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Block 3 Biochemistry > DNA Replication And Repair > Flashcards

DNA Replication And Repair Flashcards

1
Q

Replication of DNA manner

A
  • semi-conservative

- each strand serves as a template for a new strand

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2
Q

Meselson and Stahl experiment

A
  • Bacterial DNA was labeled with heavy isotope 15 N as source for nitrogen for several generations
  • hybrid DNA of 14 N and 15 N was observed leading to the semi-conservative consensus
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3
Q

Origin of replication

A
  • point of initiation of DNA replication

- eukaryotic chromosomes have 1000-2000 separate origins of replication per chromosome

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4
Q

Bidirectional replication

A
  • two replication forks (sites where DNA replication will occur) proceed in opposite directions from the origin of replication
  • evidence for bidirectional replication is the theta structure of radioactively labeled DNA (E. Cole chromosome) during replication
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5
Q

DNA replication direction

A

-proceeds only in 5’ to 3’ direction

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6
Q

Primers

A
  • DNA replication is initiated from pre-existing primers

- they are short sections of RNA which are complementary to the template strand and contains a free 3’ OH group

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7
Q

Leading strand replication

A

-is continuous (the new DNA is synthesized uninterruptedly from a single RNA primer)

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8
Q

Lagging strand replication

A
  • proceeds from multiple primers and results in forming short DNA sequences which are eventually joined
  • synthesis is discontinuous
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9
Q

Supercoiling

A

-DNA molecules during replication creates torsional strain which must be removed for replication to proceed

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10
Q

DNA polymerase catalyze what rxn?

A

(DNMP)n +dNTP—>(dNMP)n+1 +PPi—>2 Pi

-only deoxyribonucleotide triphosphates can serve as substrates

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11
Q

Template and primers of pro and eukaryotic cells

A

-both strands of the parent DNA molecule serves as template and small fragments of RNA serves as primers

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12
Q

RNA polymerase (primase)

A
  • necessary for the synthesis of the RNA primers required for DNA replication
  • requires a free 3’OH terminal from which to start
  • adds an oligonucleotide from 10-60 bases to serve as primers
  • more primers required for lagging strands
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13
Q

Okazaki fragments

A
  • shoot sections of primer RNA plus DNA which forms on the lagging strand
  • humans have shorter Okazaki fragments than bacteria
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14
Q

Helicases

A
  • carry out unwinding of DNA

- bind to ss-DNA and require ATP which is hydrolyzed in order to drive enzyme function

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15
Q

Helicases in E. coli

A

-rep protein and the proteins dnaB+dnaC which are part of the replisome

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16
Q

Effects of supercoiling

A
  • closed loops of DNA can increase or decrease torsional strain on the molecule by varying the amount of supercoiling
  • supercoils are introduced into DNA when a closed circular duplex is twisted around a central axis
  • unwinding of DNA creates positive supercoiling which must be removed by topoisomerases
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17
Q

Topoisomerases

A
  • catalyze interconversion of different topological isomers of DNA
  • relieve tension ahead of the replication fork which is introduced via unwinding of the DNA strands
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18
Q

Type I DNA topoisomerase

A
  • makes a break or nick in one strand of DNA helix and passes the other strand through the break to relax the supercoil
  • break is then resealed by the enzyme
  • does not require ATP
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19
Q

Type II topoisomerases

A
  • aka DNA gyrase
  • produce an enzyme-bridged break in both strands of DNA
  • another region of duplex DNA is passed through the gap by the enzyme, thus two supercoils are removed in one step
  • requires ATP
  • break is then rejoined
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20
Q

Topoisomerase II and drugs

A
  • important anti-cancer drugs such as adriamycin and etoposide
  • Ciprofloxacin (Cipro) is a widely used antibiotic which is active against gyrase
  • antifungal, antiparasitic and antiviral agents are being directed at topoisomerases
  • targets gram + and - , little resistance
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21
Q

Single stranded binding proteins (SSB)

A
  • keep the separated strands as single strands
  • displaced and reused during replication
  • affinity for ss is 1000x greater than ds
  • proteins bound to ss-DNA would be protected from ss specific nucleases
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22
Q

DNA polymerase III and DNA polymerase I

A
  • major enzyme involved in DNA replication, responsiable to growing DNA strand until 5’ ribonucleotide of the primer of the previously synthesized precursor fragments is reached and can go no further
  • DNA polymerase I takes over since it acts as an exonuclease and removes the RNA primer as it lays down deoxyribonucleotides in the same place
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23
Q

DNA polymerase III is processive

A
  • once DNA polymerase III is bound to the template it probably never dissociates until the entire chromosome has been replicated
  • DNA polymerase is active as a holoenzyme composed of 7 subunits
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24
Q

How many DNA polymerase function at a time?

A

-two DNA polymerase molecules function at each replication fork, this 4 in a replication bubble

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25
Q

Enzymatic activities of DNA polymerase I

A

1) it possesses a 5’->3’ exonuclease activity and starts cutting out the RNA primer one nucleotide at a time
2) as it removes primer is fills in with the dNTP matching the exposed DNA template
3) possesses a 3’->5’ exonuclease activity whose main function is editing or proofreading (recognizes improper hydrogen bonding), since it will cleave off any unpaired 3’ terminal nucleotide

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26
Q

Replication error rate

A
  • wrong base is incorporated about once in 10,000 elongation steps (10-4 error rate)
  • error rate of exonuclease activity of DNA polymerase I is about 10-3
  • combined error rate is 10-7, once in every 10 million bases the wrong one will end up being incorporated into to the new DNA molecule
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27
Q

DNA Ligase

A
  • seals the nick b/t the fragments of newly synthesized DNA (necessary for both DNA replication and DNA repair)
  • E. coli ligase requires NAD for activity, eukaryotes require ATP
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28
Q

DNA ligase catalyze?

A
  • synthesis of a phosphodiester bond b/t a 3’ OH and a 5’ phosphate group, as long as both groups are termini of adjacent-base paired deoxynucleotides
  • enzyme cannot bridge a gap (the enzyme cannot fill in a missing nucleotide)
29
Q

Replisome

A

-large protein complex that carries out DNA replication, starting at the replication origin

30
Q

DNA polymerase alpha

Location and function

A
  • nuclear
  • synthesis and priming of lagging strand
  • formation and extension of RNA primers
31
Q

DNA polymerase beta

Location and function?

A
  • Nuclear

- DNA repair

32
Q

DNA polymerase gamma

Location and function?

A
  • mitochondrial

- replicates mitochondrial DNA

33
Q

DNA polymerase delta

Location and function?

A
  • nuclear
  • synthesis of leading strand
  • DNA polymerase III?
34
Q

DNA polymerase epsilon

Location and function?

A
  • nuclear

- DNA repair

35
Q

How are histones dissociated from the nucleosome during DNA synthesis?

A
  • weakened through acetylation and phosphorylation

- makes the histones more negative and weakens their association with DNA

36
Q

Number of Initiation sites in eukaryotes

A
  • perhaps 2000 per chromosome rather than one

- eukaryotes replicate at about 500-5000 base pairs per minute

37
Q

Origin recognition complex (ORC)

A
  • to activate an origin, ORC must bind at origins of replication sequences
  • Helicase and SSB proteins prepare the region for insertion of DNA polymerase-primase
  • licensing factor must also be bound near each origin to ensure the origin is used only once per cell cycle
38
Q

PCNA (proliferating cell nuclear antigen) function?

A
  • serves as a clamp which is assembled with DNA pol gamma and epsilon to ensure processivity.
  • antibodies to PCNA are used clinically to examine the degree of cell proliferation in a tissue sample
39
Q

Pol alpha and RNase H

A
  • RNA primers are synthesized and extended by pol alpha
  • RNA primers are removed by RNAse H (RNA primer is attached to DNA so will only remove RNA) and the gaps filled through further DNA synthesis
40
Q

Old and New histones?

A
  • old histone octamers are not disassembled

- newly synthesized histone octamers associate with one branch only of the replication fork

41
Q

How does a cell decide to begin DNA replication (in S-phase)?

A

-proteins called cyclins regulate key steps in the cell cycle, including initiation of DNA synthesis in S-phase

42
Q

Cyclins

A

-control cyclin-dependent kinases (CDKs) at various times in the cell cycle

43
Q

Cyclins A and E

A

-synthesized to control the onset of S-phase DNA synthesis by activating kinase CdK2

44
Q

CdK2-cyclin E/A

A

-complex phosphorylates pRb (retinoblastoma protein) causing dissociation of hyperphosphorylated pRb to activate E2F transcription factor

45
Q

E2F

A

-turns on many genes to activate DNA synthesis such as DNA pol alpha

46
Q

Inhibitors of DNA replication role?

A
  • block activation of cyclic-dependent kinases (CdKs)

- ex. Radiation

47
Q

Physical agents that can cause DNA damage

A

-high temperatures, radiation of different wavelengths but particularly short-wave (240-300nm) ultraviolet (UVB) and x-rays

48
Q

Chemical agents that cause DNA damage?

A

-methylating agents, nitrous acid, nitrosamines, acridine dyes

49
Q

How do DNA damaging agents act?

A
  • by altering the structure of DNA and causing disruption or normal hydrogen bonding of complementary base pairs
  • some cause breaks in phosphate backbone
  • cause a spectrum of different damages to DNA
50
Q

Altered bases DNA damage
-thymine dimer is best know example
2 types?

A
  • pyramidine dimers

- deamination

51
Q

Pyrimidine dimers
Mechanism?
Consequences?

A
  • covalent linkage of two polynucleotide chains of DNA
  • ultraviolet radiation is a common cause of dimer formation
  • hydrogen bonding of the thymine to their paired adenines is disrupted and results in inhibition of advance of the replication fork
52
Q

Deamination

A

-chemically induced or spontaneous loss of an amino group results in conversion of cytosine to uracil or conversion of adenine to hypoxanthine

53
Q

Depurination

A
  • spontaneous loss of a purine

- occurs at a rate of about 10,000 purines per day per cell->called an apurinic site in DNA

54
Q

Strand breaks

Ss and ds?

A
  • ss- chemical and radiation (DNA ligase can repair)

- ds- chemical-particularly anticancer drugs (more lethal than single strand breaks)

55
Q

Photoreactivation

A
  • mechanism only operates on pyrimidine dimers

- E. coli enzyme which catalyzes photoreversal of pyrimidine dimers is called photolyase. (Does not exist in mammals)

56
Q 
Excision repair (molecular scissors)
-what does it require?
A
  • repair pathway for removal of bulky chemical modifications of DNA and pyrimidine dimers
  • requires: an endonuclease to nick the DNA (cut), an enzyme (polymerase) to replace the damaged section of DNA (patch) and a DNA ligase to form phosphodiester bond (seal)
57
Q

When spontaneous deamination of cytosine to uracil, what is the enzyme?

A
  • Uracil DNA glysocylase->hydrolyzes the bond between uracil and deoxyribose resulting in removal of uracil from the DNA
  • results in a apyrimidinic site in the DNA and subsequent recognition by a specific endonuclease.
  • small gap is made in the damaged strand by endonuclease which is repaired by DNA polymerase and ligase
58
Q

SOS repair

A
  • post replication repair
  • is error prone
  • last ditch effort
  • induced in response to high DNA damage levels
59
Q

Xeroderma pigmentosum (XP)

A
  • increased sensitivity to sunlight
  • due to defects in the repair of ultraviolet light-induced damage to the DNA
  • eight different genetic loci of the disease have been identified
60
Q

XP variants

A
  • individuals who poses clinical symptoms of xeroderma pigmentosum but who have normal excision repair activity
  • have mutations in repair DNA polymerase n
61
Q

Xeroderma Pigmentosum
Sensitivity?
Cancer?
Symptoms?

A
  • ultraviolet radiation
  • skin carcinomas, melanomas
  • skin and eye photosensitivity
62
Q

Ataxia telangiectasia
Sensitivity?
Cancer?
Symptoms?

A
  • Gamma radiation
  • lymphomas
  • ataxia, dilation of blood vessels in skin, chromosome
63
Q

Fanconi’s anemia
Sensitivity?
Cancer?
Symptoms?

A
  • cross linking agents
  • leukemias
  • hypoplastic pancytopenia, congenital anomalies
64
Q

Bloom’s Syndrome
Sensitivity?
Cancer?
Symptoms?

A
  • ultraviolet
  • leukemia’s
  • photosensitivity
65
Q

Cockayne’s syndrome
Sensitivity?
Cancer?
Symptoms?

A
  • ultraviolet
  • various tumors
  • neurological defects, dwarfism
66
Q

Cockayne’s Syndrome (CS)
Lacking?
Mutated protein?

A
  • lack transcription helicases used in repair during gene transcription (known as transcription coupled repair)
  • mutated CSB protein does not allow DNA damaged genes to be repaired during transcription (causes loss of mRNA production, more severe phenotype than XP)
67
Q

Loss of DNA repair pathway

A
  • may underlie tumor formation in HNPCC (hereditary nonpolyposis colorectal cancer)
  • culprit genes are inc=valves in mismatch repair and mutation in these genes could predispose an individual to this type of cancer
  • most commonly inherited genetic diseases and this genetic defect accounts for around 10% of colorectal cancer cases
68
Q

Hereditary breast and ovarian cancers

A
  • BRCA1 and BRCA2 genes are linked

- code for recombination repair proteins linked to Fanconi’s Anemia type

Block 3 Biochemistry (3 decks)

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