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BIOC4332: Biochemistry II > Exam I > Flashcards

Exam I Flashcards

1
Q

What are the components of a nucleotide?

A

Base, phosphate, sugar

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2
Q

Nucleic acids contain what kind of sugars?

A

Pentose

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3
Q

What is the difference between ribose and deoxyribose?

A

Ribose is found in RNA and has two -OH groups attached to the 2’ and 3’ carbons.

Deoxyribose is found in DNA and has one -OH group attached to the 3’ carbdon.

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4
Q

What is the difference between a purine and pyrimidine? Give examples of bases.

A

Purines consist of two rings (Ex. Adenine and guanine)

Pyrimidines consist of one ring (Ex. Cytosine, thymine, uracil)

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5
Q

What’s the difference between thymine and uracil?

A

A methyl group

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6
Q

What is the name of the bond that connects the sugar to the base?

A

N-glycosidic bond (Bond between C and N, loss of water)

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7
Q

What is the name of the bond that connects the sugar to the phosphate?

A

Phosphodiester bond (Bond between the “phosphonyl” P from the phosphate and the O from the sugar)

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8
Q

What is the energy of a hydrogen bond?

A

~1 kcal/mol

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9
Q

Where are the hydrogen bonds located in DNA?

A

Between the bases (A to T, C to G)

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10
Q

What base pair is stronger and why?

A

G and C pairs are stronger because they have three H-bonds. A to T only have two H-bonds.

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11
Q

DNA normally exists in __ form.

A

B

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12
Q

DNA is a ___-handed helix.

A

right

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13
Q

What are some characteristics of right-handed helixes?

A

~10 bp/turn, 3.4 nm per turn, 2 nm wide

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14
Q

Why do minor and major grooves in DNA exist?

A

The strands run antiparallel to each other, so the N-glycosidic bonds are not equally opposite.

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15
Q

Why are the phosphates in the phophodiester bonds of the two chains in DNA not diametrically opposite?

A

To minimize repulsion between negative charges

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16
Q

Nucleic acid information can be “read” by what?

A

DNA binding proteins

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17
Q

The alpha helix of a HTH (helix-turn-helix) domain can fit where in DNA?

A

The major groove

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18
Q

The alpha helix of a ZFP (zinc finger protein) can fit inside where in DNA?

A

The major groove

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19
Q

Beta sheets can fit inside where in DNA?

A

The major groove

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20
Q

The amino acid side chains of DNA binding proteins can form _____ with the bases in the double helix.

A

H-bonds

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21
Q

What is multivalency?

A

Multiple contacts increasing binding strength

(More H-bonds = Overall interaction is strong)

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22
Q

If you stretched it out, how long would all the DNA in a human cell be?

A

1 meter

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23
Q

Why do DNA dyes have a positive charge?

A

Nucleic acids have a negative charge.

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24
Q

The accuracy of hybridization depends on what? Explain.

A

The temperature of annealing

Only stable hybrids form at higher temperatures (No mismatches).

Imperfect base pairing happens at lower temperatures (Has mismatches)

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25
Q

Explain FISH.

A

FISH stands for fluorescence in situ hybridization.

The FISH “probe” is a complementary sequence + fluorescent dye (FL).

FISH applications include detection of translocations in cancer and analysis of gene localization.

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26
Q

Explain gel electrophoresis.

A

DNA is chopped up into smaller pieces using a restriction enzyme.

DNAs of different lengths migrate proportionally to their length. The shortest DNAs move fastest.

DNA is stained with intercalating dye to give a brighter signal.

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27
Q

Explain Southern & Nothern blots.

A

Watch a video.

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28
Q

What is recombinant DNA?

A

Artificially created DNA that combines sequences that do not occur together in nature

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29
Q

Why clone DNA?

A

To identity genes, express genes, mutate genes, and sequence genomes

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30
Q

What is the difference between endonucleases and exonucleases?

A

Endonucleases hydrolyze DNA in the interior of double helices.

Exonucleases hydrolyze DNA from the ends.

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31
Q

Restriction enzymes are what kind of nuclease enzyme? What do they do?

A

They’re deoxyriboendonucleases that recognize specific DNA sequences and hydrolyze a phosphodiester bond at specific sequences.

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32
Q

Many restriction enzymes recognize DNA palindromes. What are palindromes?

A

DNA palindromes read the same forward and backward.

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33
Q

Restriction enzymes leave what kind of ends? What does this allow for?

A

They leave overhanging, sticky ends, allowing for more specificity because they can base pair.

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34
Q

What is the function of ligase?

A

Covalently joins two DNA fragments (Usually in DNA repair)

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35
Q

Where do restriction enzymes come from?

A

They’re made by bacteria to defend themselves from foreign DNA.

Bacteria protect their DNA by methylating the related recognition site to prevent their hydrolysis.

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36
Q

What’s a polylinker?

A

Multiple cloning site; a DNA segment with several unique sites for restriction enyzmes

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37
Q

What are genomic libraries?

A

Collection of chimeric plasmids that include all DNA in a genome

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38
Q

What is the function of reverse transcriptase?

A

It’s a DNA polymerase that uses RNA as a template.

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39
Q

Eukaryotic mRNAs end with a string of what?

A

A bases (Poly A tail)

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40
Q

By what method is an RNA:DNA hybrid converted to DNA:DNA using DNA polymerase?

A

Polymerase chain reaction (PCR)

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41
Q

Learn the steps of PCR.

A

Ok

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42
Q

What is the function of DNA polymerase?

A

Catalyzes the addition of a nucleotide unit using the principle of base complementarity

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43
Q

What is a primer?

A

It requires a chain that base pairs with the template and has a 3’ -OH on which to add

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44
Q

In Sanger sequencing, how does DNA replication stop?

A

DNA replication stops without a 3’ -OH on the sugar. This prevents strand extension.

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45
Q

What is a CONTIG?

A

A series of overlapping DNA sequences used to make a physical map that reconstructs the original DNA sequence.

Software algorithms can recognize the overlap between clones. This builds a CONTIG.

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46
Q

What are the three components useful for cloning vectors?

A
  1. Replication origin
  2. Selectable markers (Drug resistance, nutritional marker)
  3. Cloning site (Restriction enzyme recognition site)
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47
Q

What is the start codon?

A

AUG

48
Q

What are some stop codons?

A

UAA, UAG, UGA

49
Q

What is the coding strand?

A

The DNA strand that has codons in their DNA form

50
Q

What is the template strand?

A

The strand read by RNA polymerase to make a complementary RNA strand

51
Q

Only __% of our genome encodes for proteins.

A

1

52
Q

25% of our genome consists of what?

A

Exons and introns

53
Q

The remaining 75% of our genome is what?

A

Spacer DNA or intergenic DNA

DNA between genes that can be unique or repetitive (Outside of DNA sequence of a gene)

54
Q

What is the function of the TATA box?

A

TATA box binding proteins recognize TATA sequence and promotes transcription.

55
Q

What are DNA-only transposons?

A

Cut-and-paste gene from one area of genome to another (Fossilized in our genome and no longer active)

56
Q

What are the two types of retrotransposons?

A

Retroviral-like: Copy-and-paste genes also fossilized in our genome

Nonretroviral: Copy-and-paste genes; some sequences are still active and jump around the genome

57
Q

What are retrotransposons derived from?

A

Viral genetic material integrating into our own genome

58
Q

Retrotransposons make up about __% of our genome.

A

40

59
Q

Nonretroviral retrotransposons are divided into what two groups? Explain.

A

LINEs: Long interspersed nuclear elements
Sequence encodes its own RNA binding protein and reverse transcriptase/endonuclease

SINEs: Short interspersed nuclear elements
Does not code for proteins and relies on L1’s reverse transcriptase

60
Q

What is the only LINE left in the human genome?

A

L1 element

61
Q

What is the most common SINE?

A

Alu

62
Q

What are pseudogenes?

A

Gene copies that have mutations that inactivate gene expression

63
Q

Where does replication begin?

A

Origin

64
Q

What are the substrates and products of a reaction with DNA polymerase?

A

Substrates: Template strand and dNTP

Products: Nascent strand and pyrophosphate

65
Q

Before DNA polymerase can bind DNA, it needs a ______.

A

primer

66
Q

How does DNA polymerase work?

A

It catalyzes the formation of phosphodiester bonds between the dNTP and growing strand.

67
Q

What are the four DNA polymerases in prokaryotes?

A

DNA polymerase I
DNA polymerase II
DNA polymerase III
Translesion polymerases

68
Q

What are the six DNA polymerases in eukaryotes?

A

DNA polymerase alpha
DNA polymerase beta
DNA polymerase gamma
DNA polymerase delta
DNA polymerase epilson
Translesion polymerases

69
Q

What is the function of DNA polymerase I?

A

DNA polymerase I: Essential; used in Okazaki fragment joining

70
Q

What is the function of DNA polymerase II?

A

DNA polymerase II: Used mainly in DNA repair

71
Q

What is the function of DNA polymerase III?

A

DNA polymerase III: Used for both leading and lagging strand replication

72
Q

What is the function of translesion polymerases?

A

Translesion polymerases: Used to polymerize over damaged bases

73
Q

What is the function of sliding clamps?

A

The clamp binds to DNA polymerase and makes it processive, keeping it bound to DNA.

74
Q

Sliding clamps are loaded by what?

A

Clamp loaders

75
Q

What is the function of primase?

A

It’s a type of RNA polymerase that creates an RNA primer on DNA, so DNA polymerase can bind.

76
Q

What is the function of DNA ligase?

A

DNA ligase joins the Okazaki fragments.

77
Q

What is the function of DNA helicase?

A

DNA helicase unwinds the DNA by circlng one of the strands and traveling along that to unwind the helix and force apart the H bonds.

78
Q

What is the function of single-strand binding protein?

A

It prevents strands from rewinding and folding up into multiple hairpin loops.

79
Q

What is the function of topoisomerase?

A

It relieves supercoiling by allowing the DNA to rotate and relieve accumulated strain.

80
Q

How does DNA polymerase make only a few mistakes?

A

Most DNA polymerases have a 3’-5’ proofreading exonuclease that removes mismatched bases.

81
Q

How is DNA replication initiated in bacteria?

A

Mutiple copies of membrane associated initator protein (DnaA) is bound to the DNA.

82
Q

What controls origin firing in prokaryotes?

A

Methylation: Hemimethylated (one strand) origins are resistant to initiation. Only fully methylated origins are competent for initiation.

83
Q

What does the protein Tus do?

A

Tus binds to the Ter sequences and acts as a funnel, allowing the replication fork to only go forward.

84
Q

Summarize the initation of DNA replication in prokaryotes.

A
  1. Origin is methylated.
  2. Initiator proteins bind.
  3. AT-rich region structurally opens up.
  4. Helicases are loaded to AT-rich region and activated.
  5. DNA primase is loaded and activated.
  6. RNA primers enable DNA polymerase activation.
85
Q

Summarize the termination of DNA replication in prokaryotes.

A
  1. Tus proteins let the replication complex pass one-way only.
  2. Both replication complexes reach the middle and can’t pass each other.
86
Q

What is the difference between eukaryotic and prokaryotic DNA in regards to replication origins?

A

Eukaryotic chromosomes are linear with multiple replication origins.

Prokaryotic DNA is circular with one origin.

87
Q

What are the two components of yeast replication origins?

A
  1. A sequence where a protein complex (ORC) binds
  2. An AT rich region that can be easily unwound
88
Q

DNA is replicated once during the __ phase of the cell cycle.

A

S

89
Q

The ORC complex is _____ and binds to thousands of locations along our chromosomes.

A

conserved

90
Q

Name two ways that re-replication is prevented.

A
  1. Origins are first “licensed” by the assembly of a pre-replication complex (pre-RC) under conditions that prevent initiation.
  2. A subset of the pre-RCs are activated (origin activation) under conditions that prevent pre-RC formation.
91
Q

Name the five steps to origin licensing.

A
  1. ORC (origin recognition complex) binds to the origin.
  2. CDC6 and CDT1 load the helicase complex constituted by a double hexamer of MCM2-MCM7.
  3. The hexameric Mcm helicase is loaded by the loaders Cdc6 (binds to ORC) and Cdt1 (binds to MCM) to form the pre-replication complex (pre-RC).
  4. Pre-RC is inactive and wrapped around double-stranded DNA in an stable complex.
  5. Cdc6 is phosphorylated and then degraded by the proteasome.
92
Q

What happens during origin activation?

A

Activation of the helicase complex requires phosphorylation of the MCM by Dbf4-dependent kinase (DDK)

93
Q

What happens during origin firing?

A

Once the MCM helicase complex is phosphorylated it “fires” splitting the complex into two sub complexes that move away from each other establishing the replication forks.

94
Q

What is replication timing?

A

The temporal order in which distinct segments of the genome are replicated.

95
Q

What are the ends of our chromosomes called?

A

Telomeres

96
Q

What is the function of ribonucleoprotein?

A

It brings its own RNA to use as a template.

97
Q

What is the function of telomerase?

A

It adds telomeric repeats (TTGGGG) to maintain telomere length.

98
Q

In humans, the mutation of telomere maintenance genes results in what?

A

Dyskeratosis congenita (DC)

Correlates with extreme telomere shortening

99
Q

Cells do what when their telomeres become too short?

A

They senesce (“dye”)

100
Q

What is the Hayflick limit?

A

The number of times a normal cell can divide

101
Q

Only __% of our DNA comprise codons.

A

1.5

102
Q

Two copies of each of your DNA molecules are not the exactly same (98-99% the same). Most of these differences are inconseqential and are called what?

A

Polymorphism

103
Q

Most polymorphisms are what?

A

Single base pair differences called SNP (single nucleotide polymorphism)

104
Q

What is an example of transition and transversion?

A

Transition: A:T to G:C (Purine for purine)

Transversion: A:T to T:A (Purine for pyrimidine)

105
Q

What are CNVs or copy number variations?

A

Segmental duplication; variations of kb length segments in chromosomes

106
Q

What is a translocation?

A

The moving/exchanging of chromosome sections

107
Q

What’s a missense mutation?

A

The substitution of a codon for another that codes a different amino acid

108
Q

What’s a nonsense mutation?

A

Substitution of a codon encoding for an amino acid with a stop codon

109
Q

What’s a frameshift mutation?

A

Insertion or deletion of a nucleotide in which the number of deleted base pairs is not divisible by three

110
Q

Causes of mutations?

A
  1. Chemical environment of cells (Oxygen/oxidation)
  2. Environmental insults (UV light, x-rays)
  3. Enzymatic mistakes
111
Q

What are the repair mechanisms for lesions affecting a single strand?

A
  1. Mismatch repair (MMR)
  2. Base excision repair (BER)
  3. Nucleotide excision repair (NER)
  4. Translesion polymerases
112
Q

What are the repair mechanisms for lesions affecting both strands (double strand breaks)?

A
  1. Homologous recombination (HR)
  2. Non-homologous end joining (NHEJ)
113
Q

What happens when there is U in DNA?

A

Uracil DNA glycosylase detects and removes it when present.

114
Q

What is the mechanism of BER?

A

Poorly matched bases are recognized and rotated out of the helix, recognized by glycosylase.

115
Q

How do translesion polymerases work?

A

The sliding clamp releases DNA polyemerase, and translesion DNA polymerase is loaded and bypasses the DNA damage, continuing synthesis.

116
Q

What are the three functions of homologous recombination?

A
  1. Assists in DNA repair
  2. Links sister chromosomes to properly segregate them between self and daughter cells
  3. Source of DNA exchange and genetic diversity
117
Q

What do RecA and Rad51 do?

A

They use the 3’-end of the broken DNA to invade the homologous duplex in HR. They can take a broken DNA end and search for a homologus duplex.

BIOC4332: Biochemistry II (7 decks)

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