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MOLECULAR BIOLOGY > WEEK 9 (DNA Repair) > Flashcards

WEEK 9 (DNA Repair) Flashcards

1
Q

Describe the components of the Nucleus

A

NUCLEAR ENVELOPE - formed from two concentric membranes & defines nuclear compartment

INNER NUCLEAR MEMBRANE - contain proteins that act as binding sites for the chromosomes and provide anchorage for the nuclear lamina

NUCLEAR LAMINA - protein filaments that provide structural support for the nuclear envelope

OUTER NUCLEAR MEMBRANE - membrane similar composition & therefore is continuous with the ER membrane

NUCLEAR PORES - form the gates which all molecules enter or leave the nucleus

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2
Q

Describe how proteins enter the nucleus through Nuclear pores

A

Proteins directed from the cytosol into the nucleus contains NUCLEAR LOCALISATION SIGNAL which consists of one or two short sequences containing several POSITIVELY CHARGED LYSINES/ARGININES & is recognised by nuclear import receptors.

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3
Q

What is a nuclear pore?

A

A large, elaborate structure composed of a complex of about 30 different proteins that line the nuclear pore which contain EXTENSIVE, UNSTRUCTURED REGIONS in which the polypeptide chains are LARGELY DISORDERED

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4
Q

What are Nucleic acids?

A

Macromolecules constructed out of long chains of monomers called NUCLEOTIDES and function primarily in the storage and transmission of genetic information but may also have structural or catalytic roles

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5
Q

What are the two types of Nucleic acids found in living organisms?

A

Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA)

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6
Q

Describe the structure of RNA

A
  • a pentose sugar (RIBOSE)
  • a nitrogenous base
  • a phosphate group
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7
Q

What is the difference between ribose and deoxyribose?

A

Ribose contains a HYDROXYL GROUP bonded to the SECOND CARBON ATOM whereas deoxyribose has a HYDROGEN ATOM rather than a hydroxyl group attached to the SECOND CARBON ATOM

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8
Q

What is the difference between Purines and Pyrimidines?

A

Pyrimidines are smaller molecules consisting of a SINGLE RING and Purines are larger consisting of TWO RINGS

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9
Q

Describe Thymine

A

A pyrimidine with an extra methyl group

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10
Q

At the time Watson and Crick performed their analysis, which forms of DNA were known?

A

A-DNA and B-DNA

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11
Q

Describe A-DNA

A
  • slightly more compact than B-DNA
  • right-handed helix
  • orientation of the bases is tilted and displaced laterally in relation to the axis of the helix
  • contains one more base pair per turn than B-DNA
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12
Q

Which other DNA right-handed helices have been discovered?

A
  • C-DNA (found under even greater dehydration conditions than those observed during the isolation of A- and B- DNA)
  • D-DNA
  • E-DNA
    (D-DNA and E-DNA occur in helices lacking guanine in their base composition)
  • P-DNA (when DNA is artificially stretched)
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13
Q

What is distinguishable about Z-DNA?

A

A left-handed double helix

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14
Q

What did Jerome Vinograd and his colleagues discover?

A

They discovered that two closed, circular DNA molecules of identical molecular mass could exhibit very different rates of SEDIMENTATION during CENTRIFUGATION. Furher analysis indicated that the DNA molecule sedimenting more RAPIDLY had a more COMPACT shape because the molecule was twisted upon itself (SUPERCOILED DNA)

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15
Q

Explain the reasoning behind Supercoiled DNA during centrifugation

A

Because supercoiled DNA is more compact than its relaxed counterpart, it occupies a SMALLER VOLUME and moves RAPIDLY in response to a centrifugal force or an electric field

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16
Q

What are Topoisomerases?

A

Enzymes that change the supercoiled state of a DNA duplex. They change the TOPOLOGY of DNA.

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17
Q

Name the different RNA types and Primary functions

A
  • mRNA (messenger RNA) - TRANSLATION/PROTEIN SYNTHESIS
  • rRNA (ribosomal RNA) - TRANSLATION/PROTEIN SYNTHESIS
  • tRNA (transfer RNA) - TRANSLATION/PROTEIN SYNTHESIS
  • hRNA (heterogenous nuclear RNA) - PRECURSORS & INTERMEDIATES OF MATURE MRNAs & OTHER RNAs
  • scRNA (small cytoplasmic RNA) - SIGNAL RECOGNITION PARTICLE & TRNA PROCESSING
  • snRNA (small nuclear) - MRNA PROCESSING
  • snoRNA (small nucleolar) - RRNA PROCESSING/METHYLATION
  • regulatory RNAs (siRNA, miRNA) - REGULATION OF TRANSCRIPTION & TRANSLATION
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18
Q

Which RNAs are catalytic?

A
  • rRNA (ribosomal RNA)
  • scRNA (small cytoplasmic RNA)
  • snRNA (small nuclear)
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19
Q

What are important nucleotides in the body and what are their functions?

A
  • ATP (Adenosine Triphosphate) which is an energy carrier
  • GTP (Guanine Triphosphate) binds to a variety of proteins (called G proteins) and acts as a switch to turn on their activities
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20
Q

Describe In situ molecular hybridisation

A

Nucleic acid probes are often used to identify COMPLEMENTARY SEQUENCES since an RNA molecule will hybridise with the segment of DNA from which it was transcribed. Single-stranded DNA or RNA is added (a probe) and hybridisation is monitored. [The nucleic acid that is added may either be RADIOACTIVE or contain a FLUORESCENT LABEL to allow its detection.

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21
Q

What can hybridisation reactions be used to detect and characterise?

A

Nucleotide sequences using a particular nucleotide sequence as a probe

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22
Q

Describe the process of creating a hybrid strand of DNA/RNA

A
  • The two strands of a DNA molecule are DENATURED by heating to 100 degrees Celsius; At this temp, complementary base pairs that hold the DOUBLE HELIX strands together are DISRUPTED and the helix rapidly dissociates into TWO SINGLE STRANDS
  • DNA denaturation is reversible by keeping the two single strands of DNA for a PROLONGED PERIOD at 65 degrees Celsius (DNA RENATURATION/HYBRIDISATION)
  • If an RNA transcript is introduced during the renaturation process, the RNA competes with the coding DNA strand and forms double stranded DNA/RNA hybrid molecule
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23
Q

What is Fluorescent In Situ Hybridisation (FISH)?

A

A technique used to identify chromosomal locations housing specific genetic information

24
Q

What are the properties of FISH?

A
  • a short fragment of DNA that is complementary to DNA in the chromosomes’ centromere regions has been hybridised
  • Fluorescence occurs only in the centromere regions and thus identifies each one along its chromosome
  • The red fluorescence is produced by PROPIDIUM IODIDE
  • has greatly enhanced the field and enabled more precise determination of the presence and frequency of GENETIC ABNORMALITIES
25
Q

What are the advantages of FISH compared to standard cytogenic analysis?

A
  • Can be used to identify genetic changes that are too small to be detected under a microscope
  • does not require cell culture
  • can be applied directly on fresh or paraffin-embedded tissues for rapid evaluation of interphase nuclei
26
Q

What are the stages of FISH?

A

1) Green fluorochrome-labeled probe is denatured
2) Tissue or cell component on glass slide is denatured to create single-stranded DNA
3) Denatured probe is applied to the slide/tissue. Slide is incubated to allow HYBRIDISATION. Wash off non-hybridised probe.
4) Detection of fluorescent hybridisation signal by FLUORESCENT MICROSCOPY

27
Q

Describe how FISH is used for Bacterial pathogen identification

A

1) Sample of infected tissue is collected from the patient and COMPLEMENTARY OLIGONUCLEOTIDE is synthesised for suspected pathogen with FLUORESCENT tag chemically attached
2) Chemically treat tissue sample to make the membranes of all cells permeable to the fluorescently tagged oligonucleotide
3) Add fluorescently tagged complementary oligonucleotide to sample. The fluorescently tagged oligonucleotide will bind only to the pathogenic DNA.
4) Plate sample and observe under microscope; the pathogenic cells will fluorescence.

28
Q

What did Elizabeth Blackburn discover?

A

Telomerase

29
Q

What is Telomerase?

A

Telomerase is a cellular reverse transcriptase that helps to provide genomic stability in highly proliferative normal, immortal, and tumor cells by maintaining the integrity of the chromosome ends called the telomeres

29
Q

What problems do the presence of linear DNA “ends” on eukaryotic chromosomes make?

A
  • The double-stranded “ends” of DNA molecules at the termini of linear chromosomes resemble
    the double-stranded breaks (DSBs) that can occur when a chromosome becomes broken internally as a result of DNA damage. Such double-stranded DNA ends are recognized by the cell’s DNA repair mechanisms that join the “loose ends” together, leading to chromosome fusions and translocations. If the ends do not fuse, they are vulnerable to degradation by nucleases.
  • During DNA replication, DNA polymerases cannot synthesise new DNA at the tips of single-stranded 5 ends.
30
Q

Where is Telomerase typically active?

A

In germ cells and adult stem cells; it isn’t active in adult somatic cells.

31
Q

Describe the importance of Telomerase

A

After replication is completed, the RNA PRIMERS are removed. The gaps left are filled by DNA POLYMERASE and sealed by LIGASE (the gaps have free 3’-OH groups available at the ends of the OKAZAKI FRAGMENTS for DNA POLYMERASE to initiate synthesis). Gaps left at the 5’ ends of the newly synthesised DNA cannot be filled by DNA POLYMERASE since no free 3’-OH groups are available so is filled by TELOMERASE

32
Q

Is Telomerase prokaryotic or eukaryotic?

A

Eukaryotic

33
Q

Why can’t DNA polymerase fill the gaps at the 5’ ends of the newly synthesised DNA?

A

Since no free 3’-OH groups are available for the initiation of synthesis

34
Q

What are the properties of Telomerase?

A
  • Ribonucleoprotein
  • Contains a short piece of RNA that is essential to its catalytic activity (TELOMERASE RNA COMPONENT (TERC))
35
Q

What is the function of TERC (TELOMERASE RNA COMPONENT)?

A
  • Serves as a GUIDE to proper attachment of the enzyme to the telomere
  • Acts as a TEMPLATE for synthesis of its DNA complement
36
Q

What is Reverse transcription?

A

Synthesis of DNA using RNA as a template

37
Q

What is the name of the catalytic subunit of Telomerase?

A

TELOMERASE REVERSE TRANSCRIPTASE (TERT)

38
Q

Describe the components of Telomerase

A
  • Telomerase RNA component (TERC)
  • Telomerase reverse transcriptase (TERT)
  • A number of accessory proteins
39
Q

What is Recombination?

A

The rearrangement of DNA sequences by the breakage and rejoining of chromosomes or chromosome segments

40
Q

What is the importance of recombination?

A
  • Major source of genetic diversity through sexual reproduction
  • Essential for the exchange of genetic information during meiosis
  • Important for the repair of damaged DNA
41
Q

What happens in DNA repair?

A
  • Proofreading
  • Mismatch repair
  • Nucleotide excision repairs
42
Q

What happens in “Proofreading”?

A

DNA POLYMERASE reads the newly added base before adding the next one, so a correction can be made

43
Q

What happens in “Mismatch repair”?

A

The incorrectly added base is detected after REPLICATION. The MISMATCH REPAIR PROTEINS detect this base and remove it from the newly synthesised strand by nuclease action. The gap is then filled with the CORRECTLY PAIRED BASE.

44
Q

What happens in “Nucleotide excision repairs”?

A

In normal cells, thymine-thymine dimers are EXCISED and REPLACED.

This repair mechanism is often employed when UV exposure causes the formation of thymine-thymine dimers. When exposed to UV, thymines lying adjacent to each other can form THYMINE DIMERS.

45
Q

Describe Xeroderma Pigmentosum

A

A condition in which thymine dimerisation from exposure to UV is not repaired; exposure to sunlight results in SKIN LESIONS.

It is an AUTOSOMAL RECESSIVE DISORDER in which there is a decreased ability to repair DNA damage. Mutations in repair genes have been known to cause cancer.

[signs appear in infancy or early childhood & affects the eyes and areas of skin exposed to the sun]

SIGNS AND SYMPTOMS:
- problems involving the NERVOUS SYSTEM
- severe sunburn after a few minutes in the sun
- redness & blistering
- freckling
- dry skin (XERODERMA)
- skin colouring (PIGMENTATION)
- multiple skin cancers

46
Q

What is the reaction in DNA replication catalysed by DNA ligase?

A

Formation of a phosphodiester bond between the 3’-OH of one Okazaki fragment and the 5’-phosphate of the next on the lagging strand

47
Q

Which enzymes remove supercoiling in replicating DNA ahead of the replication fork?

A

Topoisomerases

Explanation: Strand separation creates TOPOLOGICAL STRESS in the helical DNA structure which is relieved by the action of topoisomerases

48
Q

DNA unwinding is done by ____________

A

Helicases

49
Q

Which mechanism will remove uracil and incorporate the correct base?

A

Base excision repair

Explanation: Base excision repair is responsible for removing small, non-helix-distorting base lesions from genome

50
Q

Which protein is required for connecting Okazaki fragments?

A

DNA gyrase

51
Q

What is the protein responsible for the formation of RNA primers?

A

Primase

52
Q

Because DNA polymerase III can only act from 5’ to 3’ direction, continuous strand growth can be achieved only along one of the template strands (Leading strand) and strand growth along the other strand must occur discontinuously resulting in the production of a series of short sections of new DNA called _____________________

A

Okazaki Fragments

53
Q

______________ is the primary prokaryotic replicators polymerase that can proofread DNA and fix incorrect base pairs due to its _____________________

A

DNA polymerase III

3’-5’ exonuclease function

54
Q

What are Exonucleases?

A

Key enzymes involved in many aspects of cellular metabolism and maintenance and are essential to genome stability by cleaving DNA from free ends

MOLECULAR BIOLOGY (11 decks)

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