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Isaak: Biochemistry MBS > Lecture 2: DNA Replication/Cell Cycle > Flashcards

Lecture 2: DNA Replication/Cell Cycle Flashcards

1
Q

Origin of Replication

A

An initiation site for DNA replication.

Not understood what constitutes a human origin of replication.

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2
Q

Helicase

A

An enzyme that disrupts the hydrogen bonds that hold duplex DNA together.

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3
Q

SSB Protein

A

Single strand binding protein. Present to prevent reannealing.

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4
Q

RPA

A

Human single strand binding protein.

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5
Q

ORC

A

Origin recognition complex.

A group of proteins that are required for identification of the origin of replication.

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6
Q

A syndrome resulting from a DNA helicase deficiency, increasing risks for leukemia.

A

Bloom’s Syndrome.

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7
Q

Werner’s Syndrome

A

A premature aging disease associated with a defective helicase.

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8
Q

DNA Primase

A

Synthesizes about a 10 nt RNA primer.

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9
Q

DNA Polymerase

A

Adds nucleotides sequentially to the 3’ termini of growing chains.

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10
Q

What direction do polymerases move in?

A

5’ to 3’.

“Downhill.”

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11
Q

What is the specific mechanism DNA polymerases use add nucleotides?

A

Catalyze the nucleophilic attack of the 3’-OH of the preceding deoxyribose on the innermost phosphorous atom of a correctly base paired deoxyribonucleotide triphosphate (dNTP). Upon formation of a phosphodiester bond, pyrophosphate (PPi) is released.

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12
Q

dNTP

A

Deoxyribonucleotide triphosphate

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13
Q

PPi

A

Pyrophosphate (P207, 4-)

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14
Q

What are the three polymerases that participate in higher eukaryotic DNA replication?

A
  • Pol α-primase complex
  • Pol ε
  • Pol δ
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15
Q

Leading Strand

A

Polymerized continuously by pol ε behind DNA helicase

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16
Q

Polymerase α - primase complex

Pol α - Primase complex

A

Come back to this!***

Pol α is the only known polymerase to be associated with a primase. This specific complex plays a critical role in the initiation of every Okazaki Fragment.

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17
Q

Polymerase ε

Pol ε

A

Catalyzes polymerization of the LEADING strand.

Is considered a “high fidelity” polymerase due to it’s 3’ to 5’ exonuclease “proofreading” activity.

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18
Q

Polymerase δ

Pol δ

A

After the RNA primer (laid by Pol α - Primase complex) is removed by an exonuclease, this enzyme catalyzes polymerization of the LAGGING strand by forming Okazaki fragments.

Considered a “high-fidelity” polymerase.

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19
Q

Exonuclease

A

Cleave individual nucleotides from the ends of polynucleotide chains.

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20
Q

PCNA

A

Proliferating Cell Nuclear Antigen

Acts as a “clamp” to hold Pol ε and Pol δ onto the DNA.

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21
Q

RFC

A

Replication Factor C

Helps to load Pol δ and Pol ε onto DNA.

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22
Q

Lagging Strand

A

Overall growth of the strand is in the 3’ to 5’ direction, but all polymerases move in the 5’ to 3’ direction.

Made up of Okazaki fragments.

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23
Q

Lagging strand fragments that are due to the 5’ to 3’ nature of polymerases working in a 3’ to 5’ direction.

A

Okazaki Fragment

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24
Q

After activation by formation of an enzyme-AMP (from ATP) complex, this enzyme then catalyzes the covalent linking of each Okazaki fragment by inserting an activated phosphate group.

A

DNA Ligase

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25
Q

DNA Polymerase γ

Pol γ

A

Responsible for human mitochondrial DNA synthesis.

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26
Q

DNA Polymerase β

Pol β

A

Is believed to play a role in certain repair events.

27
Q

Potent inhibitor of the DNA polymerase encoded by herpes simplex viruses.

A

Acyclovir

28
Q

AZT

A

Azidothymidine

Potent inhibitor of the reverse transcriptase encoded by the HIV virus.

Limited due to toxicity; it is a substrate for DNA polymerase γ and disrupts mitochondrial DNA synthesis.

29
Q

List the four phases of mitosis.

A
  1. G1 (cell growth)
  2. S (synthesis and DNA replication)
  3. G2 (cell growth)
  4. M (mitosis)
30
Q

Protein Kinases

A

Enzymes that add phosphate groups to other molecules; phosphorylators.

31
Q

CDK

A

Cyclin-dependent protein kinases. Made up of a kinase and a cyclin.

Closely regulate progress through the cell cycle.

32
Q

Cyclin

A

Any of a number of proteins associated with the cycle of cell division; thought to initiate certain processes of mitosis.

33
Q

p53

A

A protein that functions at the G1/S checkpoint to help prevent cells from entering into S phase if they contain damaged DNA. The DNA will then be repaired.

Alternatively, if the DNA cannot be repaired, apoptosis may occur.

Therefore, p53 is a tumor-suppressing protein. A defective p53 protein may cause cell proliferation (cancer).

34
Q

Li-Fraumeni Syndrome

A

Associated with individuals who inherit mutant forms of p53; these people are at risk of developing tumors at multiple sites.

35
Q

How do enzymes involved in mismatch repair identify the proper “wild-type” versus the mutant?

A

In E. coli, it has been shown to be “methylation” of adenine residues in the wild type.

Newly replicated DNA strands lack these methyl groups.

36
Q

What are the 3 mismatch repair proteins in E. coli?

A
  • Mut S
  • Mut H
  • Mut L
37
Q

Mut S

A

Recognizes mismatched bases. (Seeks)

Mismatch repair protein in E. coli.

38
Q

Mut H

A

Recognizes the d(GATC) site and subsequently catalyzes the incision. (Hacks)

Mismatch repair protein in E. coli.

39
Q

Mut L

A

Unknown function, but is thought to provide a protein-protein interface between Mut S and Mut H.

(Lack of understanding)

Mismatch repair protein in E. coli.

40
Q

After incision by Mut H in mismatch repair in E. coli, what are the following steps?

A
  1. “Unzipping” by DNA helicase and an exonuclease.
  2. SSB binds.
  3. Filled in by a DNA polymerase (DNA pol III, E. coli specific.)
  4. DNA ligase to suture.
  5. Methylation.
41
Q

hMSH

A

human Mut S homologue

42
Q

hMLH

A

human Mut L homologue

43
Q

Trinucleotide Repeat Disorder

A

Genetic disorders caused by trinucleotide repeat expansion, a kind of mutation where trinucleotide repeats in certain genes exceed the normal, stable threshold, which differs per gene.

44
Q

Huntington’s Disease

A

People inherit a Huntington’s gene containing CAG repeats above the normal range. They run a rusk of developing Huntington’s Disease.

Inverse relationship with age of onset and number of repeats. Genetic anticipation.

45
Q

Excision Repair

A

The removal of a “faulty” DNA segment and its replacement by DNA synthesis.

46
Q

Name the four steps of excision repair.

A
  1. Incision.
  2. Excision.
  3. Resynthesis.
  4. Ligation.
47
Q

Which step of excision repair is specific to the type of damage?

A

Step #1, incision.

48
Q

uvrABC Complex

A

The protein/enzyme complex that performs the incision step of excision repair of UV-damage (DNA thymine dimer formation) DNA in E. coli.

49
Q

What is the purpose of studying repair systems in E. coli?

A

To demonstrate that many of the same enzymes that function in DNA replication are often involved in DNA repair.

50
Q

What skin disease results from biochemical defects in the excision repair process of UV-damaged DNA in humans?

A

Xeroderma Pigmentosum.

People with this condition generally die from metastases of malignant skin tumors before the age of 30.

51
Q

Which nitrogenous base spontaneously deaminates to form uracil?

A

Cytosine.

Though this mutation happens at a low rate, it is significant.

52
Q

Which enzyme removes uracil from DNA in humans?

A

Uracil-DNA glycosidase.

53
Q

Name the three common types of mutations:

A
  1. Substitutions
  2. Deletions
  3. Insertions
54
Q

Substitution

A

Mutation that exchanges one base for another.

55
Q

Insertion

A

Mutations in which extra base pairs are inserted into a new place in the DNA.

56
Q

Deletion

A

Mutation in which a section of DNA is lost, or deleted.

57
Q

Name three types of chemical mutations.

A
  1. Deamination
  2. Alkylation
  3. Intercalation (by polycyclic hydrocarbons)
58
Q

Proflavin and Ethidium Bromide are what types of chemical mutagens?

A

Intercalators.

59
Q

Radiation

A

Exposure of cells to ionizing radiation causing the removal of electrons from molecules. Can alter the structure of DNA.

60
Q

What repair system repairs alkylations and spontaneous cytosine–>uracil reactions?

A

Base excision repair.

61
Q

What is the nucleotide excision repair system used for?

A

Removal of bulky adducts (thymine dimers), likely from UV damage.

62
Q

What repair system fixes replication errors, insertions, and deletions?

A

Mismatch repair system.

63
Q

Mutations in hMSH or hMLH may cause what pathology?

A

Hereditary non-polyposis colon cancer (HNPCC).

64
Q

Name three trinucleotide repeat disorders.

A
  1. Huntington’s Disease
  2. Myotonic Dystrophy
  3. Fragile X Syndrome

Isaak: Biochemistry MBS (11 decks)

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