Molecular Biology Exam 3

What is a Water-Mediated Point Mutation?

hydrolysis leading to deamination (removal of an amino group from compound) causing a change in a single DNA nucleotide such as Cytosine being deaminated to Uracil

What is a Nitrous Acid Induced deamination?

when deamination is caused by other chemicals and environmental pollutants

What is Depurination?

hydrolytic attack of N-b-glycosyl bond between base and pentose breaking the connection between the DNA backbone and the base creating an abasic site

What is Oxidative damage?

when irradation and aerobic metabolism are continually producing reactive oxygen causing removal of bases and strand breaks

What is Alkylation?

addition of an alkyl group to atoms in nucleotide bases or phosphodiester backbone

What is Ionizing radiation?

when DNA is damaged from UV light such as gamma rays and X rays causing single and double strand breaks

How does the Ames test work?

is used to identify potential genotoxic chemicals; mutant strain of salmonella that can’t produce histidine is mixed liver extract from rat and placed on agar plate with medium lacking histidine and filter paper is placed on the plate and if chemical is genotoxic it will mutate cells allowing them to grow on the histidine negative plate (have now mutated to now produce histidine)

What is genotoxic?

DNA reactive compound that causes changes in genomic DNA

What is a auxotroph?

a mutant organism that requires a particular additional nutrient which the normal strain does not

What is a reversion mutation?

a secondary mutation that reverses the effect of an initial mutation in a gene

What’s the similarities of BER (Base Excision Repair) in bacteria and eukaryotes?

• use DNA glycosylase to recognize and remove the damaged base

• Forms an AP site after the base is removed

• Uses AP endonuclease to cut the DNA backbone at or near the AP site

• Uses DNA polymerase to add the correct nucleotide

• Uses DNA ligase to seal the remaining nick in the DNA backbone

• Restores the correct DNA sequence and DNA integrity

What’s the differences of BER (Base Excision Repair) between bacteria and eukaryotes?

1. Bacteria

• uses bacteria DNA glycoslyases

• uses DNA polymerase I as main repair polymerase

• LigA as bacterial DNA ligase

• Uses endonuclease IV and exonuclease III as AP endonuclease

• Is less complex (no nucleus) and has more simpler organization

2. Eukaryote

• uses eukaryotic DNA glycolases

• uses DNA polymerase b in short-patch BER for main repair polymerase

• uses DNA ligase III or DNA ligase I as DNA ligase based on pathway

• uses APE1 as AP endonuclease

• organizes DNA into chromatin inside a nucleus and is more complex with chromatin and other repair proteins

What’s the difference between NER (Nucleotide Excision Repair) vs BER (Base Excision Repair)?

1. NER

• repairs large bulky lesions by removing DNA from either side of them

• does not require recognition of the damaged nucleotide

• uses excinuclease

2. BER

• repairs damaged nucleotides that cause only small DNA distortion and nicked DNA that does not have ligatable junctions

• needs recognition of the damaged nucleotide

• uses different glycoslylases to recognize damaged nucleotides

What does MMR (mismatch repair) do?

fixes error in DNA replication; can repair single nucleotide mismatches and loops made up of up t0 4bp

uses the parental strand to repair the newly synthesized strand

1. Recognizes mismatch using MutS

2. Identify the new DNA strand using MutL

3. Cut out the mistake using exonucleases and MutH (endonuclease) at GATC site

4. Replace the missing DNA using DNA polymerase to fill in the correct nucleotides using old strand as template

5. DNA ligase seals the kink back up

(eukaryotes use MSH and MLH/PMS proteins instead)

What does Double Stranded DNA repair do?

Two types

1. Non-homologous end joining (NhHEJ): takes the two broken ends of the DNA and “glues” them back together; done when cells have entered the cell cycle and repair needs to be fast (leads to death if not fixed)

2. Homologous Recombination: uses the other chromosome as a template to repair the break site; if maternal and paternal chromosomes are different in this DNA region it can lead to loss of heterozygosity

What does Nucleotide Excision Repair do?

repairs large bulky regions; disease such as cancer can develop if not fixed

1. DNA is damaged and forms a bulky region

2. UvrA₂ UvrB complex scans for DNA damage and separates DNA strands forming a single stranded DNA bubble that contains the lesion

3. UvrA dissociates leaving UvrB tightly bound to damaged DNA

4. UvrB recuits the exinuclease UvrC which cuts the DNA on both sides of the damage

5. the helicase UvrD removes the 12-13 nucleotide DNA fragment

6. Pol I synthesizes the new DNA and ligase seals the nicks

eukaryotes use XCP to scan for abonromalities and recruits XPB and XPD in place of the UvrA₂ UvrB complex; RPA binds to the DNA bubble and positions nucleases XPF (3’ side) and XPG (5’) side and they both nick the DNA and helicase removes the 24-32 nucleotide fragment, Pol replaces DNA and ligase seals the nicks

What does Base Excision Repair do?

repairs damaged nucleotides that cause only small DNA distortion and nicked DNA that does not have ligatable functions; if BER fails damaged bases remain which can cause replication errors, mutations and possibly cancer

1. DNA gycolsylase cleaves nucelotide base from pentose leaving an apurinic or acrymidinic site (AP)

2. Single stranded DNA is cleaved by AP endonuclease at abasic site creating nicked DNA

3. DNA segment is removed and replaced by Pol I

4. DNA ligase seals nick

For eukaryotes step 3 is divided into long and short patches

Long Patch

3. pol replaces ~10 nucleotide creating a DNA flap

4. flap endonucleases removes nucleotides and DNA ligase seals nick

Short Patch

3. Pol B removes and replaces damaged nucleotide, ligase seals nick

What are the similarities between DNA replication and transcription?

• nucleic acid synthesis in the 5’ to 3’ direction

• nucleic acid template strand required

• major steps are initiation, elongation, and termination

What are the differences between DNA replication and transcription?

1. Replication

• initiation requires a primer

• whole genome copied

• both DNA strands are template

2. Transcription

• defined section of genome transcribed

• only one DNA strand serves as a template

How are template strands determined?

by the promoter

What are the steps of transcription?

1. RNA polymerase binds to the promoter forming a closed complex in which the bound DNA is intact

2. The DNA is partially unwound downstream of the transcription start site forming the transcription bubble and an open complex

3. Transcription is initiated in the open complex creating a conformational change that converts the complex to the form required for elongation

4. Elongation starts causing promoter clearance (transcription moving along DNA away from promoter). This creates the tightly bound elongation complex

What are the principles of transcription?

1. In the transcription bubble RNA polymerase uses DNA template strand to make RNA

2. DNA enters the RNA polymerase and is unwound. The pin keeps the DNA single stranded

3. RNA exits the RNA exit channel

4. rNTP enter the RNA polymerase via the rNTP entry channel

What is the structure of of bacterial RNA polymerase?

1. Two alpha subunits: determinants for RNA polymerase assembly; binds to DNA at promoters (non-sequence specific) and upstream of promoters (sequence specific); binds to regulatory factors

2. Sigma factors: involved in recognition of specific promoter sequences

3. Beta subunit: has part of the active center that is responsible for RNA synthesis; non-specifically interacts with DNA and RNA

4. Beta prime subunit: same as beta subunit

5. Omega subunit: facilitates assembly and stabilization of RNA polymerase

What is the function of the bacterial promoter?

specific DNA region that initiates transcription and determines which is the template strand; upstream of gene

What is the lac operon expirement?

lac promoter in e. coli expresses genes in the lac operon which encodes for genes for lactose metabolism; lactose activity is measured by the enzyme converting the colorless x-gal into a blue molecule and level of blue is measured

What is DNA footprinting?

method used to determine region of DNA bound to a protein (such as a sigma factor)

1. randomly cleave DNA samples with DNase1

2. Isolate labled DNA fragments and denature, only labeled strands are detected in next step

3. Separate fragments by polyacrylamide gel electrophoresis and visualize radiolabeled bands on x-ray film

What are the steps of Initiation?

1. The RNA Polymerase holoenzyme binds to promoter creating a closed complex (with sigma factor binding directly to promoter)

2. The closed complex spontaneously converts to an open complex

3. The DNA relaxes as the DNA duplex is melted, with a DNA loop entering the open complex

4. If the promoter is cleared than sigma factor leaves the RNA polymerase, and transcription proceeds

What are the steps of elongation?

1. Enzyme leaves promoter region after synthesizing a short chain

2. RNA polymerase unwinds a small region of the double-stranded DNA creating a strucuture called transcription bubble

3. Template reads 3’ to 5’ (RNA)

4. RNA exists the RNA exit channel

5. rNTP enter the RNA polymerase via the rNTP entry channel

What are the steps of Termination?

Has two types:

1. p (rho) dependent: template strand codes for the rho utilization (rut) site which is CA-rich sequence

• rho factor is a helicase

• rho helicase binds to mRNA at the rut site

• rho helicase (powered by ATP hydrolysis) moves toward RNA polymerase

• helicase unwinds RNA-DNA duplex, releasing transcript

2. p (rho) independent: RNA transcript has a self-complemnetary sequence permitting a hairpin centered 15 to 20 nucleotides before the end of the RNA

• three A residues in the template strand that are transcribed into U resides near the 3’ end of the hair pin

• formation of the hairpin stalls RNA polymerase causing disruption in the A-U base pair in the RNA-DNA hybird

• RNA transcript dissociates from polymerase

How is DNA transcribed from DNA to RNA?

1. Determine which is the template strand

2. Read the template strand 3’ to 5’ base pairing for your 5’ to 3’ RNA

3. Label the 5’ and 3’ ends of the RNA