Uiuc Mcb 250 Exam 3

Front 1

Define mutant, mutation, and mutagen

Back 1

Mutant: an organism that differs from the parent or wildtype

Mutation: a stable and heritable change in the DNA sequence

Mutagen: a chemical or physical agent that induces a mutation

Front 2

What are the 2 types of point mutations?

Back 2

Transition and Transversion

Transition point mutations: substitution from a purine to a different purine (A to G, G to A), or a pyrimidine to a pyrimidine (T to C, C to T).

Transversion point mutations: substitution from a purine to a pyrimidine or vice versa.

Front 3

Which of the 2 types of point mutations is most common?

Back 3

Transition point mutations

Front 4

What 3 types of point mutations are discussed with respect to mutation consequences in the peptide sequence?

Back 4

Silent Mutations: a mutation that cannot be detected without sequencing the gene.

Missense Mutations: a mutation that changes the codon to specify a different amino acid.

Nonsense Mutations: a mutation that changes the codon to a STOP codon

Front 5

What are the two general types of oligonucleotide sequence mutations?

Back 5

Insertions and Deletions, which result in a reading frame shift.

Front 6

What are the three main factors which cause spontaneous mutation?

Back 6

Errors in Replication
-Tautomerization or incorrect proofreading

The inherent chemical instability of DNA
-Deamination of C
-Depurination (hydrolysis of the N-glycosidic linkage)

Oxdative Damage
-Oxygen reacts with DNA causing modified bases or single and double stranded DNA breaks

Front 7

Does a spontaneous mutation caused by tautomerization lead to a mutation?

Back 7

Sometimes it does. Initially, however, this is more correctly known as a base pair mismatch. If it is corrected before replication, no mutation will be inherited. However, if the mismatch is not repaired, one progeny of the cell will receive a chromosome with a mutation.

Front 8

General, where can methyl-directed mismatch repair be found?

Back 8

Eukaryotes DO NOT use methyl-directed mismatch repair. However, some prokaryotes, E. coli in particular, do.

Front 9

What is MutHLS?

Back 9

MutHLS is the protein complex which removes mismatch repairs in some prokaryotes, specifically discussed in E. coli methyl-directed mismatch repair.

Front 10

How does MutHLS mismatch repair work?

Back 10

MutS scans along the DNA independently until it finds a kink in the DNA. It then recruits MutL.

MutL is the linker protein which, once bound to MutS, binds to a MutH.

MutH binds to and is activated by MutS and MutL. Once activated, MutH, bound to a hemimethylated DNA site somewhere on either side of the mismatch. (This means MutH can act in either 5'-3' or 3'-5', and note that the distance could be anything from 50 to 1000 base pairs)

MutH

Front 11

Why is Deamination important in the study of cellular mutations and how does it work?

Back 11

Cytosine can deaminate forming Uracil, which is not recognized as a DNA mismatch because uracil is not supposed to exist in DNA. Thus it results in a mutation in one of the cell's progeny.

Front 12

What is an "abasic" site?

Back 12

An abasic site is a site on a DNA strand which lacks a base! Abasic sites are formed where purine's N-glycosidic bonds are broken under acidic conditions.

What can polymerase do? Nothing, it can't create a base pair with no template base.

Front 13

What can oxidative damage do to DNA?

Back 13

It can cause ss or ds DNA breaks.

It can also create modified bases with different altered base pairing properties!

Front 14

What cause induced mutations?

Back 14

UV Light
Chemical Modification of DNA
Ionizing radiation
Intercalating Agents

Front 15

How does UV Light cause mutations?

Back 15

UV light activates thymine bases to form at the point of overlap of the double bonds. This formed a thymine dimer.

Polymerase doesn't know what to do at a thymine dimer.

Front 16

How does chemical modification result in mutations?

Back 16

Some chemical modifications can change bases such that new base pairs form, such as ethylating the oxygen of guanine leads to a G-T basepair.

Other chemical modifications can also change the donor/acceptor configuration so significantly that there are no possible base pairs, stalling replication

Front 17

How does Ionizing Radiation cause mutations?

Back 17

Ionizing radiation can directly damage DNA or create oxygen radicals which then damage DNA.

Front 18

How do Intercalating Agents cause mutations?

Back 18

Intercalating Agents such as Ethidium Bromide fit in between DNA base pairs where they are stabilized by ring stacking interactions. This causes the insertion of an extra base, possible resulting in a frame shift.

Front 19

What are the 4 mechanisms of DNA repair in prokaryotes discussed?

Back 19

Base Excision Repair
Nucleotide Excision Repair
Recombination Repair
SOS - Trans-lesion DNA Synthesis

Front 20

How does Base Excision Repair work?

Back 20

A glycosylase removes the incorrect base from the DNA strand, resulting in an abasic site.

An AP endonuclease removes the apurinic or apyrimidinic nucleotide.

DNA Pol I and Ligase then show up to fill in the site and seal the nick.

Front 21

How does Nucleotide Excision Repair work in general?

Back 21

The bad base along with surrounding nucleotides are removed. DNA pol I and Ligase then finish the job.

Front 22

How does SOS - trans-lesion DNA synthesis work in general?

Back 22

Polymerase doesn't know how to work with lesions, so the cell removes lesion DNA and fills it in with random DNA. This could result in a functional chromosome or a dead cell, but at least it has a chance of survival.

Front 23

Describe Nucleotide Excision Repair in detail!

Back 23

UvrAB scans DNA for mismatches that distort the DNA backbone.

Once a distortion is found, UvrB melts a region around the mismatch and UvrA leaves.

Then, UvrC is recruited to the site and makes nicks on both sides of the distorted DNA.

Then, UvrD (helicase II) comes in and removes the nicked DNA fragment.

Finally, DNA pol I and Ligase come in to finish the job.

Front 24

How does Thymine Dimer Repair work in detail?

Back 24

DNA photolyase recognizes the thymine dimer and binds to it. Then, when activated by visible light, photolyase repairs the thymine dimer break.
(Humans dont have photolyase)

Front 25

How does SOS - Translesion Synthesis work?

Back 25

DNA pol III goes along replicating ssDNA strand until it meets a lesion.

DNA pol III and its beta clamp leaves and a DNA Pol IV or V come in and place random DNA bases down on non-template strand of DNA where template strand is undetermined.

DNA pol IV or V then leaves and DNA pol III and beta-clamp are recruited to finish the non-template strand.

Front 26

How does dsDNA Break repair by homologous recombination work in E. coli?

Back 26

a dsDNA break is recognized by RecBCD (helicase and nuclease) complex, which ENDOnuclease cleaves away chunks of DNA at both 3' and 5' ended ssDNA until it recognies a chi site, where is changes conformation. This conformation change results in no more cutting of 3' end and more active cutting of 5' ended ssDNA.

RecBCD recruits RecA to the 3' tail of the DNA. the Rec A is ABSOLUTELY necessary for recombination. (Mutationto RecA stops all homologous recombination). RecA recognizes homology and catalyzes 3' end strand invasion/duplex formation.

Once both 3' ends have found their homologous regions, DNA replication and finally ligation occurs to form two holiday junctions.

At this point, RuvAB come together to facilitate branch migration. RuvB is a special helicase and RuvA binds to the holiday junction.

Then, RuvC cuts the holiday junction at a random orientation, which is most likely resolved by ligase.

Front 27

What are epichromosomal elements?

Back 27

Epichromosomal elements: additional DNA molecules which may or may not be essential, but are important for the proper functioning of the cell. (i.e. mitochondrial DNA)

Front 28

What are transposons? What are some important features of transposons?

Back 28

Transposons: parasitic DNA elements that must become part of a chromosome, plasmid, or phage genome in order to propagate.

o Transposons cannot replicate on their own! They are incapable of independent
replication!
o Transposons must be part of another self-replicating DNA.
o That is, the transposon cannot exist as a free-standing piece of DNA like a plasmid.

Front 29

What is the difference between a plasmid and a chromosome?

Back 29

A chromosome is a replicon that codes for ESSENTIAL FUNCTIONS.

Plasmids, on the other hand, encode for genes that are useful under certain conditions.

Front 30

What are the two classifications of plasmids?

Back 30

High copy number and low copy number. High copy number plasmids are partitioned randomly between child cells. Low copy number plasmids are directly partitioned.

Front 31

What is the difference between vertical and horizontal gene transfer?

Back 31

Vertical transfer: inheritance via direct descent

Horizontal transfer occurs when the bacterium A transfers DNA to bacterium B, which
may or may not have the gene or perhaps has a different allele.

Front 32

What is transformation?

Back 32

Transformation is gene transfer between cells mediated by “naked” DNA.

The only thing required is free DNA and a cell capable of picking it up.
o Transformation is an active process: there is dedicated machinery for this.

Non-replicating DNA (DNA lacking its own mechanism of replication) must be stabilized by
homologous or non-homologous recombination.

Front 33

What are the two types of transformation?

Back 33

Artificial and Natural

Front 34

Describe, step by step, transformation in Bacillus.

Back 34

• Step 1: dsDNA enters the environment (is added to the medium in which the cells are growing).
• Step 2: The DNA binds to binding proteins on the surface of the cell.
o These proteins are part of the transformation machinery.
• Step 3: A nuclease (more machinery) degrades one strand while the other strand is pumped into
the cell.
• Step 4: recA binds to the single-stranded DNA, then it searches for a region of homology where it
invades the chromosomal DNA. This process is homologous recombination.
o Whenever there is ssDNA in the cell, the recA protein is looking for it.
o The result is a “D-loop” where the ssDNA is attached.
• Step 5: Part of the original DNA is then cut out.
• Step 6: The new ssDNA is now officially integrated and the gap filled with a complementary
strand created by DNA polymerase and sealed by DNA ligase.
• Note: because this process uses homologous recombination, the incoming DNA must share a
significant portion of homology for this to work!

Front 35

What is trandusction?

Back 35

Transduction is gene transfer between cells mediated by bacteriophages – the bacteriophage vectors the
DNA.

Front 36

Why is transduction important for gene transfer?

Back 36

Out of the billions of viruses made from a bacteria, some will by chance have a gene with a promotor region from the host cell. If this virus attacks another cell, the DNA will infect the host cell and be transposed into the host cell's genome.

Front 37

What is conjugation?

Back 37

Conjugation is gene transfer between cells mediated by direct transfer of DNA from one cell (donor) to
another (recipient).

Front 38

What are some important features of transposons?

Back 38

Transposons are infectious within a cell.
Transposons are not revertable at a relevant frequency.
Transposon mutations can be polar.

Front 39

What characteristics are common to all prokaryotic transposons?

Back 39

o They are all dsDNA.
o They are flanked by inverted repeats (IRs).
o The repeats are variable between various types/flavors of transposons, but every
transposon of a particular type has the same set of repeats.
o Generally, transposons contain one or more genes between the repeats.

Front 40

Define transposable element.

Back 40

Transposable element: any segment of DNA able to transpose

Front 41

Please define the two types of transposable elements.

Back 41

o Insertion sequence: the simplest transposable element which does not confer recognizable
phenotypes (they don’t bring anything in), though they can disrupt a gene depending upon
location of insertion. This type of transposable element codes only for the ability to transpose.
o Transposons: confer recognizable phenotypes in addition to coding for the ability to transpose. These go beyond self-replication to the actual alteration of the phenotype of the host organism.

Front 42

Does an insertion sequence code for any genes?

Back 42

The insertion sequence does not code for observable phenotypes. It only codes for transposase, the enzyme that controls the transposition event.

Front 43

How do insertion sequences vary?

Back 43

Since they all have the same three regions, IR -> transposase gene -> IR, then there can be variation between the insertion sequences in the junk DNA between the inverted repeats, as well as the length and sequence of the inverted repeats.

Front 44

What are the two types of transposons?

Back 44

Class I or composite transposons and Class II transposons.

Front 45

Please describe composite transposons.

Back 45

Composite Transposons, or Class I transposons, are a segment of DNA flanked by two copies of an IS element. Each IS element is itself flanked by two IRs.

Front 46

What is a good example of a composite transposon?

Back 46

Tn5. Tn5 codes for resistance for kanamycin in its center DNA segement, and of coarse transposase in the IS segments.

Front 47

Please describe Class II transposons.

Back 47

Class II transposons are a segment of DNA flanked by IR sequences, which are NOT part of IS sequences.
Class II transposons do NOT have IS sequences.

Front 48

What is a good example of a Class II transposon?

Back 48

Tn3, which codes for resistance to penicillin. This transposon has a res site, and genes codes outwards from the res site towards the IRs.

Front 49

Can transposons code for anything other than antibiotic resistances?

Back 49

Yes, one composite transposon in particular, Tn1681, codes for Enterotoxin.

Front 50

Where are transposons found in DNA?

Back 50

Generally, transposons are randomly found throughout DNA. However, some transposons are only found in specific sites, such as Tn7 which is found in one site in E. coli.

Front 51

What is required for transposition?

Back 51

Cis-acting DNA sites and Trans-acting proteins.

The Cis acting DNA sites are the IR sequences near the transposon genes and the target site.

The Trans acting protein is transposase.

Front 52

What is a major difference between transposase and serine/tyrosine residue based recombinases?

Back 52

Transposases do not covalently bond to DNA! -> no DNA-transposase adduct.

Transposase uses a DDE (D=Aspartate, E=Glutamate) at the active site (no serine or tyrosine).

Transposases use the the OH grp from a water molecule to attack the phosphate group of the DNA molecule they nick.

Front 53

What sites of transposase are conserved across vastly different forms of life?

Back 53

The DDE active site

The protein-DNA binding site (hydrogen bonding) (alpha helices surrounding active site)

The protein-protein contact regions (beta sheet around active site)

Front 54

Is there anything special about the target site of transposition?

Back 54

Generally no. Although, the number of bases which are duplicated at a target site varies based on the IS sequence.

After the target site DNA is duplicated, it can be seen that they are DIRECT repeats which flank the inverted repeats of the insert sequence/transposon.

Front 55

What are the two fundamental methods of transposition?

Back 55

Replicative and Non-replicative transposition.

Front 56

Please describe the process of replicative transposition using a specific transposon.

Back 56

Tn3 uses replicative transposition mechanism. Two of the genes Tn3 codes for are tnpA='a transposase' and tnpR='a resolvase'.

The transposase recognizes the IRs on the ends of Tn3 and nicks near the IRs. The transposase also binds to a random site on DNA and nicks the DNA at a distance set by the length of the target sight required.

Then, the free ends of the transposon are joined to their respective free ends of the chromosome.

Then DNA pol I and ligase come in to fill in the gaps and seal the nicks.

The result is a fused DNA molecule of the original transposon plasmid and the target DNA molecule, which now contains two copies of the transposon.

Resolvase is of the serine/tyrosine site-specific recombinase family.

Resolvase binds to the res site in each copy of the transposon Tn3, and uses site specific recombination techniques to cut and fuse the original tranposon plasmid and target DNA molecule back together. Except, now both the target DNA molecule and the transposons plasmid have a copy of the transposon.

Front 57

Please describe the process of non-replicative transposition using a specific transposon.

Back 57

Tn5 uses the non-replicative transposition mechanism.


The transposase recognizes the IRs on the ends of Tn5 and nicks near the IRs. The transposase also binds to a random site on DNA and nicks the DNA at a distance set by the length of the target sight required.

Then, the free ends of the transposon are joined to their respective free ends of the chromosome.

Then, the transposon regions anneal and nicking occurs on the transposons plasmids last two connected strands to free the plasmid (with a ds DNA break) from the transposon.

Ligase then seals the nicks in the host DNA molecule.

To repair the transposase's old plasmid, which is now a dsDNA linear fragment, an copy of the plasmid containing the transposon must have been made or be present in the cell. If so, then homologous recombination (ds break repair) occurs as seen previously leaving the transposon's old plasmid with a brand new copy of the transposon (and back in circular form), ready to replicate or non-replicate again.

Front 58

How is transposition regulated?

Back 58

tnpA, the transposase in Tn5, has two start codons; one at the beginning of the mRNA, and one about 1/3 of the way from the beginning of the mRNA. If the ribosome translates from the first start codon, TnpA is made. If the ribosome translates from the second start codon, Inh is made. Inh actively inhibits transposase.

Thus, the more transposase that is present or manufactured in the cell, the more inh will inhibit it. This causes an equilibrium of very low transposition frequency when transposase has been present in the cell long enough for inh to be present in high concentrations.

Front 59

Why are transposons so important?

Back 59

They can cause insertions, large scale deletions, and inversions of and in host DNA.

They also have very few host-range limitations or restrictions for carrying genes between bacteria. This combined with the fact that they can carry anything from new catabolism sytems to antibiotic resistance and toxins makes transposons a very important subject.

Front 60

How does transposition lead to deletion mutations and inversions?

Back 60

Homologous recombination proteins within a cell can catalyze recombination events between transposons as homologous regions of DNA.

If the transposons are direct repeats, then the DNA molecule is resolved in one functional DNA molecule and one non-functional DNA molecule (each with one copy of the transposon).

If the transposons are inverted wrt each other, than cell inverts all the DNA in between and including one of the transposons.

Front 61

What are the two types of eukaryotic transposons?

Back 61

Classical Transposons - may be autonomous or non-autonomous

Retrotransposons

Front 62

How does the Ac/Ds system work?

Back 62

The Ac transposons carry the genes required for transposition.

The Ds transposons are mutated Ac transposons which do not carry functional genes required for transposition.

Front 63

What is the drosophila melanogaster p-element system transposition system?

Back 63

the p-element is a classical autonomous transposon. Thus, transcription of transposon mRNA leads to translation of transposase and transposase inhibitor (as seen in prokaryotes).

Sperm cell of drosophila melanogaster carries almost no cytoplasm or extra proteins at all. Only carries nutrients and mechinery required to travel and fertilize egg cell. So, no inhibitor or transposase proteins would be present in this cell, even if the genes were.

The egg cell, however, would have both transpoase and its inhibitor if it carried the transposon.

So, if the female has the transposon, very little transposition will occur.

If the female does not have the transposon, but the male does, lots of transposition will occur (leading to mutations).

Front 64

What are the two types of retrotransposons?

Back 64

LTR-containing: These are flanked by long terminal repeats (LTR).
Orientations are in the form of inverted repeats.

Non-LTR: Lack LTRs. Flanked by short DIRECT repeat sequences

Front 65

How does LTR transposition occur?

Back 65

A promotor region in the transposon is recognized by RNA polymerase which then codes an mRNA version of the transposon.

Then reverse transcriptase, coded by the transposon, converts the RNA into a dsDNA segment.

The mRNA is translated into virus-like structural proteins, reverse transcriptase, and integrase.

These get packaged with an mRNA copy of the transposon into a virus-like particle formed by the proteins from the mRNA.

Reverse transcriptase then copies the mRNA into dsDNA.

Integrase then binds to the dsDNA and signals the cell to transport the complex into the nucleus.

The integrase then inserts the dsDNA copy of the transposon into the host DNA somewhere.

Front 66

What are some examples of LTR retrotransposons?

Back 66

Copia elements in Drosophila melanogaster.

Ty elements in Saccharomyces cerevisiae

Front 67

How do Non-LTR transposons work?

Back 67

an mRNA copy of the transposon is made.

The mRNA is translated into a peptide chain with two major domains, a reverse transcriptase and an endonuclease.

The endonuclease cuts the domains into two functioning proteins.

The proteins then bind to an mRNA transcribed from the transposon, which signals the cell to transport this complex into the nucleus.

Once in the nuclease, the reverse transcriptase makes a dsDNA copy of the mRNA.

The endonuclease then inserts the dsDNA copy into the host DNA somewhere.