Biochem Unit 4, Lectures 4-6

Tyrosine recombinase cleaves _________ strand(s) of DNA at a time, whereas serine recombinase cleaves ___ strand(s) at a time

1; 4

What are excinucleases?

A type of endonuclease that cleaves out large segments of DNA. Often used in excision of pyrimidine dimers

Describe the eventual result of deamination of cytosine to uracil through one cycle of replication

After one cycle of replication, one of the new DNA parent/daughter strands will be normal. However, in the strand that uses uracil as the template, a G will be substituted for an A, resulting in mutation

Which of the following enzyme in base excision repair is NOT required to repair depurinated nucleotides? A: Endonuclease B: Glycosylase C: Ligase D: DNA polymerase

B

Which exonucleases generates 3' overhangs? A: 3'-to-5' exonuclease B: 5'-to-3' exonuclease

B: 5'-to-3'

Describe how recombination of V, J, (D, for heavy chain) and C segments fosters antibody diversity.

Both light and heavy chains have multiple V, J, and C segments (heavy chains also have D segments). Through the process of recombination, one type of each segment is selected in both the light and the heavy chains. Since each segment differs from all others (i.e. one V segment is different from another V segment), we end up with lots of different possible combinations of segments. This in turn, creates a wide variety of antibodies, each highly specific for a different antigen

Cytosine is often methylated at what position? Is methylation at this position mutagenic?

C5; No it is not mutagenic if it is methylated at this position

Describe the process of the Base-excision repair pathway

DNA glycosylase cleaves the glycosidic bond between the base and the ribose ring (this cleaves off the base). Next, an endonuclease recognizes the abasic site and cleaves the portion of DNA that is lacking a nucleotide base. Next, DNA polymerase adds nucleotides to replace the gap left by removing the damaged/mutated nucleotides. Finally, DNA ligase seal the nick in the phosphodiester backbone left by DNA polymerase

Incorrect base incorporation during DNA replication is corrected by what repair mechanism(s)?

DNA polymerase proofreading or mismatch repair

True or false: Nucleotide base deamination does not allow for non-standard base pairing

False: It does!

True or false: Transposable elements comprise only a small portion of the human genome

False: Transposable elements make up ~45% of the human genome

True or false: DNA polymerase extends DNA in the 5'-to-3' direction. This means that new nucleotides are added to the 5' end of DNA.

False: this means that new nucleotides are added to the 3' end of DNA

What is the difference between DNA methylation and DNA alkylation

In DNA methylation: Only a methyl group is added; nucleotide base pairing is not affected. Conversely, in DNA alkylation either a methyl or ethyl group is added; can disrupt nucleotide base pairing, causing mutations

In Mismatch repair and nucleotide excision repair, DNA polymerase is used to extend how many strands of DNA? What about in repair by homologous recombination?

In both mismatch repair and nucleotide excision repair, DNA polymerase is used to extend one strand of DNA (the lone damaged strand). Conversely, in repair by homologous DNA recombination, DNA polymerase is used to extend two strands of DNA (double-strand breaks)

If recombination-recognition sequences are oriented head to head, what type of mutation can occur?

Inversion

Define transition mutations

Mutations that involve the conversion of purine to purine or pyrimidine to pyrimidine changes

If a transposon is inserted into an exon, it will disrupt ___________ function. Conversely, if a transposon is inserted into an enhancer or repressor, it will disrupt ________________

Protein function; gene expression

Define pyrimidine dimers

Pyrimidine dimers are a type of structural DNA damage caused when pyrimidines base pair with themselves along the same DNA strand (i.e., T-T). These pyrimidine dimers result in "kinks" in the DNA helix

What class of enzyme can catalyze recombination between sequences of DNA?

Recombinases

Which class of enzymes is responsible for recombining different combination of V, D, C, and J segments in antibodies, and where do these enzymes bind to the sequence?

Recombination Activating Gene (RAG) enzymes are responsible for recombining different combinations of segments into unique antibodies. RAG enzymes bind at specific recombination signal sequences (RSS).

Define depurination

Removal of the purine group from a nucleotide

Broadly describe the process of serine recombination

Serine recombinase is an enzyme bound by serine, which contains a hydroxyl group. The hydroxyl group of serine initiates nucleophilic attack on the opposite strand of DNA (cleaves four strands simultaneously, i.e, two strands from each duplex). This process frees up hydroxyl groups at the cleavage sites, and allows the enzyme to rotate the helices relative to one another. Ligase stitches the reoriented helices back together, resulting in two newly recombined helices

After the mismatch repair enzymes cleave the daughter strand an exonuclease is recruited to the daughter strand. What does it do?

The exonuclease binds at the location where the daughter strand is cleaved. Next the exonuclease "chews" away at the DNA daughter strand until it reaches the mismatch. Once it reaches the mismatch, it eliminates the misincorporated nucleotide

What gene encodes beta-galactosidase?

The lacZ gene encodes beta-galactosidase

Why does cell growth pause when cells are switched from a glucose media to a lactose media? Note: This lag in cell growth is referred to as the transition period

The lag in cell growth during the transition is due to the cells turning on expression of Beta-galactosidase, which is not normally produced in glucose media; it takes the cells time to produce the Beta-galactosidase protein

What is the major factor in determining whether DNA methylation (or ethylation) will be mutagenic?

The position at which the methyl (or ethyl) group is added. If the methyl/ethyl is added in a position that does not disrupt base pairing, it will not be mutagenic. However, if the methyl/ethyl is added at a position that disrupts base pairing, it is mutagenic

True or false: Depending on the cleavage plane utilized to resolve a Holliday junction, you can achieve different recombination outcomes

True

True or false: E. Coli can metabolize either glucose or lactose

True

True or false: Pyrimidine dimer damage is often caused by UV light

True

True or false: Recombination can occur between virtually any homologous sequences

True

True or false: There is a Holliday junction in the tyrosine recombination pathway, but not in the serine recombination pathway

True

True or false: Transposable elements can affect gene expression and can even cause mutations that result in disease

True

True or false: Uracil bases are not found in DNA

True

True or false: Viral DNA can incorporate into the bacterial genome via recombination between specific sequences

True

True or false: Nucleotide deamination occurs spontaneously

True: As we age, our genomes become more and more deaminated

True or false: Some DNA bases are normally methylated at a position that does NOT disrupt base-pair hydrogen bonds

True: However, it is important to keep in mind that sometimes methylation DOES occur at a position that dirupts Watson-Crick H-Bonds. This methylation is mutagenic

Deamination of Guanine results in what?

Xanthine

How many potential cleavage planes are there to resolve Holliday junctions?

2: The DNA can be curved in either the vertical or horizontal plane

True or false: Mismatch repair enzymes cleave only the 3' end of the mismatch in the daughter strand

False: Mismatch repair enzymes can cleave either the 3' end or the 5' end of the non-methylated daughter strand

True or false: It is abnormal for DNA to become methylated within a cell over time

False: Most DNA in a cell is methylated. In fact, only very recently synthesized daughter strands will not be methylated

True or false: Most proteins bind the DNA minor groove

False: Most proteins bind the DNA major groove

True or false: The relative orientation of sequences recognized by recombinase enzymes is irrelevant to the recombination outcome

False: Relative orientation of sequences recognized by recombinases DICTATES the recombination outcome

True or false: Allolactose produced by beta-galactosidase results in a decrease in beta-galactosidase levels

False: The presence of the enzyme product (allolactose) stimulates production of the enzyme

In regard to DNA methylation, which strands of DNA will be methylated following replication?

For a short period following replication, the template/parent strand is methylated whereas the new/daughter strand is not

Deamination of adenine results in what?

Hypoxanthine

Let's say we have a double-stranded break in the DNA. What is employed as a template to fix it?

If a DNA double-strand break is present, the homologous strands of DNA are used as a template for repair. Keep in mind that we have two copies of each DNA helix in our cells because our chromosomes are diploid

What is the general function of methyltransferases

Methyl transferases are responsible for demethylating nucleotides that were methylated in mutagenic positions

Define transversion mutations

Mutations that involve the conversion of purine to pyrimidine or pyrimidine to purine

What are retrotransposons?

Retrotransposons are a class of transposable elements that utilizes an RNA intermediate, which activates reverse transcriptase to copy the transposable element.

How do beta-galactosidase levels change when cells are switched from a glucose medium to a lactose media? Explain

There is little beta-galactosidase in glucose media; levels increase when switched to lactose. Beta-galatosidase is not necessary in a glucose media because the function of the enzymes is to break down lactose into glucose and galactose (sometime via an allolactose intermediate). However, in lactose, the presence of the enzyme's substrate (lactose) increases enzyme levels

Describe the example of antibody diversity that relates segments to shirts and pants

Think of the V segment (could also be J, D, or C) as a shirt and the J segment (could also be the C, D, or V segment) as pants. You have lots (thousands) of different shirts and pants in your wardrobe. This means that there are lots of combinations for lots of different outfits. unique outfit combinations can be thought of as unique antibody combinations

What does the term "abasic mean"?

This means that the base has been removed

Describe the five-step process by which homologous recombination repairs DNA double-strand breaks (long answer)

1. A double-strand break in one of the two homologs is converted to a double-strand gap by the action of exonucleases. strands with 3' ends are degraded less than those with 5' ends, producing 3' single-strand extensions 2. An exposed 3' end pairs with its complement in the intact homolog. The other strand of the duplex is displaced 3. The invading 3' end is extended by DNA polymerase plus branch migration, eventually generating a DNA molecule with two crossovers called Holliday intermediates (junction) 4. Further DNA replication replaces the DNA missing from the site of the of the original double-strand break 5. Cleavage of the Holliday intermediates by specialized nucleases generates either of the two recombination products. One set of products contains non-cross over products while the other possible set contains cross over products

What are four chemical changes to bases in a DNA molecule that can result in mutation?

1. Base alkylation 2. Deamination 3. Depurination 4. Pyrimidine dimer formation

What are the two ways in which incorrect base incorporation during DNA replication can be corrected for?

1. DNA polymerase proofreading 2. Mismatch repair

Recombination between homologous sequences can occur in what two circumstances?

1. In response to DNA damage 2. Spontaneously during meiosis

What serves as the template for DNA repair in the following three circumstances? 1. Mismatch repair 2. Homologous recombination 3. Nucleotide excision repair

1. Mismatch repair: Opposite strand of DNA in the SAME duplex serves as the template for DNA polymerase 2. Homologous recombination: Homologous DNA in ANOTHER duplex serves as the template for DNA polymerase 3. Nucleotide excision repair: Opposite strand of DNA in the SAME duplex serves as the template for DNA polymerase

What are two ways in which mismatch repair enzymes distinguish between the parent and daughter DNA strands?

1. The parent/template strand is methylated whereas the newly synthesized daughter strand is NOT methylated 2. The Newly-synthesized daughter strand is cleaved, whereas the parent strand is not cleaved

Broadly speaking, what are the two purposes of homology-directed recombination?

1. To repair DNA breaks 2. To generate genetic diversity during meiosis (crossing over)

Describe the six-step process of transposition by DNA transposons

1. Transposase binds to short DNA sequences 2. Transposase oligomerizes on DNA to form a "synaptic" complex 3. Transposase cleaves adjacent DNA via phosphoryl transfer reactions, excising the transposase + transposon DNA 4. The Transposase-DNA complex binds to target DNA 5. 3'-OH groups of the transposon DNA attacks the target DNA, integrating the transposon DNA 6. DNA polymerase and ligase seal the gaps

Name three examples of sequence specific ("site-directed") recombination that is catalyzed by enzymes.

1. V(D)J recombination in the immune system 2. Tyrosine and serine recombinases 3. DNA "cut and paste" transposons

How many total strands are involved in a Holliday Junction?

4

What is a double-stranded break?

A double stranded break refers to DNA damage in which both strands in the helix are broken

Describe the eventual result of depurination (specifically, a depurinated adenine) after one cycle of DNA replication

After one cycle of replication, one of the new DNA parent/daughter strands will be normal However, in the strand that used the depurinated A as a template, the new parent/daughter complex will be missing an A-T base pair.

Describe how allolactose stimulates beta-galactosidase expression

Allolactose stimulates beta-galactosidase expression by removing a transcriptional repressor from the operator of the lacZ gene that produces beta-galactosidase

Outline the general structure of an antibody, including the V, J, D, and C segments

Antibodies are composed of two "chains", a light chain and a heavy chain. Sequence diversity is greatest at the tips of the chains. The light chain contains multiple V, J, and C segments, whereas the heavy chain contains multiple V, D, J, and C segments.

Antibodies recognize highly specific _____________

Antigens

Depurination (N-glycosidic bond between base and pentose cleaved) is corrected what what repair mechanism?

Base excision repair (no DNA glycosylase needed)

Deamination (loss of exocyclic amino groups) is corrected by what repair mechanism?

Base excision repair employing DNA glycosylases

Deaminated or depurinated based can be removed via what process?

Base-excision repair

Lactose is metabolized by what enzyme?

Beta-galactosidase

DNA polymerase fails to recognize that an incorrect nucleotide has been incorporated, resulting in a mismatch. Which repair pathway will correct this mistake? A: Base excision repair B: Direct repair by methyltransferases C: Mismatch repair D: Nucleotide excision repair

C

What are the two classes of transposable elements?

Class 1: Retrotransposons Class 2: DNA transposons

In the final step of V(D)J recombination, what molecules are responsible for joining the recombination signal sequences back together to eliminate the sequence between V and J?

DNA non-homologous end-joining (NHEJ) proteins

Exonucleases work to eliminate misincorporated nucleotides from the daughter strand of DNA by "chewing away" all the DNA from the mismatch repair enzyme cleavage site through the misincorporated nucleotide. This results in a gap in the daughter strand DNA. How is this problem solved?

DNA polymerase fills in the gap created by the exonuclease, and then DNA ligase is recruited to correct for the "nick" left by DNA polymerase.

In homologous recombination repair of double strand-breaks in DNA, why is the 3' over hang created?

DNA polymerase works 5'-to-3', which means that nucleotides are added to the 3' end of DNA. The 3' overhang will base pair with the homologous sequence and recruit DNA polymerase, which will add new nucleotides to the 3' end and newly synthesized DNA will cover the break

If recombinase-recognition sequences are oriented head to tail, what mutation can occur?

Deletion or insertion is possible

Why is DNA glycosylase not needed to repair depurinated nucleotides?

Depurinated nucleotides do not have a base (they are abasic). Thus, DNA glycosylase is not need to repair depurinated nucleotides. Since there is no base, there is no glycosidic bond for glycosylase to cleave

Base alkylation is corrected by what DNA repair mechanism?

Direct repair involving methyltransferases

Pyrimidine dimers (bond forms between adjacent T (or C) nucleotides) are corrected by what repair mechanism(s)?

Direct repair or nucleotide excision repair promoted by DNA photolyases

How are endonucleases different from exonucleases?

Endonucleases cleave from within a strand of DNA, where exonucleases removes nucleotides from the end of a DNA strand

Define epigenetics

Epigenetics describes a change in gene expression not caused by change in DNA sequence, which can result in heritable changes in gene expression

Describe the general process by which pyrimidine dimers are excised from the DNA

Excinucleases, a type of endonuclease, recognizes pyrimidine dimers and excises damaged DNA. This results in a gap which is filled by DNA polymerase, and finally, the nick is sealed by DNA ligase

True or false: In step 1 of homologous recombination repair of DNA double-strand breaks, exonucleases degrade strands with 3' end more than those with 5' ends, producing 5' single-strand extensions

False: Exonucleases degrade strands with 3' ends LESS than those with 5' ends, producing 3' single-strand extensions

True or false: In V(D)J recombination, after water nicks the DNA, the 5'-OH group on the nicked strand initiates nucleophilic attack on the opposite DNA strand

False: In V(D)J recombination, after water nicks the DNA, the 3'-OH group on the nicked strand initiates nucleophilic attack on the opposite DNA strand

True or false: using DNA non-homologous end-joining proteins is not a viable mechanism to repair a DNA double-strand break

False: It is!

Describe an instance in which DNA methylation is normal as well as an instance when it is mutagenic

Normal: For a short period of time following DNA replication, the template strand is methylated and the new strand is not Mutagenic: A nucleotide is methylated so that it can no longer base pair which the correct Watson-Crick base. Instead it pairs with the wrong nucleotide, causing mutation

Describe the experiment that led McClintock to discover transposons.

Normally, in maize, if all cells are normal, you get a red kernel. However, a point mutation in the cells can cause the kernels to be yellow. McClintock found that there were also "mosaic" kernels that showed characteristics of both the normal pigmented kernel, as well as the mutated yellow kernel. She discovered that the mosaic kernels were the results of a transposable element that was inserted into the gene that causes pigmentation. This resulted in a mutation that leads to the mosaic kernel, which is effectively a mix of the normal and mutated phenotypes

Describe the process by which Recombination Activating Gene (RAG) enzymes recombine different V and J segments into unique antibodies (long answer)

RAG enzymes bind to recombination signal sequences (RSS), and catalyze a phosphoryl-transfer reaction by activating water, which nicks the DNA (Nick= one strand of DNA is broken), freeing up a 3'-OH. The 3'-OH on the nicked DNA then attacks the opposite strand of DNA, resulting in the formation of a DNA hair pin. After the DNA hairpins are formed, they are then broken (forming what looks like a double-strand break) to fuse the V and J segments together. The RSS then join together to eliminate the sequence between V and J. The joining together of these non-homologous ends is carried out by DNA non-homologous end-joining (NHEJ) proteins, which repair double-strand breaks in the DNA by simply fusing the broken ends of the DNA together

Deamination of 5-methylcytosine results in what?

Thymine

What is the one nucleotide base in DNA that cannot be deaminated?

Thymine. It doesn't have an amine group to remove

How can transposable elements generate genetic diversity?

Transposable elements can cause genetic changes by disrupting gene expression if they are inserted into a gene coding region or a sequence that regulates gene transcription

Define transposons

Transposons are mobile DNA elements that move around genomes

True or false: Not all chemical modifications of a nucleotide are mutagenic

True: Some chemical modifications of a DNA base are normal

Broadly describe the process tyrosine recombination

Tyrosine recombinase is bound by a hydroxyl group. The hydroxyl group of tyrosine initiates nucleophilic attack on one strand of DNA. . The same thing happens on the other DNA strand in the duplex. The nucleophilic attack on the DNA frees up a hydroxyl group on the nicked strand. The hydroxyl attack results in the formation of a Holliday junction. Next, all of the previous steps repeat in the other duplex, resolving the Holiday junction, and created to recombined duplexes

In the tyrosine recombinase recombination process, there are two _____________ (sequential/simultaneous) _____________ (single/double) stranded breaks, whereas in the serine recombinase recombination pathway, there are two _____________ (sequential/simultaneous) _____________ (single/double) stranded breaks

Tyrosine: Sequential; single Serine: Simultaneous; double

Deamination of cytosine results in the formation of what nucleotide?

Uracil

In V(D)J recombination what molecule is responsible for "nicking" one strand of DNA, and allowing for 3'-OH nucleophilic attack on the opposite strand of DNA?

Water

Does adding a methyl at O6 in guanine result in a mutation?

Yes, adding a methyl at position O6 in guanine will disrupt base pairing, resulting in a transition mutation

Fill in the blank: E. coli can use either glucose or lactose. Lactose is broken down by ________________ into ______________ and _____________. Sometime, ___________________ (same as blank 1) generates an intermediate called _____________, which the enzyme then breaks down into ____________ and _______________

beta-galactosidase; glucose; galactose; beta-galactosidase; allolactose; glucose; galactose