Genetics Exam 4 Study Guide

Do spontaneous mutations require an agent?

No

3 ways: how do spontaneous mutations usually come about?

a. Errors in DNA rep

b. Errors in DNA proofreading

c. Spontaneous changes in the chemical structure of a nucleotide base

What are the 2 kinds of spontaneous DNA replication errors/strand slippage results? For the first one, what can that cause?

a. Inc or dec numbers of nucleotide repeats in daughter DNA i. Trinucleotide repeat expansion disorders

b. Mispaired nucleotides

What is strand slippage?

DNA pol slips on the template strand or vice versa in DNA rep

What is a replisome? What is it comprised of?

a. The DNA-replicating protein complex

b. Helicase, DNA pol, primase, and more

How does strand slippage occur? What step comes after this to increase repeats?

a. The DNA pol temporarily goes away from the template, so the daughter DNA forms a temporary hairpin.

b. Replication resumes, but now everything is thrown off, so some of the repeats get re-replicated. So, overall, the number of repeats on the daughter strand increases.

What are trinucleotide repeat expansion disorders?

Genetic disorders that are caused by strand slippage making too many increased repeats beyond the threshold

What are mispaired nucleotides? What are they similar to?

a. Non-complementary base pairing/non-Watson-and-Crick base pairing

b. Third-base wobble

What is the difference between an incorporated error and a replicated error?

a. An incorporated error is the initial mispairing between nucleotide base pairs: G with T or C with A

b. A replicated error is made when the incorporated error is never fixed, so it gets replicated and converted into a mutation

What are the two types of spontaneous nucleotide base changes (just names)?

Depurination and deamination

What is depurination? What does it look like structurally? Are depurinations usually repaired before rep, and if so, what happens if they aren’t? What is a depurination lesion called?

a. Losing a purine

b. The nucleotide is broken from the backbone/sugar

c. Yes; unrepaired depurinations get replaced by an A

d. Depurination lesion: apurinic site (hole)

What is deamination? What is the difference between deaminations in unmethylated cytosine vs methylated cytosine?

a. Losing an amino (NH2) group

b. Differences in cytosines:

i. Unmethylated cytosine deamination: cytosine loses its NH2 and an O takes the NH2’s place, converting the cytosine into a uracil. The DNA picks up on this and replaces it with another normal cytosine, so all is well

ii. Methylated cytosine deamination: methylated cytosine loses its NH2 and an O takes the NH2’s place, this time converting the methylated cytosine into a thymine. This thymine can now pair with adenine, which is bad because it’s supposed to be a cytosine that pairs with guanine. This is a mutation unless fixed

What is a mutagen?

An agent that causes DNA damage leading to mutations

Is DNA actually damaged in a mutation

Yes

What are induced mutations

mutations generated by exposure to physical, chemical or biological mutagens; mutations that are produced in science experiments

What are nucleotide base analogs mutations?

They are similar to a nucleotide base and can falsely pair with one

What are deaminating agents mutations? What can they cause?

a. Removes amino groups from nuc bases

b. Can make nuc base analogs and thus cause transitions and transversions

What are alkylating agents mutations? What do they cause? What is a popular one, and what does it do?

a. Add bulky side groups like methyl groups to bases

b. The bulky side groups distort the DNA double helix shape because they’re so heavy, so the base pairing is affected

c. EMS: via alkylation, makes transition mutations (pur to pur or pyr to pyr)

What are oxidizing agents? What do they cause?

a. Takes the electrons, causing the actual substance to lose them

b. Incorrect base pairing

What are hydroxylating agents?

Add hydroxyl

What are DNA intercalating agents?

Molecules that fit between DNA base pairs, so the duplex is distorted and frameshift mutations occur

Can radiation cause DNA damage?

Yes

What’s the relationship between UV rays and radiation?

UV rays are a type of radiation. Radiation is the header

What is a photoproduct? What are two common ones/what do they feature? Are these usually repaired–if so, how/what kind, and what happens when they’re not?

UV rays (a type of radiation) target a structure, so it’s messed up: its nucleotides have too many bonds

b. Two common photoproducts:

i. Thymine dimer: two adjacent thymines form a covalent bond on their 5 and 6 carbons

ii. 6-4 photoproduct: two adjacent thymines form a covalent bond on their 4 and 6 carbon

c. Yes, they’re usually repaired: DNA repair systems can correct most pyrimidine dimers. But sometimes, they’re not repaired, and replication is disrupted–then mutations come.

What is the purpose of the Ames test? What enzyme (both names) do we use and why? How does it work? What unit of measurement is counted to evaluate mutagenicity? What does this unit of measurement look like in cases of no mutagenicity vs a lot of mutagenicity? What kinds of mutations does the Ames test work with?

a. To determine if a compound or any of its breakdown products is mutagenic

b. Liver enzyme (S9 extract) because it lets you know that, if you expose your compound to that digestive enzyme, if a mutation gets created

c. You take a his- bacteria. You expose that his- to a chemical. If the bacteria stays his-, the chemical is safe. If the chemical turns the bacteria his+, the chemical is probably a mutagen.

d. The number of reversion mutations from his- to his+

e. High reversion rate = chemical is probably a mutagen. Low reversion rate = probably safe chemical.

f. Base substitution and frameshift

What two GENERAL ways can DNA damage be repaired?

a. Directly: direct and actual repair

b. Indirectly: don’t actually repair, leaves damage unrepaired: just allows the organism to find a way around the problem

What is a damaged section of DNA referred to as?

DNA lesion

What is the most direct way to repair DNA lesions? What is one general mechanism that accomplishes this?

to reverse it; proofreading by DNA pol

What are the 4 ways of repairing DNA damage (just names)?

a. Photoreactive repair

b. Base excision repair (BER)

c. Nucleotide excision repair (NER)

d. Mismatch repair

What is photoreactive repair? How does it work? Photoreactive repair takes place in everyone but who?

a. Repairs damage caused by UV-caused photoproducts.

b. Visible light activates photolyase, which repairs the photoproducts

c. Humans

What is the most common mutagen of most organisms?

UV Radiation

What step is between a photoproduct being made by UV radiation and a mutation?

The UV radiation makes the photoproduct inhibit DNA rep

How does photolyase help a photoproduct?

It uses energy from visible light to break the bonds that produce the photoproduct

What is photolyase encoded by?

The E. coli phr (photoreactive repair) gene

What is base excision repair (BER)? What process does it utilize?

a. To repair a damaged or incorrect DNA single base, you just synthesize a new strand segment.

b. Nick translation

What role does each thing play in BER?

a. DNA glycosylase- Removes base and makes AP site

b. AP site - Place made by DNA glycosylase/where the nick is near

c. AP endonuclease - Makes the nick near the AP site

d. Nick - The single-stranded cut made by the AP endonuclease near the AP site

BER uses _____ to find and remove the incorrect base, creating ______. ______ sees this and ______; this action is _____.

a. Glycosylases

b. An AP site

c. DNA pol

d. Fills with correct nucleotides

e. Nick translation

What is nucleotide excision repair (NER)? What is often used to repair; thus, what is it also called?

a. Removing a whole strand segment of DNA damage and synthesizing a new replacement b. UV-repair

Compare BER and NER

Similarity: both include removing a broken something and synthesizing a new strand to replace it

Contrast BER and NER

Difference: BER’s broken something is a simple base. NER’s broken something is a whole strand segment. (NER is more damage and more removed.)

What is mismatch repair?

DNA base-pairs have mismatched (not A&T or C&G). So mismatch repair just takes out a segment of the (new) synthesized strand that was synthesized from that mistake and resynthesizes the taken-out segment.

Is mismatch repair a “frontline” repair mechanism or a “plan B” one? How so?

Plan B: it repairs the mismatched nucleotides that escape DNA pol proofreading

What is the role of repair enzymes in mismatch repair? How does it do this, and is this foolproof?

a. Repair enzymes in mismatch repair can tell the difference between the original, correct nuc and the incorrect, mismatched one.

b. They can tell the difference between incorrect and correct nuc because the correct/original is methylated. Sometimes they can’t tell tho, so no, not foolproof

What are the 3 main DNA damage-signaling systems/pathways (just names)? What are these referred to? What happens if they’re mutated or non-functional?

a. BRCA1, ATM, and p53

b. Tumor suppressors

c. If mutated or non-functional, can’t protect the cell from cancer-causing mutations, and then more tumors will obviously grow

Which is the most important DNA damage-signaling system/pathway? What does it do?

a. p53

b. Nurse. Pauses cell cycle before S phase to let the cell take time to repair itself, OR mediates apoptosis if there’s too much damage

What does ATM do?

Increases the level of p53 in response to DNA damage

How does p53 level correspond with DNA damage and work/stopping?

a. Low p53 = no DNA damage = cell keeps growing

b. High p53 = probably DNA damage = cell pauses til DNA damage is repaired

What is translesion DNA synthesis? Is it a first line of defense, or what?

a. Cells can bypass DNA lesions and continue DNA rep

b. NO. It is only an SOS, last ditch effort

How is translesion DNA synthesis activated? What is the quality of these? What do they do?

a. Activated by translesion DNA polymerases

b. Translesion DNA polymerases are terrible quality and are error-prone

c. Translesion DNA polymerases have no proofreading ability, but they can replicate smoothly across lesions that DNA polymerase would be stalled at

What is a double-strand break (DSB)? What can they cause? What two mechanisms can do double-strand break repair (just names)?

a. When both strands of DNA are damaged, so neither strand can be the template for repair

b. DSBs can cause chromosome instability → incomplete replication → potential cell death, mutations, and cancer

c. Nonhomologous end joining and synthesis-dependent strand annealing

What does nonhomologous end joining (NHEJ) do? Is it good or dodgy; how so?

a. Repairs double-strand breaks that occur BEFORE DNA rep

b. Dodgy. It’s really error-prone, so it ALWAYS leads to a mutation

What is synthesis-dependent strand annealing (SDSA)? Is it good or dodgy; how so?

a. Repairs DSBs that occur AFTER DNA rep

b. It is error-free

What is the final result of SDSA?

The excised DNA is replaced with a duplex identical to the sis tid

What is constitutive transcription?

Continuous transcription–no regulation is needed and gene is expressed @ constant rate

What is regulated transcription?

Gene is expression is controlled because environmental conditions change

What is a regulatory sequence?

Promoter + enhancer + repressor, etc. Non-transcribed regions around the actual gene/genes.

What is negative control of transcription–aka what is the protein involved & what happens to transcription?

Repressor protein binds, transcription dec

What are operators?

The regulatory sequence repressors bind to

What is an active site in general? In respect to negative control of transcription?

a. The part of the protein that actually binds to the DNA sequence

b. The part of the repressor protein that binds to the operator

Where is the operator located?

Beside or inside the promoter

On a structural level, how does the repressor protein (once joined to the operator in the promoter region) prevent transcription?

The repressor protein is actually sitting where RNA pol should come in, so RNA pol can’t join and initiate transcription.

What do active sites have to do with negative control of transcription?

Repressor proteins have 2 active sites (allostery)

What are the 2 active sites in repressor proteins and their general functions?

a. DNA-binding domain: binds the DNA directly

b. Allosteric domain: a molecule is bound that affects how the repressor protein interacts with the DNA

Explain the outcome of the DNA-binding domain being used in repressor proteins

The DNA is bound directly, so no transcription

Relate the DNA-binding domain and the allosteric domain, “affected”/square-rectangle-wise

The allosteric domain affects the DNA-binding domain, but the DNA-binding domain doesn’t affect the allosteric domain.

What is allostery?

When a molecule or protein binds to the (repressor) protein, so the DNA-binding domain is changed

What are effector proteins? What are the 2 types with respect to repressor proteins?

a. They are the protein that binds to the allosteric domain.

b. Two types: inducer and corepressor

What are the 2 modes of the allosteric domain in negative control of transcription? Explain each, as well as their outcomes + what happens to transcription.

a. Inducer: binds to the allosteric domain on the repressor protein and changes the conformation of the DNA-binding site to where it can’t fit the DNA sequence/operator anymore, so the repressor can’t bind to the DNA sequence/operator and transcription does occur.

b. Corepressor: binds to the allosteric domain on the repressor protein and changes the conformation of the DNA-binding site to fit the DNA sequence/operator, so the DNA-binding site can fit the DNA sequence/operator and transcription is inhibited (because now RNA pol can’t get on)

What is positive control of transcription–aka what protein is involved & what happens to transcription?

Activator protein binds, transcription inc

What does activator protein do to RNA pol?

It welcomes it

What domain(s) do activator proteins have?

Same as repressor proteins: DNA-binding domain and allosteric domain

What are the 2 modes of allosteric domain in activator proteins with respect to positive control of transcription? Include outcomes, including what happens to transcription

a. Allosteric effector compound: the DNA-binding domain on the activator protein is inactive til the allosteric effector compound joins. Once it does (binds the allosteric domain), a conformational change happens to the DNA-binding domain and now the DNA-binding domain is activated. Now it can attach to the DNA and transcription inc/gets a boost

b. Inhibitor: binds to the allosteric domain, which conformationally changes the DNA-binding domain. Now it’s inactive and can’t join the DNA, so transcription doesn’t get that boost/is dec

In short, what do each of the 2 modes of the allosteric domain in activator proteins do to transcription?

a. Allosteric effector compound: inc transcription

b. Inhibitor: dec transcription

What are regulatory DNA-binding proteins?

Just the header over activator and repressor proteins

How do regulatory DNA-binding proteins work structurally (like with nucleotides/DNA sequences)? AKA, how do regulatory DNA-binding proteins know which sequence of DNA to bind to?

Protein side chains match DNA side chains

What do alpha helices have to do with regulatory DNA-binding proteins?

Alpha helices are a “motif” (recurring thing) in the actual proteins. They’re little sticks that jut out so the proteins can stick in between nucleotides in the actual DNA

What are the 2 types of interactions that can happen with regulatory DNA-binding proteins when their alpha helices jut into nucleotides of DNA?

a. Monomeric: same polypeptide folds to form (seemingly) 2 domains

b. Multimeric: actually multiple polypeptides (dimeric, trimeric, or tetrameric)

What are homodimers and heterodimers?

In multimeric polypeptides, the polypeptides that join together may be identical (homodimers) or different (heterodimers)

What is the helix-turn-helix (HTH) motif?

The most common motif. The alpha helices of the jutting regulatory DNA-binding proteins interact with inverted repeats and stuff “turns.”

What is the difference between active sites and activator binding sites?

a. Active sites are on the protein, where they either bind to the DNA directly or have a protein come in.

b. Activator binding sites are on the DNA. They bind activator proteins

What is an operon? Using the mnemonic, what all makes it up (in order of occurrence, name + function)? How does this change in the lac operon?

a. Girl group on their way to a party, + their chaperones: a bunch of genes that do similar functions and get transcribed together + a shared regulatory sequence

b. PrOG i. Promoter: inc transcription ii. Repressor: dec transcription (not really in the operon but just imagine it, the repressor is just a protein added by a separate regulatory gene) iii. Operator: on/off switch iv. Genes: The actual genes (girl group) that are transcribed together

c. Add C before P: CAP binding site

If the r (repressor) isn’t really a part of the regulatory sequence in the lac operon, why is it in PROG?

Because the repressor in the lac operon is always expressed

How is the repressor expressed in the lac operon, functionally?

The repressor binds to lacO, but to defend, an allosteric site on the repressor emerges. Allolactose binds to that, and then the repressor pops off the operon, so now transcription can occur.

Are operons in prok, euk, or both?

only in prok

What does the lac operon digest?

Lactose

Ideally, the lac operon is turned on in what conditions?

The presence of lactose and the absence of glucose

Which is the preferred energy for E. coli?

Glucose

What is the lac operon? Plan A or plan B? How so?

a. Plan B

b. Because glucose is the preferred form of energy for E. coli, but when only lactose is available, the cell “makes do” by using the lac operon

This doesn’t have much to do with the lac operon, but you can think of ____ to help with the idea of the lac operon being a “backup plan.”

Lactate fermentation

Name the (C)PrO parts of the lac operon and define each

a. C: CAP binding site: helps RNA pol bind

b. P: promoter: binds RNA pol

c. r: just a repressor protein, not part of the lac operon, coded by lac i

d. O: lacO operator: binds to the repressor protein

What are the 3 genes (G) in the lac operon? When are they together as one and when are they separate?

a. LacZ: codes for beta-galactosidase

b. LacY: codes for permeate

c. LacA: codes for transacetylase

d. Together in transcription

e. Separate in expression

What is an inducer? What is the main inducer in the lac operon, + another one?

a. A protein that inhibits a repressor so transcription can start

b. Allolactose i. Also lactose

Under what conditions is allolactose produced?

When lactose is available

What is the relationship between the lac operon being on/off and the presence or absence of glucose/lactose–and why for each?

a. Off when there’s no lactose (because nothing to use) and off when there’s a lot of glucose (because no need to use the backup lactose-using plan when plan A is working)

b. On when there’s a lot of lactose (because hello, materials!) and on when there’s not a lot of glucose (because we’ve run out of plan-A glucose and now need plan B)

The lac operon is transcriptionally silent when what happens with glucose/lactose?

When no lactose is available or when glucose is available

What’s the main thing you need to digest lactose?

The gene for beta-galactosidase

What role does each of these play in the lac operon?

a. Beta-galactosidase i. Metabolizes lactose

b. Allolactose i. Enhancer that inhibits lac repressor from lacO, allowing the operon to turn on and produce beta-galactosidase

c. lacO i. Binds to the lac repressor and inhibits beta-galactosidase production

What happens when there’s no allolactose nor beta-galactosidase?

When there is no beta-galactosidase and thus no allolactose, the lac repressor protein binds to lacO, which prevents transcription/beta-galactosidase production

When lactose is available in the cell, what is produced? What does it do? What happens then?

a. When lactose is available, allolactose is produced

b. Allolactose inhibits the repressor from lacO

c. The repressor can’t attach to the operon, so beta-galactosidase is made… EVENTUALLY!

True or false: all you need to start making beta-galactosidase and enough genes to digest lactose is for the allolactose to inhibit the repressor. If false, what else do you need?

False; CAP