Genetics Final Price Summer 2015 UVU

What is a somatic mutation?

Genetic alteration acquired by a cell (chemical, physical, environmental damage to a cell) that can be passed to the progeny of the mutated cell in the course of cell division.

What are Indels?

Insertions or deletions of one or more base pairs. May arise when loops in single-stranded regions are stabilized by the "slipped mispairing" of repeated sequences in the course of replication.

What is polymerase chain reaction (PCR) and how does it work?

It amplifies particular stretches of DNA. How it works: a section of DNA is heated until it unzips. The temperature is lowered then left and right primers match up to their complementary sequences. The temperature is raised, polymerase is added and DNA synthesizing occurs.

How does deamination of cytosine cause mutations? What types of mutations are caused? How might this relate to the use of Thymine in DNA rather than Uracil? What is the long term effect of cytosine deamination on the G/C content of unconstrained portions of genomes (such as Introns)?

It changes the C to a U, which will be fixed to a T. It causes transition mutations. DNA polymerase proofreads and corrects the U to a T because they both base pair with A (also Uracil is only in RNA). It makes the C / G content decrease.

What is the role of Primase?

It is an enzyme that creates a short RNA sequence, called a primer, on a DNA template strand so that DNA polymerase can make a copy of that DNA strand.

Why are primers necessary for DNA replication?

Primers are required because the major DNA polymerase involved with DNA replication is unable to initiate DNA synthesis, and requires a 3' end.

What are tautomeric shifts and how do they contribute to replication errors?

Produces a transient change to an alternative form of a molecule so that is resembles another base (base becomes ionized). If it doesn't revert it may lead in the spontaneous mismatch of base pairs. Causes a transition mutation.

What are the effects of the epigenetic modification of cytosine methylation in DNA?

Reduces gene expression.

Explain the parental conflict hypothesis and how it might explain the existence of Genomic Imprinting.

The Genetic Conflict hypothesis, supposes that imprinting grew out of a competition between males for maternal resources. In some species, more than one male can father offspring from the same litter. A house cat, for example, can mate more than once during a heat and have a litter of kittens with two or more fathers. If one father's kittens grow larger than the rest, his offspring will be more likely to survive to adulthood and pass along their genes. So it's in the interest of the father's genes to produce larger offspring. The larger kittens will be able to compete for maternal resources at the expense of the other father's kittens.

Why does deletion of a group of genes on chromosome 15 only causes Prader-willi syndrome when the deletion is inherited from the father? What would happen if the same deletion were inherited from the mother?

The critical genes must come from the baby's father in order to function properly; the mother's genes in this area are "turned off" through a rare phenomenon called "genomic imprinting." This deletion in mother however is associated with Angelman's syndrome.

Which tautomer of thymine can form the most hydrogen bonds in pairing with other DNA bases?

The enol form of thymine forms three hydrogen bonds with guanine.

What is the role of Topoisomerase?

The supercoiling of DNA during replication can be created or relaxed by these enzymes (an example of a gyrase). They finish by rejoining the strands of the relaxed DNA molecule.

In adult cells that have stopped dividing, what types of repair systems are possible?

There are many repair systems that are available: direct reversal, excision repair, transcription-coupled repair, and non-homologous end-joining.

What are the two methods for repair of double strand breaks? Which of the two is preferred and why?

There are two main pathways that repair DSBs, Homologous recombination (HR) and Non-homologous end-joining (NHEJ). HR is restricted to the S and G2 phases of the cell cycle due to the requirement for the sister chromatid as a template, while NHEJ is active throughout the cell cycle and does not rely on a template. NHEJ is obviously more preferred because it can be used anytime in the cell cycle.

Why are two primers required and how must they be oriented relative to each other for PCR to work?

These are synthetic primers, and two different ones are used. Each one is complementary to a specific sequence on one of the strands. The 3'-OH ends of the primers face each other because they have annealed to opposite strands.

What is the mode of action of Base Modifiers (including UV)?

These mutagens alter a base in such a way that it will form a specific mispair. Alkylating agents such as EMS (adds ethyl group) and NG (adds a methyl group). Would result in an G-C->A-T or A-T->G-C transition in the next round of replication. UV light causes damage to Thymidine and form Thymine dimers (causes puckering and incorrect pairing).

How are X-rays mutagens?

They do all kinds of things, including breakage of the phosphodiester backbone and destruction of bases, by breaking covalent bonds.

How does 5-methyl-cytosine undermine the ability of Uracil Glycosylase to correct mutations at the locations of 5-methyl-cytosine?

Uracil glycosylase takes any uracil and turns it back to cytosine when the cytosine gets deaminated. When methyl cytosine is there, and it is deaminated, it just turns into thyamine, and uracil glycosylase skips over it.

What are Transitions?

When a base becomes ionized (such as in Base Analogs) and proofreading does not occur, and results in a purine substitute for a purine and a pyrimidine for a pyrimidine. Base switching but in the same class.

Give an example of a DNA-repair defect that leads to cancer.

XP (xeroderma pigmentosum) patients lack nucleotide excision repair and are highly prone to developing pigmented skin cancers. Indivduals with HNPCC (hereditary nonpolyposis colorectal cancer) are prone to colorectal cancer due to a loss of the mismatch repair system.

You are an expert in DNA-repair mechanisms. You receive a sample of a human cell line derived from a woman who has symptoms of xeroderma pigmentosum. You determine that she has a mutation in a gene that has not been previously associated with XP. How is this possible?

XP is a heterogeneous genetic disorder and is caused by mutations in any one of several genes involved in the process of NER (nucleotide excision repair). As you read in the text about the discovery of yet another protein involved in NHEJ through research on cell line 2BN, it is certainly possible that this patient has a mutation in a yet unknown gene that encodes a protein necessary for NER.

List the "exonuclease" activities often found in DNA polymerases.

1. 5' to 3' elongation (polymerase activity) 2. 3' to 5' exonuclease (proof-reading activity) 3. 5' to 3' exonuclease (repair activity)

Base Excision Repair

1. DNA glycolase cleave sugar bond freeing altered base and making AP site 2. Endonuclease makes cut 3. dRpase removes stretch of DNA 4. DNA polymerase synthesizes new DNA 5. DNA ligase seals nick

Differentiate between Missense and Nonsense Mutations

A missense mutation results in a different amino acid in the protein product of the gene. A nonsense mutation causes premature termination of translation, resulting in a shortened protein.

What is meant by a primer?

A primer is a short segment of RNA that is synthesized by primase using DNA as a template during DNA replication. Once primer is synthesized, DNA polymerase then adds DNA to the 3' end of the RNA.

What are telomeres?

A region of repetitive DNA at the end of a chromosome, which protects the end of the chromosome from deterioration.

If thymine enolizes while acting as a template during replication, what base-pair substitution will result?

After the first round of replication, the enol form of thymine will be paired with guanine. At the next round, the guanine will pair with cytosine to result in the TA*CG transition.

How do alkylating agents cause mutations? What types of mutations do they tend to cause?

Alkylating agents add a chemical group to the top of a G or a T. They cause them to base pair together causing a transition mutation.

What is a frameshift mutation and what can cause it?

Also called a framing error or a reading frame shift, is a genetic mutation caused by indels (insertions or deletions) of a number of nucleotides in a DNA sequence that is not divisible by three. Shifts the way the DNA sequence is read.

Why is DNA synthesis continuous on one strand and discontinuous on the opposite strand?

Because the DNA polymerase is capable of adding new nucleotides only at the 3' end of a DNA strand, and because the two strands are antiparallel, at least two molecules of DNA polymerase must be involved in the replication of any specific region of DNA.

Differentiate between Synonymous and Neutral Mutations

Both are base-pair substitutions. A synonymous mutation is one that does not alter the amino acid sequence of the protein product from the gene, because the new codon codes for the same amino acid as did the non-mutant codon. A neutral mutation results in a different amino acid that is functionally equivalent, and the mutation therefore has no known adaptive significance.

In mismatch repair in E. coli, only a mismatch in the newly synthesized strand is corrected. How is E. coli able to recognize the newly synthesized strand? Why this this ability make biological sense?

DNA in E. coli is methylated. To distinguish the old template strand from the newly synthesized strand, the mismatch repair mechanism takes advantage of a delay in the methylation of the new strand. This makes sense as replication errors produce mismatches only on the newly synthesized strand, so the mismatch repair system replaces the "wrong" base on that strand.

What are Intercalating Agents?

DNA modifier that include proflavin, acridine orange and IRC compounds. These are planar molecules that mimic base pairs and are able to slip themselves in (intercalating) between the stacked nitrogen bases in a DNA double helix. Can cause an insertion or deletion of a single nucleotide pair.

Explain how epigenetic mechanisms are used to equalize gene expression from the X chromosome in male and female mammals.

Dosage Compensation is the equalization of gene expression in males and females of a species. Because sex chromosomes contain different numbers of genes, different species of organisms have developed different mechanisms to cope with this inequality. Replicating the actual gene is impossible; thus organisms instead equalize the expression from each gene. In humans, the females (XX) silence the transcription of one X chromosome, and transcribe all information only from the one expressed X chromosome. Thus females have the same amount of expressed X-linked genes as the human males (XY) who have just the one X chromosome to express from which to transcribe and express genes.

What is transcription coupled repair? Why might it be of special value to a long-lived terminally differentiated cell that is no-longer dividing? What human genetic disease results from defects in transcription coupled repair?

Expressed genes are scanned by translocating RNA polymerases, which sensitively detect DNA damage and initiate transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes lesions from the template DNA strands of actively transcribed genes. Cockayne's syndrome.

Differentiate between Frameshift and Nonsense Mutations

Frameshift mutations arise from addition or deletion of one or more bses in other than multiples of three, thus altering the reading frame for translation. Therefore, the amino acid sequence from the site of the mutation to then end of the protein product of the gene will be altered. Frameshift mutations can and often do result in premature stop codons in the new reading frame, leading to shortened protein products. A nonsense mutation causes premature termination of translation in the original reading frame, resulting in a shortened protein.

What is Genomic Imprinting? How does this term relate to epigenetics?

Genomic imprinting is the epigenetic phenomenon by which certain genes are expressed in a parent-of-origin-specific manner. If the allele inherited from the father is imprinted, it is thereby silenced, and only the allele from the mother is expressed. If the allele from the mother is imprinted, then only the allele from the father is expressed. It is an epigenetic process that involves DNA methylation and histone methylation without altering the genetic sequence.

What are helicases and topoisomerases?

Helicases are enzymes that disrupt the hydrogen bonds that hold the two DNA strands together in a double helix. Topoisomerases are enzymes that create and relax supercoiling in the DNA double helix.

As compared to HIV, why is cellular DNA replication resistant to the effects of AZT?

Human cells have an ability to quickly repair its own DNA chain if it is broken by AZT during its formation, whereas the HIV virus lacks that ability. Also AZT has a greater affinity for HIV reverse transcriptase.

If thymine makes up 15% of the bases in a specific DNA molecule, what percentage of the bases is cytosine?

If the DNA is double stranded, A=T and G=C and A+T+C+G=100%. If T=15%, then C=[100-15(2)]/2=35%

If the GC content of a DNA molecule is 48%, what are the percentages of the four bases (A,T, G and C) in this molecule?

If the DNA is double stranded, G=C=24% and A=T=26%

What is a germinal (germ line) mutation?

Inherited genetic alterations that occur in the germ cells (i.e., sperm and eggs). Fundamental source of evolutionary diversity.

What is the role of Helicase?

It serves as an unzipper by breaking the bonds between the two DNA strands. This unzipping takes place in both directions from the replication origins, creating a replication bubble.

If the enol form of thymine is inserted into a growing chain in the course of replication, what basepair substitution will result?

It will be inserted opposite a G. During the next round of replication, one expects the thymine to revert to its keto form, and to specify the insertion of an A in the opposite strand. So the transition will be from a C-G base pair (prior to insertion) to a T-A base pair (after fixation of the mutation).

What is the role of DNA Ligase?

Joins broken pieces of DNA by catalyzing the formation of a phosphodiester bond between the 5' phosphate end of one fragment and the adjacent 3'-OH group of another.

Why does the need for a primer lead to a problem in replication of the ends of linear chromosomes? How is that problem solved?

Lagging strands requires primers ahead of the process of replication; when the last primer is removed sequences are missing. The solution involves the addition of multiple copies of a simple noncoding sequence to the DNA at the chromosome tips.

Distinguish early vs. late somatic mutations? How much will the organism be affected in each case?

Late somatic mutation affects the organism less than that that of early somatic mutation. The mutant population is at least 10 fold greater in that of early somatic mutation.

Oxidative damage can cause the formation of oxidized forms of Guanosine. What type of mutations are caused by such damage?

Mispairs with A, causing a G to T transversion.

What are Transversions?

Much like transitions except base classes switch: purine for a pyrimidine and pyrimidine for a purine.

If the four deoxynucleotides showed nonspecific base pairing (A to C, A to G, T to G, and so on), would the unique information contained in a gene be maintained through round after round of replication? Explain.

No. The information of DNA is dependent on a faithful copying mechanism. The strict rules of complementary ensure that replication and transcription are reproducible.

Give examples of how epigenetics can be influenced by environmental factors (such as nutrition, eposure to toxins, stress).

One example of an epigenetic change is DNA methylation — the addition of a methyl group, or a "chemical cap," to part of the DNA molecule, which prevents certain genes from being expressed. Another example is histone modification. Histones are proteins that DNA wraps around. (Without histones, DNA would be too long to fit inside cells.) If histones squeeze DNA tightly, the DNA cannot be "read" by the cell. Modifications that relax the histones can make the DNA accessible to proteins that "read" genes

Why does the distinction between somatic and germinal mutations breakdown in plants?

Single cell organisms have no distinction between germline and somatic tissue.

What are the roles of Single Stranded DNA Binding Proteins?

Stabilizes unwound DNA by binding to single-stranded DNA to prevent the duplex from reforming.

What is telomerase and what does it do?

Telomerase is a reverse transcriptase that can replicate backwards (adds the short repeats to the 3' ends of DNA molecule), and allows for telomeres to keep their length, however telomerases activity decreases as we age.

It is essential that RNA primers at the ends of Okazaki fragments be removed and replaced by DNA because otherwise what would happen?

The RNA would be more likely to contain errors because primase lacks a proofreading function.

If the helicases were missing during replication, what would happen to the replication process?

The absence of helicases would prevent the replication process because they are enzymes that disrupt the h bonds that hold the two DNA strands together in a double helix. This exposes lengths of single-stranded DNA that will act as the template and are required for DNA replication.

How does proof-reading by DNA polymerase help reduce the effects of tautomeric shifts?

The base mismatches are corrected by the proofreading abilities of the DNA polymerases.

The DNA polymerases are positioned over the following DNA segment and moving from right to left. If we assume that an Okazaki fragment is made from this segment, what will be the fragment's sequence? Label it's 5' and 3' ends. 5'....CCTTAAGACTAACTACTTACTGGGATC....3' 3'....GGAATTCTGATTGATGAATGACCCTAG....5'

The bottom strand will serve as the template for the Okazaki fragment so its sequence will be: 5'....CCTTAAGACTAACTACTTACTGGGATC....3'

Where does the energy for bond formation come from (the growing chain or the incoming nucleotide)?

The incoming nucleotide. The high-energy bond between the alpha and beta phosphate is broken to allow the formation of the bond between nucleotide and nucleic acid chain. ATP->ADP

What chemical mutational event is Uracil Glycosylase primarily involved in correcting?

The most common mutation is the deamination of cytosine to uracil. UDG repairs these mutations.

What type of mutation is depicted by the following sequences (shown as mRNA)? Wild-type: ....5'AAUCCUUACGGA3'.... Mutant: ....5'AAUCCUACGGA3'....

The mutant has a deletion of one base, and this will result in a frameshift (-1) mutation.

How can DNA methylation help a cell detect which base of a mis-match is likely to be the correct one so the incorrect base can be corrected?

The older DNA strand is more methylated so it can look at the newer stand (which is less methylated) and fix it.

What is the role of primers in PCR?

The role of the primers is to allow the polymer to start. Primers also give the PCR specificity. By choosing the sequence of the primers carefully they hybridize with a unique sequence in a large mix of other sequences, resulting in amplification of only one particular piece of DNA located between the two primer sequences.

What is Epigenetics?

The study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself.

How do cells use visible light to reverse damage caused by UV? What enzyme is used for this light-dependent repair process?

They capture blue light to break the UV induced cyclobutane ring of thymine dimers. Not used by placental mammals. The enzyme is Photolyase.

How do intercalating agents cause mutations? What is slippage (aka "slipped mispairing")? What changes are caused in the DNA sequence due to intercalating agents and/or slippage?

They sneak in-between nucleotides spreads them apart and causes a frameshift mutation. Slippage is where the polymerase forgets where it is and then bungees up and pairs at the wrong place causing more bases. It involves denaturation and displacement of the DNA strands, resulting in mispairing of the complementary bases.

What are bypass polymerases? How do they differ from the replicative polymerases? How do their special features facilitate their role in DNA repair?

Translesion or bypass polymerases are able to replicate past damaged DNA that otherwise would stall replicative polymerases. They differ from replicative polymerases in that they can tolerate large adducts on the bases (as they have much larger active sites that can accommodate damaged bases), they are much more error-prone (as they lack the 3' to 5' proofreading function), and they can only add relatively few nucleotides before falling off. Their main function is to unblock the replication fork, not to synthesize long stretches of DNA that could contain many mismatches.

What is the role of an Initiator Protein?

When it is time to replicate, these special initiator proteins attach to the DNA at regions called replication origins. These regions are characterized by a weak bond between the two DNA strands.

How does the cell respond to cope with the damage caused by Aflatoxin? Can the cellular response lead to "collateral damage"?

When the cell catches that error it will go to that site and when polymerase III approaches the blank spot it skips it and then polymerase V will tack on any base. This causes problems because it would NOT be the correct sequence!

If a mutation that inactivated telomerase occurred in a cell (telomerase activity in the cell=zero), what do you expect the outcome to be?

Without functional telomerase, the telomeres would shorten at each replication cycle, leading to eventual loss of essential coding information and death. In fact, there are some current observations that decline or loss in telomerase activity plays a role in the mechanisms of aging in humans.

What human genetic disease results from defects in nucleotide excision repair? Based on the number of complementation groups shown in the figure (see lecture notes on mutation and/or repair) how many proteins are found in the repair complex?

Xeroderma pigmentosum (really sensitive skin to sunlight) , 4

A certain compound that is an analog of the base cytosine can become incorporated into DNA. It normally hydrogen bonds just as cytosine does, but it quite often isomerizes to a form that hydrogen bonds as thymine does. Do you expect this compound to be mutagenic, and, if so, what types of changes might it induce at the DNA level?

Yes. It was cause CG to TA transitions.

Consider the following wild-type and mutant sequences: Wild-type: ....CTTGCAAGCGAATC.... Mutant: ....CTTGCTAGCGAATC.... the substitution shown seems to have created a stop codon. What further information do you need to be confident that it has done so?

You need to know the reading frame of the possible message.

Nucleotide Excision Repair

1. Recognition of damaged base 2. Assembly of multiprotein complex at the site 3. Cutting of the damaged strand several nucleotides upstream and downstream of the damage site and removal of these nucleotides

What are the major strategies for repair of mutations or DNA damage?

1. Repair/reversal of damage 2. Excision & replacement of missing/damaged base 3. Excision & replacement of a damaged segment

What part (which carbon) of the sugar at the end of the growing chain is the attachment point of the next nucleotide?

3' carbon

Which DNA polymerase exonuclease activity is involved in proof-reading?

3' to 5' activity

Which part (carbon) of the incoming nucleotide is attached to the growing chain?

5' carbon

What is the "direction" of DNA synthesis?

5' to 3'

Which DNA polymerase exonuclease activity is involved in the removal of primers?

5' to 3' activity

A certain acridine-like compound generates only single insertions. A mutation induced with this compound is treated with the same compound, and some revertants are produced. How is this outcome possible?

A +1 frameshift mutation can be reverted by two further single insertions so that the reading frame is re-established.

What is the mode of action of Base Analogs?

A chemical that can substitute for a normal nucleobase in nucleic acids. They are categorized in two separate groups, purine analogues and pyrimidine analogues. This results in a change in one base pair of DNA, specifically a transition mutation.

What is the role of the Origin of Replication?

A sequence of DNA at which replication is initiated on a chromosome, plasmid or virus. Larger DNA's may have multiple origins of replication at one time.

Differentiate between Transitions and Transversions

A transition mutation is the substitution of a purine for a purine or the substitution of a pyrimidine for a pyrimidine. A transversion mutation is the substitution of a purine for a pyrimidine, or vise versa.

How does AZT inhibit HIV replication?

AZT inhibits the enzyme (reverse transcriptase) that HIV uses to synthesize DNA, thus preventing viral DNA from forming.

Acridine orange is an effective mutagen for producing null alleles by mutation. Why does it produce null alleles?

Acridine orange causes frameshift mutations and frameshift mutations often result in null alleles.

How does Aflatoxin damage DNA? What type of mutation (Transition or transversion) is caused by Aflatoxin?

Aflotoxin causes an AP site (apurinic or apurimidic). An empty spot in DNA where a purine or a pyrimidine has been removed. It causes a transversion mutation.

What is the role of DNA Polymerase?

An enzyme that assembles the DNA nucleotides into polynucleotides using a preexisting strand of DNA as a template during DNA replication. It also proofreads the newly constructed strand of DNA to catch mis-paired bases and to fix them.

What is the role of Uracil Glycosylase in DNA repair?

An enzyme that reverts mutations in DNA using base excision repair.

In Base Excision and Nucleotide Excision Repair initially how many nicks in the backbone are required for each?

Base Excision requires one nick and Nucleotide Excision Repair two nicks.

How do base analogs lead to replication errors? What types of mutations do they cause (transitions, or transversions?) Compare their mode of action to tautomeric shifts of the standard bases.

Causes substitutions for DNA bases during replication and cause transition mutations. Tautomeric shifts are the same bases that are shifted into different forms and causes spontaneous mismatch of base pairs.

Which repair pathway recognizes DNA damage during transcription? What happens if the damage is not repaired?

DNA damage that stalls transcription is repaired by TC-NER (transcription-coupled nucleotide excision repair). Humans lacking this pathway suffer from Cockayne syndrome. A consequence of this defect is that a cell is much more likely to activate its apoptosis (cell suicide) pathway. Affected individuals are very sensitive to sunlight and have short stature, the appearance of premature aging, and a variety of developmental disorders.

What are Okazaki fragments, and why are they necessary in relation to bi-directional DNA replication?

DNA is copied in these shorts segments on the lagging strand of DNA and are later joined by DNA ligase.

What would happen if DNA synthesis were discontinuous on both strands?

DNA synthesis might take longer.

What base is most sensitive to UV and what effects does UV have on that type of base?

Thymine. It causes a thymine dimer where thymine binds to itself (another thymine).

What are the main classes of mutagens?

Transitions, Transversions and Indels.

Look at this website on Epigenetics and play the slide slow

http://www.pbs.org/wgbh/nova/body/epigenetic-‐mice.html

Watch this video on Epigenetics (53 minutes)

https://www.youtube.com/watch?v=D44cu7v9x1w

Watch this video on Epigenetics (13 minutes)

https://www.youtube.com/watch?v=M4boKud1MRk