BIOC 406 Exam 3 (General)

What is single strand damage?

The “everyday” damage that is the more common, less catastrophic type where only one side of the double helix is affected, so the other strand acts as a backup template for repair.

What are the causes of single-strand damage?

Oxidative stress, UV radiation, spontaneous hydrolysis, replication errors

What are double-strand breaks?

The dangerous damage where both strands are severed simultaneously, where the chromosome is essentially snapped in two.

What are the causes of double-strand breaks? (DSBs)

Ionizing radiation, chemotherapy drugs, replication fork collapse, free radicals

What is BRCA1 and BRCA2?

These are tumor suppressor proteins that help repair double-strand breaks (DSBs) via homologous recombination (HR) that help recruit repair machinery and process the broken DNA ends

What is Nucleotide Excision Repair (NER)?

A DNA repair mechanism what fixes bulky lesions due to UV-induced thymine dimers

How does Nucleotide Excision Repair (NER) work?

Cuts out ~25-30 nucleotide patch and has Ligase seal it

What is Base Excision Repair (BER)?

A DNA repair mechanism that fixes single damaged/modified bases (e.g. oxidized bases)

What are the key players in Base Excision Repair (BER)?

Glycosylase, AP endonuclease, Polymerase, Ligase

What is Mismatch Repair (MMR)?

A DNA repair mechanism that fixes mismatched bases as well as small insertions/deletions (indels)

What are the key players in Mismatch Repair (MMR)?

MutS, MutL, MutH, Polymerase, Ligase

What is Non-Homologous End Joining (NHEJ?)

A DNA repair mechanism that fixes double-strand breaks by quickly gluing ends together

What are the key players in NHEJ?

Ku proteins, DNA, PKcs, Ligase IV

What is Homologous Recombination (HR)?

A DNA repair mechanism that fixes double-strand breaks by using the sister chromatid template

What are the key players in Homologous Recombination (HR)?

BRCA1, BRCA2, RAD51

How does CRISPR-Cas9 work?

It cuts both strands of DNA (double-strand break) and as long as the cell is provided with a template DNA, it will use HR to copy the template and insert its sequence. Without a template, the cell uses error-prone NHEJ which causes gene knockout insertions/deletions.

What is the issue with tautomerization?

Refers to the concept where a base temporarily shifts to an alternative chemical form (e.g. adenine acting like guanine) which causes a mispairing during replication.

How to fix tautomerization?

DNA Polymerase’s 3’→5’ proofreading exonuclease detects and removes mis-incorporated bases caused by tautomerization.

True or False: For single stranded repair, the BRCA1 and BRCA2 proteins are critical.

False

True or False: Homologous repair (HDR) mechanisms are used during CRISPR-Cas9 engineering when a template DNA is present

True — HDR naturally responds to DNA double-strand breaks by copying information from a homologous template. CRISPR-Cas9 takes advantage of this mechanism.

True or False: Ligases are used for Base Excision Repair, Nucleotide Excision Repair, and Non- Homologous End Joining (NHEJ)

True

True or False: DNA mismatch repair mechanisms can fix short deletions and insertions

True

True or False: Tautomerization are repaired by Proofreading functions of DNA Polymerases

True

True or False: Human DNA Polymerase performs fewer proofreading events.

False

True or False: The DNA Repair mechanisms in yeast are simpler, leading to more errors in DNA Replication

False

True or False: The speed of DNA Polymerase is faster in yeast which results in fewer mutations.

True

True or False: The Helicase activity of yeast is faster, allowing for greater accuracy.

False

What is the speed of Human DNA Polymerase?

~50 nucleotides/second

What is the speed of RNA Polymerase?

~20-40 nucleotides/second

True or False: RNA Polymerase are slower than DNA Polymerase

True

True or False: Convergent evolution has resulted in similar RNA and DNA Polymerase active sites

True

True or False: Active sites of both Polymerases have Mg2+ ions mediating catalysis of the 3’ ribose sugar.

True

True or False: Topoisomerases and helicases are needed for both DNA Replication and Transcription

True

What does Topoisomerase do during DNA replication?

It cuts and rejoins DNA to relieve positive supercoil tension created by Helicase prior

What does Topoisomerase do during DNA transcription?

I and II relieves the positive supercoiling ahead and negative supercoiling behind RNA Polymerase

What does Helicase do during DNA replication?

The MCM complex unwinds the double helix at the replication fork and creates two single-stranded templates

What does Helicase do during DNA transcription?

The TFIIH complex (specifically the XPB and XPD subunits) open up the DNA double helix at the promoter during initiation. Without it, RNA Polymerase II cannot access the template strand and transcription wouldn’t occur.

What is Splicing

The process of pre-mRNA removing Introns via a spliceosome so that the exons would join together to create specific proteins

Where is DNA in prokaryotes?

Cytoplasm

Where is DNA in eukaryotes?

Nucleus W

What is the DNA structure of prokaryotes?

Circular and naked

What is the DNA structure in eukaryotes?

Linear and wrapped in histones

What is the DNA size in prokaryotes?

~4 million bp

What is the DNA size in Eukaryotes?

~3 billion bp

What are the types of RNA Polymerases in prokaroytes?

There’s just one

What are the types of RNA Polymerases in eukaryotes?

Pol I, II, III

What is the promoter sequence in prokaryotes?

TATAAT + TTGACA

What is the promoter sequence in eukaryotes?

TATA box + other elements

What is/are the transcription factor(s) in prokaryotes?

Sigma factor

What are the transcription factors in eukaryotes?

TFIIA, B, D, E, F, H (large pre-initiation complex

What are the unique characteristics of eukaryotic transcription that differs from prokaryotes?

Splicing, 7-methylguanosine 5’ cap added, Poly-A tail 3’ cap added

What are the unique characteristics of prokaryotic transcription that differs from eukaryotes?

Transcription is simultaneously coupled with translation

Is eukaryotic mRNA polycistronic or monocistronic?

Monocistronic: each gene gets its own separate mRNA

Is prokaryotic mRNA polycistronic or monocistronic?

Polycistronic— multiple genes are transcribed together into one long mRNA, where each protein has its own start codon within the same mRNA

True or False: In prokaryotes, splicing occurs co-transcriptionally

False — splicing only occurs in eukaryotes.

True or False: There are three RNA Polymerases in prokaryotes.

False — Eukaryotes are the ones that have three RNA polymerases. Prokaryotes only have one.

True or False: Prokaryotes and Eukaryotes have a TATAAT sequence at its promoter that is required for RNA Polymerase recruitment.

True — The eukaryotic equivalent of the TATAAT sequence is the TATA box, which indirectly is the one that recruits RNA polymerase

True or False: The TFIID component of Eukaryotes contains the Helicase activity

False — The TFIID, the promoter recognition factor, binds to the TATA box and is what bends the DNA that recruits other transcriptional factors. It has nothing to do with Helicase. What is being mistaken is the TFIIH which uses helicase to unwind DNA and begin transcription.

True or False: Prokaryotic and Eukaryotic mRNA are polycistronic.

False — only prokaryotic mRNA is polycistronic.

Put these statements in order:

I. After RNA Pol II has synthesized ~20-30 nucleotides of new mRNA, the 5’ end of RNA is detected which makes a capping enzyme add a 7-methylguanosine cap that protects the mRNA and is marked for ribosome recognition.

II. TFIIH’s XPB subunit uses ATP hydrolysis to open PIC, allowing helicase to unwind the DNA and create a transcription bubble where RNA Polymerase II can now access the template strand.

III. TFIID (that contains TBP) lands on the TATA box. TBP bends DNA ~80 degrees which creates a platform for TFIIA, TFIIB, RNA Pol II, TFIIF, TFIIE, and TFIIH to assembling, forming the Pre-Initiation Complex (PIC).

IV. TFIIH’s kinase domain then phosphorylates the C-Terminal Domain (CTD) of RNA Pol II at either Serine 5 (makes RNAP II break free from the promoter) or Serine 2 (go into elongation mode).

III, II, IV, I

True or False: There are more exons in the human genome than introns.

False — Exons only make up ~1-2% of the human genome whereas introns make up ~24%. The average human genes may have barely more exons, but at the whole genome scale (pseudogenes, repetitive intron-derived elements, overall genomic composition), introns dominate overwhelmingly.

True or False: DNA methylation and histone N-terminal tail acetylation are found within transcriptionally inactive areas of the genome.

False — DNA methylation is indeed a gene silencer, but N-terminal tail acetylation, aka Histone acetylation, is a gene activator.

True or False: The polyadenylation signal AATAAA in the RNA signals for endonuclease cleavage and addition of the poly(A) tail by poly(A) polymerase.

False — The mechanism itself is correct, but the actual polyadenylation signal in mRNA is in the RNA sequence (not the DNA sequence like in the statement). So the actual polyadenylation signal in mRNA is AAUAAA.

True or False: Mediators promote chromatin condensation and gene repression.

False — A mediator is a large co-activator complex of ~26 subunits and acts as a physical bridge between distant enhancers and the RNA Pol II machinery at the promoter, relaxing chromatin and promoting gene activation.

Is DNA methylation a gene silencer or gene activator?

Gene silencer

Is Histone acetylation a gene silencer or gene activator?

Gene activator (HAT adds it)

Is Histone deacetylation a gene silencer or gene activator?

Gene silencer (HDAC removes it)

Is H3K4 methylation a gene silencer or gene activator?

Gene activator

Is H3K27 methylation a gene silencer or gene activator?

Gene silencer

Is H3K9 methylation a gene silencer or gene activator?

Gene silencer

List the correct order of polyadenylation:

I. AAUAAA appears in nascent mRNA

II. Endonuclease cleaves mRNA ~10-30 nt downstream

III. Poly(A) Binding Proteins stabilize the tail

IV. RNA Pol II transcribes past end of gene

V. CPSF recognizes AAUAAA

VI. Poly(A) Polymerase adds ~200 adenines to new 3’ end

IV, I, V, II, VI, III

What is S-Adenosyl Methionine (SAM)?

AKA the universal methyl group donor — Structurally an Adenosine + Methionine joined together where the methionine part that carries the methyl group (CH3) gets transferred by an enzyme onto their target, turning SAM into SAH (S-Adenosyl homocysteine).

What enzymes use SAM?

N7MTase for translation initiation (5’ cap on mRNA), DNMTs for gene silencing (cytosine methylation), and HMTs for chromatin regulation (Histone tail mark methylation)

True or False: Chromatin remodelers need SAM to move nucleosomes.

False — Chromatin remodelers use the energy from ATP hydrolysis to reposition nucleosomes accordingly so DNA transcription can be activated or deactivated.

What do HATs do?

opens chromatin and activates the gene

What do HDACs (Histone Deacytlases) do?

Closes chromatin and silences a gene

What do HMT methylation on H3K4me3 do?

Activates the gene

What do HMT methylation on H3K27me3 do?

Silences the gene

What do DNMT methylation on DNA do?

Permanently turn off a gene