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Basic Principles Of Medicine 1-FTM 1 > DNA replication > Flashcards

DNA replication Flashcards

1
Q

What is DNA replication?

A

The co-ordinated duplication of DNA during the process of cell division

  • provides the transmission of hereditary transmission
  • DNA replication in bacteria takes approx. 40 minutes, but in eukaryotes can vary from 1.4 hours in yeast to 24 hours in cultured animal cells
  • DNA replication is semi-conservative
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2
Q

What are the required components of DNA synthesis?

A
  1. All four dNTPs (deoxynucleotide triphosphate) -building blocks of the DNA molecule
  2. A fragment of DNA to act as a template
  3. DNA polymerase
  4. Magnesium ions (Mg2+) -required for DNA polymerase activity
  5. A primer providing a free 3’ OH group
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3
Q

What are the roles and properties of DNA polymerase?

A
  1. DNA polymerase catalyzes the formation of a phosphodiester bond between 3’ OH bond group of the deoxygenation group of the deoxyribose on the last nucleotide and the 5’-phosphate of the dNTP precuesors
  2. The deoxynucleoside 5’ triphosphate provides the energy source for the reaction.
  3. At each step in the lengthening the new DNA chain, DNA polymerase finds the correct precursor dNTP that can form a complementary base pair with the nucleoside on the template stand of the DNA

Pyrophosphate is liberated as a result of this incorporating a single base

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4
Q

What is the function of Azidothymine (AZT)(Zidovudine)?

A

Antiviral Deoxythymidine analogue(viral transcriptase inhibitor)

Host cellular kinases add 3 phosphates

It is utilize by the viral reverse transcriptase

Upon incorporation into the dsDNA chain termination occurs due to lack of 3’ OH

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5
Q

What is Azidothymine (AZT)(Zidovudine) used for clinically?

A
  • Prolong life of HIV infected individuals

- reduce mother to baby transmission by more than 20%

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6
Q

What is didanosine (ddi)?

A

An antiviral (viral transcriptase inhibitor) deoxyadenosine analogue

Host cellular kinases add 3 phosphates

Utilized by the viral reverse transcriptase , upon incorporation in the dsDNA chain termination occurs due to lack of 3’ OH

Specificity for infected cells lies in the fact that these drugs have a 100-300 fold greater affinity for incorporation by reverse transcriptase than eukaryotic DNA polymerase

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7
Q

What is didanosine used for clinically?

A

Used in treatment of mainly HIV in combination with other anti-virals

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8
Q

What is acyclovir?

A

Antiviral (viral DNA polymerase inhibitor )Doexyguanosine analogue

Viral kinase (viral thymidine kinase)add some Phosphate to acyclovir(acyclovir mono-phosphate) host cellular kinase adds two more phosphates

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9
Q

What is acyclovir mainly used to treat?

A

High specificity for herpes simplex & varicella zoster

Herpes simplex can causes conjunctivitis and cold sores

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10
Q

Why is acyclovir activated in infected cells?What is the effect of this?

A
  1. Viral thymidine kinase= much more effective in catalyzing 1st phosphorylation event than host thymidine kinase
  2. Only infected cells possess the viral kinase therefore acyclovir is only activated in infected cells
  3. Causes chain termination due to lack of 3’ OH 30x more potent against viral DNA polymerase than host
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11
Q

What is tenofivir?

A

Antiviral nucleotide base analogue

Doexyadenosine monophosphate

Host cellular kinases add two phosphate groups to tenofovir

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12
Q

How does tenofovir works?

A

Tenofovir diphosphate competes with its natural nucleotide counterpart, deoxyadenosine 5’ -triphosphate, for incorporation into newly forming HIV DNA

-once successfully incorporated, termination of the elongating DNA chain ensues, and DNA synthesis is interrupted

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13
Q

What is the anti-viral nucleotide base analogue?

A

Tenofovir

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14
Q

What is the antiviral nucleoside analogue that inhibits DNA polymerase?

A

Acyclovir

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15
Q

What are the antiviral analogues that inhibit viral reverse transcriptase?

A

Didadenosine

Azidothymidine(zidovudine) (AZT)

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16
Q

What is cytosine arabinoside (araC)?

A

Cytosine nucleoside analogue , ribose sugar is replaced by arabinose

In cells it’s rapidly converted to cytosine arabinoside triphosphate , which acts as a substrate for several DNA polymerases

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17
Q

How does cytosine arabinoside (araC)/adenosine arabinoside) function molecularly?

A

AraC has a 3’ OH group to act as an acceptor for further chain elongation. However the OH group at position 2 prevents polymerase adding the next dNTP due to stereo resistants —> chain termination

There is also an adenosine analogue called adenosine arabinoside

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18
Q

What is araC used for clinically?

A

Important in the therapy of leukemia’s, in particular acute myeloid leukemia and lymphoma

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19
Q

How is repair of base misincorporation done by DNA polymerase?

A

DNA polymerase occasionally makes a mistake which may result in mutation, in such cases.

The 3’ to 5’ proofreading exonuclease activity of DNApolymerase clips off any unpaired residues at the primer terminus. It continues this activity until a base paired 3’-OH terminus is encountered

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20
Q

Bacterial genomes are usually _________ and contain a _______ ORI — ____________ replication

A

Circular

Single

Theta

21
Q

What are origins of replication?

A

Where DNA replication is initiated at distinct sites called ORI

22
Q

Why in E. Coli, once the replication pr9cess has started, it c9ntinu3s until completion?

A

Because the the replication process is only regulated up until completion

23
Q

What type of replication is used in various bacterial virus (bacteriophage) and plasmids that have a DNA genome?

A

Rolling circle replication

24
Q

Summarize rolling circle replication

A

Unwibding and replication

Nucleases cuts one strand of circular DNA

  • Nucleotides added to 3’ end, displacing other end
  • Other strand copied discontinuously
25
Q

What type of DNA replication is carried out by eukaryotic chromosomes?

A

Linear replication as we have many replication origins and no termini

26
Q

Why do eukaryotes have multiple ORI?

A

Multiple origins of replication are required, in order to replicate the large genomes that are found in eukaryotes in a timely fashion

Human haploid genome= about 3.4 million base pairs
Average size of chromosomes= 100,000,000 bp

Therefore, if there was a single origin of replication on each human chromosome it would take 830 hours for replication to complete

The number of origins of replication increases in organisms containing large genomes

27
Q

Summarize the enzymes involved in DNA replication in prokaryotes

A
  1. Initiator proteins (DNaA protein): binds to origin of replication and breaks hydrogen bonds between bases.
  2. DNA helicase (DNaB): opens helix and binds primase to form primosome
  3. Helicase inhibitor (DNaC): delivers helicase to DNA template
  4. DNA primase: an RNA polymerase that synthesizes a RNA primer on the lagging strand to enable DNA polymerase to synthesize DNA strand
  5. DNA polymerase 1: remove RNA primer and replace with DNA
  6. DNA polymerase III: synthesis of leading and lagging strands
  7. Single stranded binding protein (SSB): binds to single stranded DNA in the replication bubble and prevents it from reannealing or forming secondary structure
28
Q

What is the significance if OriC?

A

The OriC contains DNA sequences that are A-T rich. Binding between A-T is the weakest compared to G-C bonding thus the OriC facilitates easier melting abd strand separation of the DNA molecule

29
Q

Describe the structure of OriC

A

The origin of replication of E. coli has a length of 245 bp and contains tandem array of three nearly identical 13-nucleotide sequences and four 9 nucleotide sequences that act as binding sites

30
Q

Explain the model of initiation of replication at E. coli Ori C

A
  1. Multiple copies of initiator proteins (DNaA) bind to the 9-men’s at the origin
  2. Strand separation occurs at the region 9f 13 mer sequences
  3. Helicase inhibitor (DNAC) protein delivers helicase (DNAB) to the template
  4. Helicase clamps around each single strand DNA
  5. Helicase proceed to unwind the DNA in opposite away from the origin
31
Q

Single stranded DNA tends to re-anneal and f9rms secondary structures, how is this resolved?

A

Single stranded binding proteins (SSB proteins )

SSB proteins bind to single stranded DNA in the replication fork. The cooperative binding of these proteins prevent reannealing and straightens out the DNA template to facilitate the DNA polymerization process

32
Q

Describe the model for the formation of a replication bubble at a replication origin in E. coli and the initiation of the new DNA strand

A
  1. Initiator proteins bind to replication origin
  2. DNA helicase binds to initiator proteins
  3. Helicase loads onto DNA
  4. Helicase denatures helix and binds with DNA primase to form primosome
  5. Primase synthesizes RNA primer, which is extended as DNA chain by polymerase
33
Q

Why is DNA synthesis on the lagging strand discontinuous ?

A

Daughter DNA strands are synthesized in the 5’ to 3’ direction, therefore the DNA synthesize on the lagging strand must be made initially as a series of short DNA molecules called Okazaki fragments

34
Q

What is the main function of DNA polymerase 1?

A

Replaces the RNA primer(RNAseH and FEN-1 protein in eukaryotes) with DNA

35
Q

What is the main function of DNA polymerase III?

A

Synthesizes the Okazaki fragments, as well the leading strand

36
Q

How are Okazaki fragments joined?

A

DNA ligase

Seals the gap between adjacent DNA fragments to form a longer, covalently continuous chain.

Ligase utilizes ATP as energy source for this reaction

37
Q

Briefly contrast leading and lagging strand synthesis

A

Leading strand synthesis- requires 1 primer event

Lagging strand synthesis- each okazaki fragment requires a separate primer

38
Q

Wj@t are the events that follow Okazaki fragments ?

A
  • DNA polymerase 1 (prokaryotes) or RNAseH &FEN-1 protein (eukaryotes) removes the primer
  • Gap filled by a DNA polymerase
  • Ligase joins the adjacent fragments
39
Q

How is DNA polymerase III loaded and maintained on the single stranded DNA template, and unloaded when it reaches double stranded DNA (e.g. the next Okazaki fragment)?

A

Solution: Clamp protein

Clamp protein tightly holds the DNA polymerase onto the template for synthesis of long templates (increases expressivity), and releases DNA polymerase when it stalls at a region of double stranded DNA

40
Q

For a bacterial replication fork moving at 500 bp per second, the prenatal DNA helix ahead of the fork must rotate at 50 revolutions per minute. This. Generates positive supercoils ahead of the replication fork

A

Solution: swivel the DNA using topoisomerase enzymes

41
Q

What is the purpose of DNA topoisomerase II (Gyrase)?

A

This enzyme introduces negative supercoils into the DNA. This reduces the positive supercoils introduced by the opening of the DNA.

It also aids in the separation of the DNA during replication and transcription

42
Q

What is ciproflaxcin? What is it used for? How does it work?

A

Thus us a quinolone drug that inhibits bacterial DNA Gyrase

It is used in the treatment of respiratory and urinary tract infections and can be used to treat anthrax

43
Q

List the major differences between eukaryotes abd prokaryotes DNA replication

A
  1. Origins of replication
  2. Polymerases
  3. Removal of primers
  4. Compartmentalized into the S phase
  5. Telomerase and the replication of the ends of eukaryote chromosomes

Depending on the organism & cell type, replication origins occur every 3-309 kbp (an average human chromosome has several hundred replication origins)

44
Q

Briefly compare prokaryote and eukaryotes DNA replication

A
  1. Single origins of replication in prokaryotes, multiple origins of replication in eukaryotes
  2. RNA primer in prokaryotes is removed by DNA polymerase I, RNA primer is removed by RNAseH and FEN- 1
  3. Have different DNA polymerases
45
Q

Explain the differences between prokaryotic and eukaryotic DNA polymerases

A

Prokaryotic DNA polymerase-
-DNA polymerase I- removal of RNA primer and replacement with DNA

-DNA polymerase III- synthesis of leading and lagging strands - main replication enzyme

Eukaryotic polymerase a:

  • Synthesizes RNA primer on leading and lagging strand
  • Subunit possesses primase activity and initiates DNA synthesizes
  • Low processivity- falls off after synthesizing a short DNA segment
  • No exonuclease activity

Polymerase-delta:

  • synthesizes DNA from leading and lagging strands
  • Main replicative enzyme
  • High processivity- can synthesize long stretches of DNA
  • 3’-5’ exonuclease proofreading activity

Other polymerases- B and y are involved in DNA repair and mitochondria genome replication, respectively

46
Q

What is Camptorhecin ?

A

An antic@nicer drug, binds to and inhibits topoisomerase I activity. The result is DNA breakage.

47
Q

What is etoposide?

A

An anti cancer drug, inhibits the activity of topoisomerase II

48
Q

What is actinomycin D/ Dactinomycin?

A

The phenoxybenzamine ring intercalates between adjacent guanine-cytosine bases

The polypeptide chains extend along the minor groove of the helix, thereby stabilizing the drug DNA complex

49
Q

How does antinomycin D prevent DNA replication?

A
  • Preventing the formation of regions of ssDNA

- also, inhibits RNA transcription (as it prevents strand separation)

Basic Principles Of Medicine 1-FTM 1 (39 decks)

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