in 3’ - 5’ direction.Has sliding clamp - high processivity (>500k)Fast (250-1000 nucleotides/sec)

Question 12

What is a processivity/sliding/DNA clamp?

Answer 12

A protein that binds DNA polymerase to the template strand.Pol III has, Pol I does not.

Question 13

What does DNA ligase do?

Answer 13

It binds the Okazaki fragments together once the RNA primer has been replaced with DNA by DNA Pol I.

Question 14

What does telomerase do?

Answer 14

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.

Question 15

What does topoisomerase do?

Answer 15

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.

Question 16

What does reverse transcriptase do?

Answer 16

Catalyzes the synthesis of DNA from an RNA template.

Question 17

How does DNA Polymerase create the phosphodiester bond?

Answer 17

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.

Question 18

What does DNA polymerase require in order to start replicating DNA?

Answer 18

RNA primer

Question 19

What direction does DNA synthesis occur in?

Answer 19

5’ to 3’. Duh.

Question 20

List the steps occurring during replication of the leading strand.

Answer 20

1. 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

Question 21

List the steps occurring during replication of the lagging strand.

Answer 21

1. 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

Question 22

List a few causes of DNA mutation.

Answer 22

UV light, reactive oxygen species, chemicals

Question 23

What three things need to happen in order for cancer to occur?

Answer 23

1. Mutation in gene of self proliferation.2. Repair mechanisms misses mutation or overwhelmed by amount of mutations3. Self destruction pathways cannot be activated.

Question 24

What are some examples of human DNA repair diseases?

Answer 24

Cockayne’s syndrome - impaired nervous system, photosensitivity, premature aging.Xeroderma Pigmentosum - damage by sunlight cannot be fixed

Question 25

Thymine dimers:Cause? How repaired? Consequences of repair failure?

Answer 25

UV lightNucleotide excision repairMalformed helix causes Pol III to fall off, Pol II takes over = preponderance of errors

Question 26

Bulky chemical adducts:Cause? How repaired? Consequences of repair failure?

Answer 26

Chemotoxic binding of large molecules to basesNucleotide excision repairChanges base pairing

Question 27

Double strand breaks:Cause? How repaired? Consequences of repair failure?

Answer 27

UnknownNon homologous end joiningCan lose up to 1/2 of a chromosome

Question 28

Uracil in DNACauses? How repaired? Consequences of repair failure?

Answer 28

Cytosine is deaminatedBase excision repairCan cause problems with replication/ transcribing DNA + recognition sites of transcription enzymes

Question 29

What is DNA mismatch?

Answer 29

When the new base does not match appropriately with the template base.

Question 30

Describe the basic steps of mismatch repair.

Answer 30

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. 

Question 31

Describe base excision repair (BER). 

Answer 31

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.

Question 32

What lesions does base excision repair fix?

Answer 32

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

Question 33

What is nucleotide excision repair (NER)?

Answer 33

A process that removes DNA lesions that distort DNA structure and result in blocking polymerase movement.

Question 34

What are the basic steps of NER?

Answer 34

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.

Question 35

What is recombinatorial repair?

Answer 35

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.

Question 36

What are the basic steps of recombinational repair for double break in DNA?

Answer 36

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.

Question 37

What is non homologous end joining?

Answer 37

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.

Question 38

What allows replication to continue in the face of DNA lesions that other repair pathways fail to remove?

Answer 38

Damage bypass or tolerance. Sliding clamp jumps over mutation. Leads to genomic instability.