BIOMG 3320 Prelim 2

Why do cells need a way to repair their DNA

Because DNA is constantly attacked by the environment and there are errors that happen during DNA replication that are not always repaired. If this damage becomes permanent, it may lead to cell death, altered function, or cancer

What can DNA damage lead to?

DNA damage can lead to cancer, But we can also use DNA damage to induce more damage in cancer cells to kill them/cure cancer (i.e. radiotherapy & chemotherapy)

What are many cancers caused by?

Many cancers are caused by defective repair of DNA

Is DNA damage relatively common?

Yes, DNA damage is relatively common. It is estimated that every cell may experience up to 10^5 spontaneous DNA lesions per day.

What are some of the common cases of DNA damage?

Deamination (replacing amino group with carbonyl || Deaminated Cytosine turns into Uracil || Deaminated 5-Methylcytosine turns into Thymine || Depurination, which leads to a loss of a base || Crosslinking of bases, especially upon exposure to UV light || Reactive chemicals, which can react with DNA to cause damage

What are mutagenic chemicals also known as?

Mutagenic chemicals are also known as carcinogens

How can the potential of a chemical to induce DNA damage/mutation be measured by?

The Ames Test which is a specialized bacterial strain

What is the Ames Test used for?

The Ames Test is used to test for carcinogens, based on their mutagenicity

Describe the Ames Test and rationale behind it

Salmonella bacteria need histidine to grow. However, some Salmonella bacteria strains contain a missense or frameshift mutation in a gene required for histidine biosynthesis. || If you plate Salmonella bacteria with a mutated histidine gene and wait for them to grow, only about 25 out of 10^9 will spontaneously mutate to be able to grow. || However, if you expose a Salmonella bacteria to a mutagen, the mutagenic chemical will revert the missense or frameshift mutation, allowing cells to grow on medium lacking histidine. Thus, the rate of mutation to the histidine gene will be higher and more colonies will grow.

Finish this: the more mutagenic a chemical is,...

The more bacterial colonies will be formed

Is the bacteria used in the Ames Test defective of DNA repair?

No, the bacterium used in the Ames test is not defective of DNA repair.

What are the two categories of DNA damage?

Single strand breaks or double strand breaks. Each type requires a different DNA repair system/pathway

What is the general mechanism for single strand damage repair?

First recognize the offending base(s). Second, remove the offending base(s). Third, repair the resulting gap with a DNA polymerase and DNA ligase.

What are the principle types of single strand repair?

Mismatch repair and Base Excision repair

Is Mismatch Repair single strand or double strand repair

Single strand repair

How does the repair system in Mismatch Repair know which strand to repair?

Methylation of a DNA strand distinguishes the parent strand from the newly synthesized strands.

Does the newly synthesized strand or parent strand remain unmethylated and for how long?

The newly synthesized strand remains unmethylated for a short window of time.

Can you distinguish the new strand from the parent strand once the new strand becomes methylated?

No, you cannot. You only have the short window of time to distinguish the two strands

What is hemimethylated DNA

One strand (parent) is methylated and the other strand (newly synthesized) is not

What is the mechanism for mismatch repair?

When there is a mismatched base in a newly synthesized DNA strand, the MutSMutL complex, with ATP hydrolysis??, scans DNA for mistakes in both directions. || 2. The MutSL complex forms around the DNA at the mismatch and moves along the DNA to MutH bound at the hemimethylated site || 3. MutH cleaves the unmethylated stand (MutH is a site specific endonuclease and is inactive until it encounters a hemimethylated DNA site. || 4. DNA helicase separates the DNA into single strands at the nick site, with ATP hydrolysis and SSB protein protects the single strand regions, and exonuclease... || 5. DNA polymerase III adds new nucleotides and ligase seals DNA

Does Mismatch repair require ATP?

Yes

What is MutH and how does it become activated?

MutH is a site specific endonuclease and is inactive until it encounters a hemimethylated DNA site. It cleaves the unmethylated strand to...

Which DNA polymerase functions in Mismatch repair and what does it do?

DNA Polymerase III adds new nucleotides in Mismatch repair to prepare for ligase to seal the DNA

Is Base Excision Repair single or double strand repair?

Base excision repair is single stranded repair

What does Base-Excision repair do?

It repairs sites where U has been added by mistake

Does Base-Excision repair use ATP?

Some steps of base excision repair do not directly require ATP, but the ligation step typically requires ATP-dependent DNA ligase.

Describe the Base-excision DNA repair pathway

First, DNA glycosylase scans DNA for mistakes and cleaves the N-Beta-Glycosidic bond at the mistake || This results in an AP site (apurinic or apyrimidic site): a site with a missing base || Second, AP endonuclease cleaves the phosphodiester bond, creating a nick || Third, Nick translation occurs where DNA polymerase I uses 5' to 3' endonuclease activity to remove nucleotides at and downstream on nick and add new nucleotides || Finally, DNA ligase seals the nick.

What is recombination?

Recombination is the rearrangement of genetic information within or between DNA molecules

What are the 3 types of DNA recombination

1. Homologous recombination || 2. Site-Specific Recombination || 3. Transposons

What is homologous recombination?

Homologous recombination is an exchange of DNA strand between a pair of homologous duplex DNA sequences

What is site-specific recombination?

site-specific recombination requires a defined sequence motif, but with only limited sequence homology?

What are DNA transposons?

DNA transposons are DNA sequences that "jump" at specific sequences or randomly

Who discovered transposable elements while studying maize?

Barbara McClintock

What is nonhomologous recombination and how was it discovered?

It is recombination that allows the movement of transposable elements, "jumping genes". It was first observed in maize where reddish streaks on the corn grains were caused by transposons

Describe the mechanism of Homologous Recombination

1. Alignment of 2 homologous DNA molecules || 2. Introduction of breaks in the DNA || 3. 5' end processing, which results in the formation of 3' ssDNA tails. This is done via recBCD, which has helicase/exonuclease activity, and processes the 5' ends to generate overhanging single stranded 3' ends. || 4. Strand invasion and formation of heteroduplex DNA || 5. DNA synthesis and ligation || 6. Cleavage and resolution of Holiday Junction

What is recBCD and what does it do?

recBCD has helicase/exonuclease activity and it processes the 5' ends of DNA (after formation of DSBs) to generate overhanging single stranded 3' ends.

What does heteroduplex formation do?

It "checks" sequence homology

What protein promotes strand invasion

RecA protein promotes strand invasion

What is the RecA protein central to

HR:recombinase

How does the RecA protein promote strand invasion?

First, the RecA protein binds tightly and cooperatively to the sugar-phosphate backbone in the single stranded DNA regions || Second, it mediates the search for matching sequences || Third, it catalyzes the strand exchange of homologous DNA molecules, with ATP

Does the process of RecA promoting strand invasion require ATP? If so, which step?

Yes, the catalysis of the strand-exchange of homologous DNA molecules requires ATP

What happens during the DNA synthesis and ligation step of Homologous Recombination?

The ends are extended using homologous strands as templates, which leads to the formation of double Holiday junctions

What are Holiday Junctions

a four-way, branched nucleic acid structure that forms during genetic recombination, where two double-stranded DNA molecules exchange segments.

How are Holiday Junctions resolved in Homologous Recombination (last step?

First, the holiday junction is rotated from the closed formation to the open formation || There are 2 types of resolution, which involve cleavage by specialized nucleases at opposing DNA sites

What does Vertical Junction Cleavage lead to ?

It leads to 2 separate dsDNA with nicks made up wholly of original DNA. As such, no chromosomes cross over

What does Horizontal Junction Cleavage lead to?

It leads to 2 dsDNAs with half original and half new DNA with nicks. And causes chromosomes to cross over.

What is recombination used by cells for?

For accurate chromosome segregation during division, genetic diversity within a population, and serves as DNA repair mechanisms

Describe DNA Recombination during meiosis in eukaryotes

First, homologous chromosomes align in the middle of the cell during prophase I || Second, physical links form between sister chromatids from the two homologous pairs. || Thus, it allows for diversity in the haploid gametes

What is Nondisjunction and what does it result in?

Nondisjunction is unequally distributed chromosomes and it could happen during meiosis I or meiosis II. It results in aneuploidy.

What is aneuploidy and what is the relationship between it and age?

Aneuploidy is a condition where a cell has the wrong number of chromosomes. The older the maternal age, the higher the chance of (producing gametes with) aneuploidy

What can failure of homologous chromosomes to form covalent crossovers during meiosis result in?

It can result in aneuploid gametes

What is homologous recombination in bacteria used primarily for?

It is primarily a DNA repair process

How can DNA recombination be used as a powerful tool in gene manipulation?

By creating a DNA fragment with two ends homologous to the genome region that you want to swap/manipulate the genome. After swapping the genome, it can allow gene knock out or transgenic variants

What are knock outs?

Deleting genes

What are transgenic variants?

Introducing modified versions of genes

Are knock outs or transgenic variants critical to study gene function

Transgenic variants are critical to study gene function

Describe the mechanism of Yeast Gene Deletion

First, design DNA fragments with the ends homologous to the gene of interest. || Second, add these DNA fragments to the yeast cells || Third, Use in vivo Homologous Recombination apparatus, forming holiday junction in 1/10^8 cells || Fourth(last), select the cells with the selectable marker which indicates the successful deletion of the gene.

What does the ability to knock out genes permit

Permits the creation of mouse models for human diseases

What does HR make possible?

Makes it possible to target and modify a specific gene or region of interest.

What are all the methods of DNA sequencing

Sanger method || Automated sequencing based on fluorescent tag || second/next generation sequencing, which includes pyrosequencing, reversible terminator sequencing, Single Molecule Real Time (SMRT) sequencing, and nanopore sequencing

What did Frederick Sanger publish in 1977?

DNA sequencing with chain-terminating inhibitors

What did Maxam and Gilbert publish in 1977

DNA sequencing by chemical degradation

What did Leroy Hood and Wilhelm Ansorge publish in 1986?

Automated sequencing based on fluorescent tag

When did the Human Genome sequencing project begin?

1990

When did Next gen sequencing begin?

2005

Describe Sanger Sequencing

First, design a primer that is complementary to the template with part of its sequence known. The primers are labeled with radioactive phosphate || Next, add dNTP and ddNTP (having H at the 3' Carbon instead of -OH), therefore once a ddNTP is added, there is no more dNTP that can be added to the strand || Third, we have four groups, each containing all dNTPs (dATP, dTTP, dCTP, and dGTP) and ONE of the ddNTPs (ddATP or ddTTP or ddCTP or ddGTP). || Fourth, we load and run the sample (from all four tubes) onto the electrophoresis gel. The closer the band is to the bottom of the gel, the shorter the DNA fragment is || Thus, the tube with the band at the very bottom of the gel is where the first nucleotide is added and synthesis is terminated || Thus, sequencing is based on chain terminating called dideoxy chain-termination sequencing.

In Sanger Sequencing, what are the primers labeled with?

Radioactive phosphate

In Sanger Sequencing, what is the purpose of the ddNTPs?

They have an H at the 3' carbon instead of -OH. This allows for no more dNTP to be added to the strand once a ddNTP is added.

What are the ingredients for Sanger Sequencing???

Four groups with all four dNTPS and only one ddNTP per group || Primer labeled with radioactive phosphate || Gel electrophoresis

Which sequencing reactions have unknown

Describe Automated Sequencing Reaction

First, in each reaction tube, primers, template of unknown sequence, DNA polymerase, all 4 dNTPs, and all 4 ddNTPS labeled with fluorescent dyes || After the synthesis, denature the dsDNA and run the gel || Next, use a detector that allows recognition of the fluorescent dye on ddNTP so we can use computer algorithms to reveal the sequence of the unknown DNA

How is automated sequencing different from sanger sequencing????

Sanger sequencing uses radioactive phosphatase while automated sequencing uses ddNTPS labeled with fluorescent dyes. Thus we cannot see different fluorescence for each different ddNTP in sanger sequencing || Automated sequencing combines all 4 ddNTPs with all 4 dNTPS, while sanger sequencing only combines 1 ddNTP with all 4 dNTPS, separating it into 4 groups each corresponding to a different ddNTP || Automated sequencing uses a computer program to resolve the results while Sanger sequencing visualizes the results on a gel.

How many base pairs can Automated Sequencing Reaction Resolve?

600-750 bp

What are the three types of second/next generation sequencing

Pyrosequencing || Reversible terminator sequencing || Single molecule real time (SMRT) sequencing

What does Reversible Terminator Sequencing use?

It uses ddNTP analogs that contain the following key parts: a base tagged with a fluorescent tag, and a 3' OH group attached to an allyl group

Does the base's fluorescent tag interfere with the binding by DNA polymerase?

No, the tag does not interfere with binding of the nucleotide by DNA polymerase

What is the purpose of the allyl group attached to the 3' OH group in Reversible Terminator Sequencing?

This group blocks the 3'OH position. Thus, when the allyl group is attached to the 3' OH group, new nucleotides cannot attach to the 3'-OH to extend the DNA chain. However, when the allyl group is removed, the 3' OH group of the growing DNA chain is exposed and synthesis can continue.

What is the mechanism of Reversible Terminator Sequencing?

First, fluorescent dNTPs with allyl groups at the 3' OH are added, and only one of the dNTPs will actually be incorporated into the DNA chain (based on Watson-Crick base pairing) || Second, all unattached dNTPs are washed away || Third, Fluorescence of the previously incorporated dNTP is observed and recorded by some detector/scanner || Fourth, a reagent that removes both allyl group and fluorescent dye is added to remove the fluorescent signal and allow the DNA synthesis to proceed at the 3' OH. || The cycle is repeated to synthesis and read the DNA sequence, one base at a time

How many bases are added and read at a time in Reversible Terminator Sequencing and why is this important

Only one dNTP is added at a time. This is important because it allows us to obtain a clear signal, after excess dNTPs are washed away after each cycle.

In Reversible Terminator Sequencing how can many templates be sequenced at the same time?

The DNA strands are attached to a chip where you can see the sequences of all template strands loaded onto the chip simultaneously. This chip has many lanes, allowing millions of DNA molecules to be sequenced at once.

Which sequencing method as significantly advanced the human genome project

The reversible terminator sequencing as it allows millions of DNA molecules to be sequenced at once (DNA strands attached to chip with many lanes)

What types of reads does the reversible terminator sequencing method usually produce?

Relatively short sequencing reads

What is the mechanism of Single Molecule Real Time (SMRT) Sequencing

SMRT sequencing only uses a single molecule of dsDNA, which can be up to 10^4 bp long. || A DNA hairpin with known sequence is ligated to either end of the dsDNA that you want to sequence, which converts the dsDNA to a large circular DNA molecule || A single molecule of DNA polymerase will be attached to the bottom of each pore || DNA is synthesized in each pore in real time by the DNA polymerase, using four kidneys of dNTPs, each one labelled with a different fluorescent dye || The dNTPs come in contact with the DNA polymerase freely, but transiently, unless the dNTP is the correct W-C bp for the extending DNA chain || If the dNTP is the correct W-C bp for the extending DNA chain, then the dNTP will stay at the DNA polymerase for a few moments before getting attached to the growing DNA strand (where signal is captured) || The fluorescent tag for SMRT dNTPS is automatically removed once a nucleotide reacts with the growing DNA chain and attaches to it

How is the fluorescent tag removed in SMRT sequencing

Since the fluorescent tag is at the 5' phosphate group, it is automatically removed once the nucleotide reacts with the growing DNA chain and attaches onto it.

When is the signal from each dNTP captured

When the dNTP with the correct Watson-Crick base pairing for the extending DNA chain comes into contact with the DNA polymerase, it stays there for a few moments before getting attached to the growing DNA strand

What happens before SMRT Sequencing

A DNA hairpin with known sequence is ligated to either end of the dsDNA that you want to sequence, which converts the dsDNA to a large circular DNA molecule

Why does a SMRT cell have many wells/pores on it (150,000 pores/cell)?

It ensures that you only see the signal at the very bottom of the wells/pores || Also note that each pores diameter is only 70nm

How are these mechanisms different vs the same?

Which sequencing methods use fluorescent dyes?

Reversible Terminator sequencing (uses a base tagged with a fluorescent tag) || SMRT sequencing (uses four kinds of dNTPS,each labeled with a different fluorescent dye)

Where does the challenge of sequencing come from?

The challenge of sequencing comes from the assembly of the sequenced fragment into a full genome.

How is assembly of non-continuous sequenced fragments done?

It is done by a computing algorithm that aligns overlapping fragments. This process uses sequencing depth and deep sequencing.

What is sequencing depth?

It is the average number of times that a specific nucleotide position in a genome is sequenced

What is deep sequencing?

It is sequencing with a depth of 100x to 1000x. This type of sequencing is especially important to note any possible single-nucleotide mutations within genomes

How does deep sequencing allow the noting of possible single nucleotide mutations within genomes?

When comparing cancerous and non-cancerous genomes from the same organism, to determine which mutation could have contributed to the cancer origin

Does Classical Sanger Sequencing introduce 4 ddNTPs into a single reaction

No, you actually need four separate reactions in separate reaction vessels, one corresponding to each specific type of ddNTP

What is the goal and subgoal of the Human Genome Project?

To sequence all 3.2 billion base pairs of human genomes. The subgoal is to sequence genomes of other organisms and develop technology

What is the Bermuda Principle?

All data relating to the HGP will be free and available within 24 hours

What is Hierarchical Shot Gun Sequencing?

It was used in the Human Genome Project || The genome is randomly divided into 30,000 segments of 100,000-200,000 bp || Then these fragments are further divided and sequenced || Then the fragments are aligned using overlapping segments

Who/What is Celera Genomics?

It intended to patent 6000 genes, spurred the HGP to finish faster, and claimed to sequence within 3 years using whole genome shotgun sequencing

What is Whole Genome Shotgun Sequencing?

It divides the whole genome into many short fragments, then sequences them, and computationally aligns the overlapping fragments to reconstruct the genome