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Molecular Genetics > Exam 2 > Flashcards

Exam 2 Flashcards

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1
Q

histones

A

major DNA binding proteins that DNA wrap around

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2
Q

micrococcal endonuclease

A
  • endonuclease from the bacterium Micrococcus
  • treat eukaryotic DNA with this enzyme
  • run product on an agarose gel

result: DNA banding pattern in multiples of 200bp

why? - nuclease cutting between regular complexes of 200np of DNA + protein

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3
Q

the nucleosome

A
  • DNA wrapped around protein core (histones)
  • the basic structure of chromatin
  • consists of ~200 bps of DNA and an octamer of histone proteins
  • is linked to other nucleosomes by Linker DNA
    -endonuclease cleaves linker DNA and releases individual nucleosomes from chromatin
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4
Q

the DNA in the nucleosome

A
  • DNA is wrapped around the outside surface of the protein octamer
  • the length of DNA per nucleosome varies for tissues or species from 154 to 260 bp
  • nucleosomal DNA is divided into
    -the core DNA (145-147bp)
    -linker DNA (7-115bp)
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5
Q

structure of the nucleosome

A
  • the nucleosome is a cylinder
  • DNA organized into ~one and two-thirds turns around the surface
  • DNA enters and exits on the same side of the nucleosome
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6
Q

protein histones in the nucleosome

A
  • small proteins rich in arginine and lysine residues
    • charges&raquo_space; it binds to the DNA
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7
Q

the histone octamer

A
  • two copies each of H2A, H2B, H3, and H4
  • core histones are HIGHLY evolutionarily conserved in eukaryotes
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8
Q

core histones

A
  • H32 - H42 tetramer + two H2A-H2B dimers
  • all histone N-terminal tails and H2A and H2B C-terminal tails extend out from the histone core
  • tails are site for covalent modification
    -important in chromatin function
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9
Q

Histone H1

A
  • H1 is associated with linker DNA
  • located at the point where DNA enters or exits the nucleosome
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10
Q

post-translational modification of proteins

A
  • protein function can be modified by enzymatically adding small molecules to the protein
  • changes protein shape
  • changes protein reactivity (+ or -), etc.
  • phosphorylation - adding PO4
  • methylation - adding CH3
  • acetylation - adding acetyl group
  • ubiquitylation - adding ubiquitin
  • Sumoylation - adding small protein SUMO

ALL of these modifications are reversible

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11
Q

nucleosomes are covalently modified

A

combinations of specific histone modifications define the function of local regions of chromatin

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12
Q

can multiple modifications sites in histones have more than one type of modification?

A

yes
- most have a single, specific type of modification, but some sites can have more than one type

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13
Q

functional effects of modifications: examples

A

acetylation of the lysine

  • reduces the positive charge on the lysine
  • causes decreased interaction with DNA
  • acetylation of histones is associated with gene activation

methylation of lysine

  • lysine retains the positive charge
  • associated with gene inactivation
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14
Q

bromodomain

A
  • proteins with the bromodomain in their structure can bind to histones that are acetylated
    -allows transcription enzymes to bind
  • proteins have different domains that can recognize acetylated, phosphorylated, etc. modified amino acids
  • this is how proteins recognize and interact with DNA
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15
Q

primary structure of chromatin

A
  • a 10-nm fiber which consists of a string of nucleosomes
  • “beads on a string”
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16
Q

secondary structure of chromatin

A
  • formed by interactions between neighboring nucleosomes
  • 10 nm strands may pack together closely to form densely packed higher levels of DNA folding
  • would allow the DNA to be accessible for transcription
  • easily reversible
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17
Q

higher order chromatin structures

A

secondary chromatin fibers

  • folded into higher-order, 3D structures that comprise interphase or mitotic chromosomes
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18
Q

chromosome

A
  • a discrete unit of the genome carrying many genes
  • each chromosome consists of a very long molecule of duplex DNA
  • plus approximately equal mass of proteins
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19
Q

bacterial chromosome

A
  • bacterial chromosome is a single large circular DNA
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20
Q

where is bacterial chromosome located?

A

nucleoid

  • the DNA is bound to proteins
  • the DNA is NOT enclosed by a membrane
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21
Q

the bacterial genome can be ___ or ____

A

relaxed or supercoiled

  • supercoiled - coiling of the circular DNA so that it crosses over its own axis many times
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22
Q

eukaryotic chromatin

A

interphase chromatin
each chromosome is a long dsDNA

  • heterochromatin - found in the edges of the nucleus and around the nucleolus
  • euchromatin - less densely packed DNA, active genes

mitosis chromatin

  • chromosomes are 5-10 times more condensed than in interphase
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23
Q

chromosome scaffold

A
  • a proteinaceous structure in the shape of a sister chromatid pair, generated when chromosomes are depleted of histones
  • eukaryotic DNA is attached to a protein scaffold
  • in metaphase chromosomes, supercoiled DNA is attached to a protein scaffold
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24
Q

chromosomes can be stained to have banding patterns

A
  • protease treatment and then staining
  • stains the chromosomes to have a series of striations, called G-bands
  • yields a characteristic banding for each chromosome
  • each band can include many hundreds of genes
  • allows us to study different regions of the chromosomes
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25
Q

polytene chromosomes

A
  • some dipterans (like Drosophila melanogaster, the fruit fly) have huge chromosomes in interphase
    -found in the salivary gland
    cells
  • generated by successive replications of a chromosome without separation of the replicated chromosomes in mitosis
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26
Q

cytological map

A
  • can label gene-specific probes to identify where specific genes are on the banding
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27
Q

puffs

A
  • sites of gene expression/activity on polytene chromosomes expand to give “puffs”
  • show that gene expression requires that the DNA must unwind
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28
Q

homologous chromosomes

A

pairs of chromosomes similar in size, shape and gene content

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29
Q

chromatid

A

one of the two DAN strands in a replicated chromosome

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30
Q

sister chromatids

A

chromatids from the same chromosomes
- join at the centromere

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31
Q

interphase

A

chromosomes replicate

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32
Q

prophase

A

chromosomes/sister chromatids condense and attach at the kinetochore to the mitotic spindle microtubules

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33
Q

metaphase

A

sister chromatids line up in center

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34
Q

anaphase

A

sister chromatids pulled to opposite opposite poles of the cell

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35
Q

telophase

A

sister chromatids in opposite poles and nuclear membrane reforms

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36
Q

cytokinesis/cell division

A

membrane pinches in middle to separate into two daughter cells
- cytoplasm is divided

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37
Q

cohesins

A

proteins that hold “glue” together sister chromatids

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38
Q

centromere

A
  • a constricted region of a chromosome (the DNA) that:
    • has unique DNA sequences and proteins not found anywhere else in the chromosome
  • is where the sister chromatids attach to the mitotic spindle microtubules
  • centromere region also contains the kinetochore
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39
Q

kinetochore

A

the proteins responsible for attaching to the spindle apparatus microtubules

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40
Q

the centromere

A

DNA is wrapped around:
- normal histone H3
- or a centromere-specific histone H3 variant, Cen-H3

  • Cen-H3 allows binding of kinetochore proteins to form the kinetochore
  • also has heterochromatin that is rich in satellite DNA sequences (repetitive DNA)
    -function of the repetitive centromeric DNA is not known
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41
Q

replication overview

A
  1. supercoiled DNA must first be relaxed
  2. initiation
  3. elongation
  4. joining and/or termination
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42
Q

telomers

A

higher organisms with linear chromosomes

  • have long series of short tandem repeated sequences called telomers
    -may be 100-1000 repeats
  • human telomere repeats are
    -TTAGGG-3’
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43
Q

why do we need telomers?

A
  • DNA replication leaves a 3’ unreplicated end on one of the replicated DNA strands
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44
Q

telomeres are essential for survival

A
  • DNA replication leaves one strand with unreplicated end
  • next round of replication results in a shorter DNA
    -due to shorter DNA template
  • and so on until genes near the ends are not replicated
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45
Q

telomeres are synthesized by telomerase

A

telomerase uses:

  • the free 3’-OH of the telomeric strand
  • its own RNA template of 3’-AAUCCC-5’
  • a reverse transcriptase
  • adds tandem repeats (5’-TTAGGG-3’ in humans) to the 3’ end at each chromosomal terminus
  • extends the ends of the chromosomes to solve the so-called end replication problem
  • telomerase uses RNA to complementary bind to end of telomer
  • then the RNA polymerase of telomerase adds the complementary dNTPs to extend the end
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46
Q

how does telomerase solve the problem?

A
  • repeat this step many times until it is long enough for the DNA polymerase to do another Okazaki fragment
  • then DNA polymerase can fill in the gap

RESULT
* extends the end of the chromosome back to how long it should be

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47
Q

telomeres are essential for survival

A

telomerase is expressed:

  • in actively dividing cells
    -stem cells
    -during development
  • not expressed in quiescent cells
    -most other cells in the body
  • loss of telomeres results in senescence
    -cell dies
  • cancer cells often have telomerase
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48
Q

telomer ends are “sticky”

A
  • can be recognized by the DNA repair enzymes as a broken chromosome
  • could be added to the end of another chromosome
  • must have a way to prevent this!
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49
Q

telomers form circular loops at the end of chromosomes

A

the protein TRF2

  • allows the 3’ telomer unit to invade into its homolog in an upstream region of the telomere
  • forms the t-loop
  • t-loop prevents DNA repair enzymes from recognizing the 3’ end as a DNA break
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50
Q

telomeric binding proteins

A

(TRF1, TRF2, Rap1, TIN2, TPP1, and POT1)

  • form the Shelterin complex
  1. function to protect the telomers from DNA damage repair
  • can result in chromosome ends sticking to other chromosomes
    2. also function to control telomer length by inhibiting telomerase
  • the more shelterins bind, the less telomerase can bind to add more DNA
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51
Q

replicon

A
  • a unit of the genome in which DNA is replicated
  • bacteria usually only have one replicon
  • eukaryotes can have many replicons
  • each replicon contain an origin for initiation of replication
    origin- a sequence of DNA at which replication is initiated
    terminus - a segment of DNA at which replication ends
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52
Q

Meselson and Stahl experiment 1958

A
  • grow organism on “heavy” 15N to label DNA
  • then grow on medium with “light” 14N
  • allow DNA replication to occur
  • isolate DNA and ultracentrifuge in dense medium
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53
Q

what meselson and stahl found

A

parental DNA
- all heavy DNA

1st generation
- all medium hybrid DNA

2nd generation
- mixture of light and medium DNA

suggests DNA is replicated semiconservatively
- resulting replicated DNA strand is one parental and one new

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54
Q

semiconservative replication

A
  • replication accomplished by separation of the strands of a parental duplex
  • with each strand then acting as a template for synthesis of a complementary strand
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55
Q

replication bubble

A

in electron microscopy:

  • a replicated region appears as a bubble within nonreplicated DNA
  • a replication fork is initiated at the origin and then moves sequentially along DNA
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56
Q

when is replication unidirectional?

A

when a single replication fork is created at an origin

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57
Q

when is replication bidirectional?

A

when an origin creates two replication forks that move in opposite directions

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58
Q

bacterial DNA is usually a singular circular replicon

A
  • bacterial replicons are usually circles
  • they replicate bidirectionally from a single origin
  • the origin of E.coli is oriC
    -245 bp in length
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59
Q

the eukaryotic cell cycle

A

cells cycle between:

  • mitotic (M) phase = when cells actually divide
  • interphase = the non-dividing phase
    -the chromosomes are generally uncoiled in euchromatin and heterochromatin
    cells spend most of their time in interphase
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60
Q

chromosome replication occurs:

A

only during interphase
- not during mitosis

DNA synthesis occurs in the S phase of interphase

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61
Q

the gap phases

A

interphase has two gap phases

  • no DNA synthesis occurs
  • G1 and G2
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62
Q

G1 phase

A

cell growth

  • everything in the cell is doubled except DNA
  • cells prepare for DNA synthesis
  • make all enzymes for DNA synthesis
63
Q

G2 phase

A
  • after DNA synthesis
  • preparing for mitosis

organelles replicate and additional cytoplasm is made in G1 and G2

64
Q

G1 checkpoint

A

progression from G1 into S phase is tightly controlled

checkpoint
a control mechanism that prevents the cell from progressing to the next stage unless specific goals and requirements have been met

  • no damage to the DNA
  • must be a certain amount of cell growth
65
Q

each eukaryotic chromosome contains many replicons

A
  • too much DNA for just one origin/replicon
  • not just DNA, but DNA + histones
  • eukaryotic replicons are 40 to 100 kb in length
  • multiple origins of replications that ultimately merge during replication
66
Q

individual replicons are activated at characteristic times during what phase?

A

S phase

  • early replicating
  • late replicating
  • see ~100 to ~300 replication foci when stained
  • eukaryotic replication takes over 6 hours to complete
    -S phase
67
Q

origin recognition complex (ORC)

A

~400 kDa protein complex

  • highly evolutionarily conserved in eukaryotes
  • binds to eukaryotic origin
  • is cis-acting
    causes DNA replication initiation on the strand of DNA to which it binds
68
Q

how is replication initiated? - experiment

A
  • inject nucleus into a Xenopus (frog) egg
  • DNA replicates
  • but it cannot replicate again
    -occurs only once
  • but if you permeabilize the nuclear membrane before injection
    -DNA will be replicated more than once
69
Q

permeabilizing the nuclear membrane

A

allows new protein made in the cytoplasm to enter the nucleus and initiate replication again

if you permeabilize before injection:

  • suggests that a protein is found in the nucleus that allows initiation of replication
  • once replication occurs, the protein is used up or inactivated
70
Q

licensing factor

A

controls eukaryotic re-replication

  • is necessary for initiation of replication at each origin
  • present in the nucleus prior to replication, but is removed, inactivated, or destroyed by replication
71
Q

licensing factor cont.

A
  • active licensing factor is present in the nucleus
  • replication inactivates it
  • new licensing factor is made in the cytoplasm
  • but it cannot get into the nucleus through the nuclear membrane
  • breakdown of the nuclear membrane in mitosis allows new licensing factor to associate with the nuclear material
72
Q

what is this licensing factor?

A
  • cyclins and cyclin-dependent kinases (Cdk)
  • they are Helicase Activating Proteins
  • Cdk enzymes must bind cyclins to be active
    -becomes the “ON-OFF” switch
73
Q

helicase

A

the enzyme that separates the strands of DNA for replication - a crucial initial step for replication

74
Q
A
75
Q

topoisomerase

A
  • relieves stress coiling
  • relaxes the supercoil
  • moves in front of the replication fork
76
Q

Type I topoisomerase

A

ssDNA nick to allow cut strand to rotate around the uncut strans

77
Q

Type II topoisomerase

A

both strands of DNA are cut and the intact strand is passed through the cut to relieve the tension

78
Q

initiation

A
  • origin recognition complex binds to the origin
  • DNA strands must be separated and stabilized
    -creates replication bubble
  • DNA synthesis is initiated at the replication forks
79
Q

elongation

A

replisome
multiprotein complex that assembles at the replication fork to undertake synthesis of DNA

  • it contains DNA polymerase and other enzymes
  • is assembled de novo (at that time and place) into the replisome complex
  • replisome moves along the DNA as the DNA is unwound
  • daughter strands of DNA are synthesized
80
Q

joining and/or termination

A
  • joining of replication forks or termination of DNA synthesis
  • separation of the duplicated chromosomes
81
Q

two types of DNA polymerases

A
  1. DNA-dependent DNA polymerase (or DNA polymerase)
  2. DNA Repair Polymerase
82
Q

DNA-dependent DNA polymerase (or DNA polymerase)

A
  • also known as DNA replicase
  • is only one subunit of a large protein assembly called the holoenzyme
  • responsible for semiconservative replication
83
Q

DNA repair polymerase

A
  • responsible for excising damaged DNA bases
  • then synthesizing new DNA to replace the excised DNA
84
Q

all DNA polymerases:

A
  • synthesize DNA antiparallel from 5’ to 3’
  • require a template that is 3’ to 5’
    -nucleotide choice is determined by complementary base pairing with the template
  • require a free 3’-OH to add nucleotides to
  • polymerase makes the phosphodiester bond
85
Q

bacterial DNA polymerases

A
  • bacterial (& eukaryotic) cells have several different DNA polymerase enzymes
  • DNA polymerase III - is the bacterial DNA polymerase
  • does semiconservative replication
86
Q

repair DNA polymerases

A

DNA polymerase I

  • repair of damaged DNA

DNA polymerase II

  • required to restart a replication fork when the process is stopped by DNA damage

DNA polymerase IV and V

  • allow replication to bypass some types of DNA damage
  • error-prone polymerases
87
Q

DNA polymerase I

A
  • only polymerase that can do Nick Translation
  • DNA polymerase I has a 5’-3’ exonuclease activity
  • can remove nucleotides in front of the polymerase
  • it can also recognize a single strand nick in the DNA
  • inserts a 5’-3’ exonuclease removes nucleotides ahead of the polymerase as it makes new DNA
88
Q

Klenow Fragment

A
  • DNA polymerase I cleaved with an enzyme

yields 2 parts:
1. larger fragment = polymerase + proofreading enzyme

  • used in the laboratory to synthesize DNA
  1. smaller fragment is the 5’-3’ exonuclease
89
Q

fidelity of DNA replication

A
  • how well does the polymerase copy the DNA?
  • substitutions = inserting the wrong base
  • frameshifts = inserting an extra nucleotide or deleting a nucleotide
  • DNA polymerases have a proofreading ability
    -due to a 3’ to 5’ exonuclease
    • is used to excise incorrectly paired bases
      -detects A-C or A-A mispairing
90
Q

DNA synthesis

A
  • dNTP binds to template base by complementary binding
  • polymerase catalyzes joining of phosphate to 3’OH of primer
  • using energy of pyrophosphate release
91
Q

DNA polymerases have proofreading ability

A
  • if wrong base is inserted and added to the chain
    -causes a warp in the chain that slows the polymerase
  • allows the 3’-5’ exonuclease to back up and remove that wrong base
  • polymerase then has the chance to add the right base
  • the fidelity of replication is improved from proofreading by a factor of ~100
92
Q

so DNA polymerase I has:

A
  • polymerase to synthesize DNA 5’-3’
  • 3’ to 5’ exonuclease for proofreading
  • 5’-3’ exonuclease that can allow Nick Translation
    -removes bases in front of the polymerase

(note: all bacterial DNA polymerases have the 3’-5’ proofreading exonuclease)

93
Q

DNA polymerases have a common structure

A

the right hand

  • DNA lies across the “palm”
    -catalytic site
  • a groove is created by the “fingers” and “thumb”
  • DNA slides through the groove as synthesis continues
94
Q

DNA synthesis differs on each DNA template

A

leading strand synthesis

  • DNA synthesis goes 5’-3’
  • DNA pol advances continuously when it synthesizes the leading strand
95
Q

problem is the “lagging strand”

A
  • DNA synthesis MUST go 5’-3’
  • as DNA synthesis on the lagging strand beings
    -the leading strand is still moving ahead
    -this leaves a gap on the lagging strand
    -so the pol must bind again near the fork and start a new strand
96
Q

lagging strand DNA synthesis is discontinuous

A
  • lagging strand is synthesized in many fragments
  • called Okazaki fragments
  • these Okazaki fragments must then be connected together
97
Q

helicase

A

separates (or melts) the DNA duplex

  • E.coli has 12 different helicases
  • most are multimeric
  • most common is a Hexamer
98
Q

helicase action

A
  • helicase encircles a ssDNA strand
  • alternates between a dsDNA binding conformation and a ssDNA binding conformation
  • this separates the DNA strands
  • requires the energy of ATP hydrolysis
99
Q

unwinding of DNA exposes ssDNA

A

must:

  • protect the ssDNA
  • prevent it from re-annealing with the other strand
  • leading strand DNA is synthesized quickly
    -no real need to protect the ssDNA
  • lagging strand DNA must wait until there is sufficient space to begin an Okazaki fragment synthesis
    -must protect this ssDNA
100
Q

single-stranded binding proteins (SSB)

A
  • SSBs bind to the lagging strand ssDNA after unwinding by helicase
  • SSB binding is cooperative
    -binding of one SSB enhances the binding of subsequent SSBs
101
Q

DNA synthesis requires a primer

A
  • all DNA pol require a 3’-OH priming end to initiate DNA synthesis
102
Q

RNA primer is provided by primase

A
  • small RNA polymerase
    -makes RNA 5’-3’
    -does not require a free 3’-OH
  • makes an RNA primer for DNA synthesis
  • associates with the oriC complex in bacteria
    -which binds to the origin
  • recognizes the origin and synthesizes ~10 bases of RNA complementary to the DNA at the origin
103
Q

priming DNA synthesis at the origin

A

requires:

  • binding of the helicase to unwind the DNA
  • binding of SSBs to protect and prevent re-annealing
  • primase binds and makes a complementary 10 bases of RNA
104
Q

coordinating synthesis of the lagging and leading strands

A
  • leading strand is synthesized continuously
  • lagging strand is synthesized discontinuously
    • polymerase must continually detach and move back to the replication fork
  • E.coli uses the same polymerase for both (circular chromosome)
    • eukaryotes use different enzymes for leading and lagging strands
105
Q

DNA polymerase III Holoenzyme (replisome) subunits

A
  • 2-DNA polymerase III catalytic cores
    -a catalytic subunit (alpha)
    -and a 5’-3’ proofreading subunit
  • 2 clamps
    -ensures processivity = that the polymerase stays with the DNA
  • a clamp-loader/dimerization complex
    -loads the DNA into the clamps
    -brings the 2 polymerase complexes together
106
Q

DNA polymerase holoenzyme assembly

A
  • a clamp loader complex assists in loading the circular clamp around the DNA strand
  • catalytic core polymerase associates with each template strand
  • the dimerization subunits of the clamp loader helps in dimerization of the 2 core polymerases
  • the clamp loader stays associated with the polymerase on the lagging strand
  • it will be needed to reload the polymerase after each Okazaki fragment
107
Q

the “sliding” clamp

A
  • the clamp forms a ring around the DNA
  • holds the polymerase strongly to the DNA
    • makes the polymerase
      highly processive =
      stays with the DNA
  • the clamp associated with the polymerase on the lagging strand dissociates at the end of each Okazaki fragment and reassembles for the next fragment
108
Q

DNA synthesis

A

see ch. 11 notes

109
Q

status at this point

A
  • leading strand continuing to replicate DNA
  • lagging strand
    -newly synthesized Okazaki fragment terminate just before the previous Okazaki fragment
    -must remove the RNA primer
    -synthesize DNA to replace the RNA primer
    -seal the last phosphodiester bond between the Okazaki fragment
110
Q

removing the RNA primer (in bacteria)

A
  • DNA polymerase III dissociates leaving a gap and the RNA primer
  • DNA pol I uses the “nick” to attach and uses its 5’-3’ exonuclease to remove the RNA primer and replace with DNA
111
Q

ligation

A
  • DNA pol I is released leaving a “nick” between the Okazaki fragments
  • DNA ligase seals the remaining phosphodiester bond at the nick
  • DNA ligase uses energy from ATP to catalyze the reaction
112
Q

DNA replication in eukaryotic cels

A

eukaryotes have 3 polymerases for DNA synthesis

  • DNA polymerase alpha/primase initiates synthesis of new strands
    -has DNA pol activity
    -and RNA pol activity for making its own primer
  • DNA polymerase ε synthesizes the leading strand
  • DNA polymerase δ synthesizes the lagging strand
  • all 3 are linked in the replisome
113
Q

eukaryotic DNA replisome is similar

A
  • helicase (MCM) binds the DNA
  • pol α/primase detaches (but remains with replisome)
  • clamo loader (RFC) mediates binding of the clamp (PCNA) to the ssDNAs
  • clamp and pol δ release to make another Okazaki fragment
  • but in subsequent okazaki fragments, pol δ continues and into the RNA primer
114
Q

primer removal and ligation

A
  • polδ displaces the RNA primer
  • leaving a flap of RNA
  • FEN1 nuclease removes the RNA primer flap
  • polδ then fills the gap
  • DNA ligase I seals the remaining nick
115
Q

what if the polymerase encounters an error or nick in the DNA?

A

DNA synthesis stops and replication fork may collapse

3 options:

  • cell death (not really acceptable)
  • skip over the lesion = lesion bypass
  • repair by recombination
116
Q

lesion bypass requires polymerase replacement

A
  • the replication fork stalls
  • the replication complex must be replaced by error-prone polymerases
    -DNA pol IV or V (for bacteria)
    -can copy through the error and incorporating the error
  • error-prone polymerases removed
  • restart DNA synthesis with primosome
    -primase and polymerase complex that restarts synthesis
  • then primosome is replaced by the replisome
117
Q

recombination to repair the lesion

A
  • excise damage
  • information from the undamaged other strand is used to repair the damaged sequence
  • DNA synthesis is restarted
  • DNA repair is used to fix the gap later
118
Q

termination of replication

A

bacteria:

  • the two replication forks meet halfway around the circle
  • ter sites that cause termination if the replication forks go too far
119
Q

termination of replication

A

eukaryotes:

  • telomer region
  • leading strand completes replication
  • lagging strand has unreplicated end
120
Q

simple repairs

A

includes:
1. direct reversal of DNA damage
2. base excision repair
- remove the base and replace with appropriate base
3. nucleotide excision repair
- remove and replace the whole nucleotide
4. mismatch repair
- requires determination of “new” and “old” DNA strands

121
Q

recombinant repairs

A
  • repair mechanism that retrieve an undamaged sequence from the other replicated DNA strand
  • nonhomologous end joining
  • translesion/error-prone repair
122
Q

replication errors

A
  • missed by the 3’-5’ proofreading exonuclease
  • results in a major distortion of the DNA strands
  • repaired by the mismatch repair system
    -if not, becomes a permanent change in the DNA
123
Q

spontaneous base deamination mutation

A

e.g. spontaneous deamination of cytosine to uracil
-can also happen to other bases

  • creates a mismatched U-G pair
  • results in a minor structural distortion of the DNA
  • uracil is preferentially removed by base excision repair
124
Q

loss or removal of a base

A

depurination

  • spontaneous loss of a base by hydrolysis
  • blocks DNA replication and transcription
  • corrected by nucleotide excision repair system
125
Q

mutagens

A
  • substances that increase the rate of mutation by inducing changes in the DNA sequence
  • either directly or indirectly
126
Q

carcinogen

A

a substance that promotes the formation of cancer

127
Q

UV light

A
  • UV light at ~260nm is absorbed by the bases
  • can cause a photochemical fusion of two pyrimidine bases side-by-side
  • thymine-thymine dimer
    -covalent bonds
  • blocks replication and transcription
  • corrected by nucleotide excision repair
128
Q

alkylating agents

A
  • includes mutagens like nitrosamines
  • found in tobacco products or produced from some food preservatives like nitrates

e.g. methylation of guanine
- creates a bulky base that distorts the DNA
- inhibits replication and transcription

129
Q

methyltransferase

A

used for direct reversal

  • transfers the methyl group to the methyltransferase
130
Q

reactive oxygen species

A
  • oxidizing agents generated by ionizing radiation or chemical agents
  • generates free radicals
  • superoxides, hydroxyl radicals, hydrogen peroxide, etc.
  • oxidation of guanine
  • causes base pairing with adenine
  • correct by mismatch repair
131
Q

ionizing radiation

A
  • gamma radiation or X-rays
  • directly ionizes the deoxyribose
    (indirectly causes reactive oxygen species)
  • causes double strand DNA breaks
  • kills rapidly proliferating cells (cancer treatment)
  • corrected by recombinant repair
132
Q

direct reversal of DNA damage

A
  • alkylating agents resulting in methylation of guanine to methyl-guanine
  • methyltransferase
    • transfers the methyl group to the methyltransferase
133
Q

base excision repair

A
  • for spontaneous base deamination
    • cytosine deamination to uracil
  • also alkylated bases
  • causes the removal of an individual damaged base
  • glycosylases cleave the bond between the deoxyribose and the base
  • results in a removal of a base
134
Q

DNA pol β

A
  • eukaryotic DNA pol specific for base excision repair
  • replaces the nucleotide
135
Q

the nucleotide excision repair system in bacteria

A
  • removes mispaired or damaged bases then synthesizes a new stretch of DNA to replace them
  • used for almost all excision repair in E.coli
136
Q

E. coli Uvr system

A
  • UvrAB dimer recognizes the damage
  • UvrA is released
  • UvrC joins UvrB
  • UvrBC cuts DNA strand above and below the damage
  • UvrD (helicase) then unwinds DNA
  • DNA pol I then binds
  • removes the damaged area as it synthesizes new DNA
  • DNA ligase seals the nick
137
Q

excision repair system - eukaryotic

A

Xeroderma pigmentosum (XP)

  • a human disease caused by mutations in any one of several nucleotide excision repair genes
  • hypersensitivity to sunlight and UV light
  • results in skin diseases and cancers
  • disease was helpful in understanding the excision repair system
138
Q

two major pathways in eukaryotes

A
  1. global genome repair
  2. transcription-coupled repair
139
Q

global genome repair

A

XPC protein detects the damage and initiates repair

140
Q

transcription-coupled repair

A
  • damage is recognized by the RNA polymerase during transcription
  • RNA pol stalls at the damage
  • the RNA pol is released
141
Q

both then use similar pathways

A
  • TFIIH transcription factor complex binds
  • helicase in complex unwinds the DNA
  • complex endonucleases cut DNA above and below damage
  • DNA pol δ/ε
    -eukaryotic replisome polymerases
  • excises and replaces the gap
  • DNA ligase seals the nick
142
Q

mismatch repair - bacteria

A
  • purpose is to repair mismatches immediately after DNA replication
  • relies on MutS complex
    • dimer that embraces the DNA
    • moves along the DNS looking for distorted DNA due to mismatched bases
143
Q

but how does MutS know which strand has the wrong base?

A
  • after DNA synthesis in bacteria
  • Dam Methylase methylates DNA at -GATC- sequences all along the DNA
  • but shortly after synthesis, new DNA is not methylated
  • so unmethylated DNA would have the mistake
    -the original template DNA would be methylated
144
Q

mismatch repair mechanism

A

see ch. 14 notes

145
Q

mismatch repair in eukaryotes

A
  • similar to E. coli
  • eukaryotic DNA is methylated BUT mismatch repair DOES NOT use methylation to identify the new strand
146
Q

how do eukaryotes recognize the correct DNA strand?

A
  • mismatch repair is coupled to the DNA synthesis machinery
  • uses nicked Okazaki fragments to ID new strands
  • also associated with clamp to determine the new strand
  • then uses a similar mechanism to remove and replace the mismatch nucleotide
147
Q

genetics

A

patterns of inheritance
- single genetic locus or just a few loci

148
Q

genome

A

an organisms complete set of DNA, including all of its genes

  • also includes mitochondrial and chloroplast genomes
  • extrachromosomal DNA and other forms of inheritance
149
Q

genomics

A

genome-wide studies via DNA or RNA sequencing + bioinformatics

150
Q

classical DNA sequencing: Sanger method

A

main principle: chain termination method based on terminating DNA synthesis one nucleotide at a time

components:
1. DNA primers (only one per sequencing rxn)
2. DNA pol
3. nucleotides (dNTPs)
4. labeled modified nucleotides dideoxynucleotides (ddNTPs)

151
Q

dideoxynucleotides (ddNTPs)

A

chain-terminating nucleotides, lacking a 3’-OH group required for the formation of a phosphodiester bond during DNA elongation.

incorporation of a dideoxynucleotide into the elongating DNA strand therefore terminates the extension, resulting in DNA fragments of varying length

152
Q

classical sanger method

A

see notes

153
Q

dye terminator sequencing

A
  • an easier way
  • sanger method based
  • non-radioactive
  • more automated
154
Q
A

Molecular Genetics (2 decks)

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