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L2 Molecular Cell Biology > DNA > Flashcards

DNA Flashcards

1
Q

What are subnuclear territories?

A

Individual chromosomes occupy distinct areas of the nucleus even in interphase - subnuclear territories

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2
Q

How are chromosomes packaged? (2)

A
  • Chromosomes are tightly coiled into chromatin
  • Chromatin resembles beads on a string, the ‘beads’ are nucleosomes
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3
Q

What is the structure of a nucleosome? (3)

A
  • Nucleosome is made of 8 core histones (2x H2A, H2B, H3, H4)
  • DNA is wound around the histone cylinder structures
  • N terminal tails of the histone subunits project out and interact with other proteins to regulate the chromatin structure
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4
Q

What is histone H1? (2)

A
  • Linker histone H1 isn’t within the nucleosome, interacts with the DNA and establishes transcriptionally silent heterochromatin
  • Rich in lysine and arginine (basic) so can bind DNA in a non-sequence specific manner, just binds to the negatively charged phosphate backbone
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5
Q

What are DNA remodelling enzymes? (2)

A
  • Remove nucleosomes to open up the DNA for replication/transcription proteins to bind
  • Chromatin is packaged to be flexible to remodelling
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6
Q

What are fractal globules?

A

‘Globules within globules’ which allow the chromatin to be condensed and decondensed without getting knotted

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7
Q

How is DNA organised in the nucleus? (2)

A
  • Transcriptionally inactive DNA is in the periphery of the nucleus (tightly packaged globules)
  • Transcriptionally active DNA (RNA transcripts) are excluded from the periphery (DNA more open)
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8
Q

What is the purpose of the specialised components of chromosomes? (2)

A
  • Facilitate reliable and complete DNA replication
  • Allow segregation of duplicated chromosomes during cell division
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9
Q

What are telomeres? (4)

A
  • Specialised repetitive DNA sequences which exist as single-stranded 3’ overhangs on the ends of chromosomes
  • Prevent loss of genetic information during replication
  • Telomerase enzyme replicates the telomeres
  • Define the ends of chromosomes and maintain genomic integrity
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10
Q

What is the centromere? (2)

A
  • Region of repetitive DNA sequences by which the chromosomes are connected to each other during mitosis
  • Binds to the kinetochore
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11
Q

What is the kinetochore?

A

Binds to the centromere and allows stabilisation of the mitotic spindle

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12
Q

How does the centromere bind to the kinetochore? (3)

A
  • Centromere contains alpha-satellite DNA repeats
  • Kinetochore inner plate binds to the centromere sequence
  • Kinetochore outer plate binds to the microtubules of the mitotic spindle
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13
Q

What is the kinetochore structure in yeast? (2)

A
  • Single nucleosome H3 is centromere-specific binds to the inner plate of the kinetochore
  • Outer plate binds to the inner plate and forms a basket-like structure around the microtubule to connect it to the centromeric nucleosome
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14
Q

What is the structure of the genome? (4)

A
  • 1% protein-coding
  • 20% introns
  • 50% transposons
  • Rest is non-repetitive DNA that aren’t introns or codons
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15
Q

What is the purpose of non-repetitive, non-coding DNA? (2)

A
  • Regulation of transcription and access to protein-coding genes
  • Some determines where and when in the body adjacent protein-coding genes are transcribed
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16
Q

What are transposons? (3)

A
  • Repeated DNA sequences which make up almost half of the human genome
  • Mobile genetic elements that jump around the genome
  • ‘Transposable elements’
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17
Q

What are the 3 types of transposons?

A
  • DNA transposons
  • Retroviral retrotransposons
  • Non-retroviral polyA retrotransposons
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18
Q

What is the action of DNA transposons? (3)

A
  • Encode transposase enzyme which allows them to move around the genome in a ‘cut-and-paste’ mechanism
  • Transposase enzymes bind to short inverted repeat sequences at the ends of the DNA transposon and cut it out and insert it elsewhere
  • Potentially mutagenic if the original sequence doesn’t re-join properly or if the sequence is inserted into an important gene
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19
Q

How were DNA transposons first discovered? (2)

A
  • Activator-dissociator DNA transposon discovered in maize
  • Dark coloured segments in maize jumped around the genome during crossing over more randomly than expected
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20
Q

What is the action of retroviral retrotransposons? (2)

A
  • The sequence is transcribed into RNA
  • DNA is reverse-transcribed using the RNA as a template and inserted into a new genomic location
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21
Q

What is the action of non-retroviral polyA retrotransposons? (3)

A
  • Sequence transcribed into RNA with a polyA tail which inserts into the genome at the target location
  • RNA is reverse transcribed back into DNA and inserted
  • Can cause disruptions resulting in haemophilia
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22
Q

Why is DNA replication semi-conservative? (2)

A
  • Double helix is separated and both strands are used as a template
  • New DNA contains one newly synthesised strand and one ‘old’ strand
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23
Q

What direction does DNA replication occur in? (4)

A
  • 5’-3’
  • New strand is antiparallel to the template strand
  • Nucleotides added to the 3’ hydroxyl end of the strand
  • Involves a nucleophilic attack on the phosphate of the incoming dNTP which results in the new nucleotide binding to the new strand and release of pyrophosphate
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24
Q

How does replication occur on the leading strand? (5)

A
  • DNA double helix is separated by DNA helicase to form the replication fork
  • DNA primase makes RNA primers
  • Primers bind to the exposed DNA
  • DNA polymerase binds to the 3’ end of the primer and extends it
  • Replication occurs continuously on the leading strand in a 5’-3’ direction
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25
Q

How does replication occur on the lagging strand? (7)

A
  • DNA double helix is separated by DNA helicase to form the replication fork
  • DNA primase makes RNA primers
  • Primers bind to the exposed DNA
  • Replication occurs discontinuously on the lagging strand in a 5’-3’ direction which forms Okazaki fragments
  • DNA polymerase extends the primer to fill the gap to the previous primer
  • Ribonuclease H removes the primer allowing DNA polymerase to fill the gap
  • DNA ligase joins the Okazaki fragments
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26
Q

How does DNA helicase work? (2)

A
  • Breaks the hydrogen bonds between the strands to form the replication fork
  • Requires energy from ATP hydrolysis
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27
Q

What is caused by mutations in DNA helicase? (3)

A
  • Bloom syndrome and Progerias e.g. Werner syndrome
  • Mutation in RECQ helicase causes premature ageing
  • Causes cell senescence
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28
Q

What is processivity? (2)

A
  • Ability of an enzyme to catalyse consecutive reactions without releasing its substrate
  • Processive enzyme = always bound to substrate
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29
Q

What is the sliding clamp? (3)

A
  • ATP-dependent sliding clamp is positioned close to the primer:template junction by a clamp loader
  • ATP is hydrolysed and clamp loader released
  • Sliding clamp enhances the processivity of DNA polymerase to increase rate of DNA replication
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30
Q

What is the sliding clamp in E.coli called?

A

PCNA

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31
Q

What is a single-stranded DNA binding protein (SSB)? (2)

A
  • Exposed DNA template strand can start to re-bind to itself forming hairpins
  • SSBs bind to single stranded DNA and prevent formation of hairpins to allow replication to continue efficiently
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32
Q

Which proteins enhance the processivity of DNA polymerase? (3)

A
  • Sliding clamp
  • SSB
  • DNA topoisomerase
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33
Q

What is DNA topoisomerase? (3)

A
  • Unwinding at the replication fork introduces superhelical tension which causes tangling
  • DNA topoisomerases relax the tension by nicking and resealing the backbone of the template strands
  • Enhances processivity of DNA polymerase
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34
Q

What are the 2 types of DNA topoisomerase?

A
  • Type I
  • Type II
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35
Q

What does type I DNA topoisomerase do?

A

Nicks and reseals one of the 2 DNA strands, no ATP required

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36
Q

What does type II DNA topoisomerase do?

A

Nicks and reseals both DNA strands, ATP required

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37
Q

What is the origin of replication?

A

Point where DNA replication starts

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38
Q

What is the origin of replication in E.coli?

A

OriC - only one in the genome

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39
Q

What is the origin of replication in yeast?

A

Autonomously replicating sequences (ARS)

40
Q

Why is initiation of DNA replication biphasic in eukaryotes? (3)

A
  • First replicator selection occurs in G1 which results in the formation of a pre-replicative complex
  • Then origin activation occurs in S phase which results in unwinding of DNA and recruitment of DNA polymerase
  • Temporal separation ensures that each origin is used once and each chromosome is replicated only once per cell cycle
41
Q

When is the pre-replicative complex formed?

42
Q

When is the pre-replicative complex activated?

43
Q

How is the pre-replicative complex formed in G1 in yeast? (4)

A
  • Origin recognition complex (ORC) binds to ARS sequence
  • Helicase-loading proteins cdc6 and cdt1 bind to ORC
  • Helicase mcm2-7 bind
  • Forms the inactive pre-replicative complex
44
Q

How are pre-replicative complexes regulated by cdks? (2)

A
  • Cdk activity is low in G1 which allows the formation of the pre-replicative complex but prevents activtion
  • Cdk activity is high in S phase Which activates the pre-replicative complex to initiate replication and inhibits formation of new pre-replicative complexes outside of G1
45
Q

How is DNA replication finished? (4)

A
  • When the primers at the end of the strands are removed by ribonuclease H it leaves 3’ single strand overhangs
  • Telomerase extends the 3’ end with TTAGGG repeats
  • The 3’ strand acts as a template so DNA primase binds a new primer and DNA polymerase fills in the gap
  • The telomere still has a 3’ overhang
46
Q

What sequence is added by telomerase?

47
Q

What is a ribonucleoprotein? (2)

A
  • Contains protein and RNA subunits
  • E.g. telomerase
48
Q

How does telomerase know to add TTAGGG? (3)

A
  • Telomerase RNA subunit has the sequence AAUCCCAAU which is complementary to TTAGGG
  • Telomerase uses reverse transcriptase activity to make DNA from its own RNA template
  • Telomere repeat sequences are synthesised in a step-wise process called the telomerase shuffle
49
Q

What sequence does the RNA template of telomerase have?

50
Q

What is the telomerase shuffle? (4)

A
  • Telomerase RNA binds to the existing telomere repeat with a 3’ overhang of AAU
  • Using reverse transcription to add TTA onto the end of the overhang
  • Telomerase shuffles 6 nucleotides forwards and uses the TTA as a template to synthesise GGGTTA using the rest of its RNA template
  • Shuffles along again, binding to TTA, repeats
51
Q

Why is DNA repair important? (2)

A
  • Genetic stability is our most robust defence against cancer
  • Only biological macromolecule to repair, all others are replaced
52
Q

What are the consequences of DNA damage? (2)

A
  • In dividing cells: errors in replication cause mutations and lead to cancer
  • In non-dividing cells: accumulation of DNA damage leads to ageing
53
Q

What are the sources of DNA damage? (2)

A
  • Endogenous sources
  • Exogenous sources
54
Q

What are endogenous sources of DNA damage? (2)

A
  • Reactions with other molecules within the cell
  • E.g. hydrolysis, ROS
55
Q

What are exogenous sources of DNA damage? (2)

A
  • Reactions with molecules from outside the cell
  • E.g. UV, X-rays, carcinogens, chemotherapeutics
56
Q

What are the 2 types of DNA damage?

A
  • Endogenous damage
  • Exogenous damage
57
Q

What is endogenous DNA damage? (4)

A
  • Depurination (abasic sites)
  • Deamination
  • Methylation
  • Replication errors
58
Q

What is exogenous DNA damage? (3)

A
  • Pyrimidine dimers
  • Double strand breaks
  • Interstrand crosslinks
59
Q

Which types of DNA damage affect one strand of the DNA helix? (5)

A
  • Depurination (abasic sites)
  • Deamination
  • Methylation
  • Replication errors
  • Pyrimidine dimers
60
Q

Which types of DNA damage affect both strand of the DNA helix? (2)

A
  • Double strand breaks
  • Interstrand crosslinks
61
Q

What is deamination? (5)

A
  • Removal of the amino group of the nucleotide by hydrolysis
  • E.g. removal of amino group of cytosine in the presence of water releases ammonia and makes uracil
  • Uracil in DNA is recognised as a thymine during replication so pairs with an adenine rather than a guanine
  • Results in a transition mutation from CG to TA (point mutation)
  • Deamination only affects one strand so only 1 of the 2 new DNA molecules are affected
62
Q

What are the 2 types of point mutations?

A
  • Transition mutation
  • Transversion mutation
63
Q

What is a transition mutation? (2)

A
  • Purine base is swapped for another purine base/pyrimidine base is swapped for another pyrimidine base
  • I.e. adenine swapped for guanine/cytosine swapped for a thymine
64
Q

What is a transversion mutation? (2)

A
  • Purine base is swapped for a pyrimidine base/pyrimidine base is swapped for a purine
  • I.e. adenine swapped for cytosine or thymine etc.
65
Q

What is a purine base? (2)

A
  • Double ring bases
  • Adenine and guanine
66
Q

What is a pyrimidine base? (2)

A
  • Single ring bases
  • Cytosine and thymine
67
Q

Why are transition mutations more likely than transversions?

A

Easier to substitute a double ring structure for another double ring structure and a single for a single

68
Q

Which point mutation is less likely to result in amino acid substitutions? (2)

A
  • Transition mutations
  • Wobble base theory
69
Q

What is the wobble base theory? (3)

A
  • If the first base in the codon is an A, the sequence has to be exactly that to code for the right amino acid
  • If the first base is a G, only the first 2 bases are essential to code for the right amino acid
  • Allows for flexibility
70
Q

What is depurination (abasic site)? (3)

A
  • N-glycosidic bond is cleaved by hydrolysis which results in the absence of a base in the DNA sequence (abasic site)
  • Results in a frameshift mutation in one of the DNA molecules during DNA replication because the missing base is skipped
  • Deletion
71
Q

Where is depurination most common?

A

At purine bases rather than pyrimidine

72
Q

What is the impact of frameshift mutations?

A

Generation of missense proteins which don’t function properly

73
Q

What are pyrimidine dimers? (3)

A
  • Photochemical reaction between pyrimidine bases results in the formation of cyclobutane rings
  • Distorts the DNA structure
  • Caused by UV light
74
Q

What kind of DNA damage is caused by UV light? (3)

A
  • Pyrimidine dimers
  • Interstrand crosslinks
  • DNA-protein crosslinks
75
Q

What are interstrand crosslinks? (2)

A
  • Incorrect bases pair with eachother within the double stranded DNA
  • Highly toxic to the cell because it blocks replication and transcription
76
Q

What are DNA protein crosslinks? (2)

A
  • Nucleotide forms a covalent bond with a protein
  • Highly toxic to the cell because it blocks replication and transcription
77
Q

What causes a double strand break? (3)

A
  • X-rays
  • Ionising radiation
  • Topoisomerase II inhibitors
78
Q

What causes a single strand break? (3)

A
  • ROS
  • Hydroxyurea
  • Camptothecin
79
Q

Which types of DNA damage are repaired by base excision repair (BER)?

A

Base damage e.g. abasic sites, deamination

80
Q

What is the mechanism of base excision repair (BER)? (5)

A
  • Mismatched base (e.g. deaminated C becomes U) is recognised during replication and is flipped out and removed by uracil DNA glycosylase
  • Results in DNA with a missing base (abasic site)
  • AP endonuclease and phosphodiesterase remove the sugar phosphate backbone at the abasic site
  • DNA polymerase adds a new nucleotide
  • DNA ligase seals the nick
81
Q

Which types of DNA damage are repaired by nucleotide excision repair (NER)?

A

Damage when more than one base is involved e.g. pyrimidine dimers caused by UV

82
Q

What is the mechanism of nucleotide excision repair (NER)? (4)

A
  • Involves excision of short patches of single stranded DNA to remove the affected bases
  • Excision nuclease creates breaks in the backbone around the damaged bases
  • DNA helicase unwinds the damaged section and removes it
  • DNA polymerase and DNA ligase fill in the gap
83
Q

What is translesion synthesis? (4)

A
  • During replication, sliding clamp moves along the template strand
  • If it encounters damage the sliding clamp dissociates from DNA polymerase and associates with translesion DNA polymerase
  • Translesion DNA polymerase puts random bases down in the damaged section
  • Normal DNA polymerase re-associates and continues as normal
84
Q

What is the problem with translesion synthesis? (3)

A
  • Lacks precision in template recognition and substrate base choice
  • Lacks exonucleolytic proof-reading activtity
  • Causes base substitutions and single nucleotide deletion mutations
85
Q

Which mechanisms repair double strand breaks? (2)

A
  • Nonhomologous end joining
  • Homologous recombination
86
Q

When in the cell cycle does nonhomologous end joining occur?

87
Q

When in the cell cycle does homologous recombination occur?

88
Q

What is the mechanism of non-homologous end joining (NHEJ)? (4)

A
  • MRN complex resections the break, producing a 3’ overhang
  • Ku70/80 heterodimer and DNA-PK assemble at the break
  • Synaptic complex forms and pulls the broken ends together so end-processing can occur (cut off 3’ overhang)
  • DNA ligase joins the break
89
Q

What is the problem with non-homologous end joining (NHEJ)? (2)

A
  • Error prone
  • Usually results in the loss of nucleotides surrounding the break site which can cause the loss of important genetic information
90
Q

What is the mechanism of homologous recombination? (7)

A
  • MRN complex resections the break, producing a 3’ overhang
  • RPA/BRCA1/BRCA2 coat the 3’ overhang with Rad51
  • This initiates strand invasion into the sister chromatid forming a Holliday junction
  • Correct sequence is transcribed from the homologous sequence in the sister chromatid template
  • Double Holliday junction forms
  • Accurate sequence is put back into the original damaged DNA via a crossing over event
  • Results in accurate repair
91
Q

Which method of double strand break repair is more accurate?

A

Homologous recombination

92
Q

When are the checkpoints in the cell cycle for DNA damage? (3)

A
  • G1
  • Entry to S phase
  • Entry to mitosis
93
Q

How is DNA damage detected? (5)

A
  • ATM/ATR are activated and bind to the site of DNA damage
  • Activates Chk1/Chk1 which phosphorylate p53 to activate it
  • p53 activates p21
  • p21 inhibits cyclin/CDK complexes to stop the cell cycle progressing
  • DNA is then repaired/cell undergoes apoptosis if repair is not possible
94
Q

What disease is associated with defects in nucleotide excision repair (NER)? (2)

A
  • Xeroderma pigmentosum
  • Increased risk of skin cancer as NER is involved in UV-induced damage
95
Q

What disease is associated with defects in double strand break repair? (2)

A
  • BRCA1/BRCA2 involved in homologous recombination
  • Defects associated with 80-90% of inherited breast cancers
96
Q

What are the features of BRCA2 deficient cells? (3)

A
  • Exhibit genomic instability
  • Sensitive to DNA damaging agents
  • Defective in homologous recombination
97
Q

How can we study DNA damage? (4)

A
  • Survival assay to examine cell line sensitivity to ionising radiation
  • Detection of markers of double strand breaks using immunofluorescence in response to damage
  • Treat cells with ionising radiation and examine using gel electrophoresis, damaged cells drag
  • Look for markers of DNA damage in Western Blots

L2 Molecular Cell Biology (13 decks)

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