Molecular Genetics Exam 2

2 sources of DNA damage

1. endogenous: Internal, metabolic pathways

2. exogenous: external factors: Radiation, chemical etc.

What are the consequences of DNA damage

damages DNA leads to mutations, cancer, aging CAM

Why is DNA repair essential

to maintain genomic stability

DNA damage responses

1. checkpoint activation (stops cell cycle)

2. DNA repair

3. Apoptosis, cell suicide

Damage: Single stranded break. List the repair and the main cause

• repair: Base excision repair

• Main cause: Radiation

Mr. Ber. made me single

Damage: Double stranded break

• Repair: Homologous recombination (Backup DNA) and NHEJ (nonhomologous end joining) (pushing two ends together

• Main cause: radiation

Mr. HR

double trouble go to mr, HR and NHEJ

Damage: Bulky adducts

• repair: NER Nucleotide excision repair. (Ner removes a WHOLE short stretch of nucleotides, unlike Ber that only does one.

• Main cause: Chemicals, carcinogens

Trying to bulk, okay Main character, take some chemicals. want it fixed, NERRR.

Damage: Base mismatches, insertions, and deletions

• Repair: Mismatch repair.

• Main cause: replication error

Damage: Base alkylation (alkyl group gets added to DNA)

Repair: Direct reversal, reverses the damage and restores orginal base

Main cause: chemicals

reasons for DNA damage

1. spontaneous damage

2. induced damage

3. replication error

SIR youve caused too much damage u must leave

Spontaneous damage

• depurination (loss of A or G bases) : point mutation

• deamination (C → U mutation) : point mutation

• Oxidative damage ( G→ T transversions) : point mutation

Induced damage

• UV radiation

• ionizing radiation (double stranded breaks)

• Chemical mutagens

Replication errors

mismatched bases

• the mutations above either cause losing a fragment of DNA or losing a mucleotide, often called a point mutation.

deamination

The loss of an NH2 group off of a nitrogenous base.

• main example of this is cytosine loses its NH2 turning into Uracil (u). U binds to A.

• Instead of C-G it turns into A-T

Hypoxanthine

Adenine, losses a NH2 bond, turning it into a Hypoxanthine. Which binds to C. SO instead of the normal A-T we get G-C. Transition mutation

Xanthine

Guanine, losses and NH2 group. However it can still bind to C, stay G-C. However it can occasionally bind to T, becoming A-T

5-methylcytosine

when 5methyl gets deamined it turns into thymine. Which is a problem because thats a normal base, and wont get recognized as a mutation. Wont be repaired. This makes CpG spots a hotspot for mutations. If cytosine gets methylated it becomes 5-methylcytosine, and then that gets deamined and turns into T. boom.

Oxidative damage

ROS = reactive oxygen species

O2-: superoxide

H2O2: hydrogen peroxide

OH. hydroxyl radical

• guanine is especially suseptible to oxidation it turns into 8-oxoguanine

• 8oxo binds with A (A slut) leading to TA binding instead of G-C

• normal binding is anti and abnormal binding is syn

UV radiation

Causes Thymine to bind to thymine causes a thymine dimer. Thats not good because it causes helix distortion and H bonding alterations

Ionizing radiation

1. causes double stranded breaks

2. lights wavelength is inversly proportional to energy

3. lights ENERGY is proportional to DNA damage

BER

Nuclotide gets removed by glycoslase, which is the bond between the base and the sugar. AP endonuclease and phosphodiasterase take out the sugar and the phosphate. DNA polymerase adds correct nucleotide. Ligase seals gap.

NER

excision nuclease removes length of DNA. DNA polymerase adds correct nucleotides to bottom strand. DNA ligase seals nicks.

NHEJ

Is error prone, can lead to the insertion or deletion of nucleotides. Just glues ends together

HR

Uses recombination specific nuclease, to correctly match nucleotides and then bind them together. More accurate

NHEJ mechanism

1. Ku70/Ku80 protein complex recognized double stranded breaks

2. DNA protein kinase and Ligase IV join the ends together

3. Errors like insertions or deletions occur.

Direct Transposition

CUt and paste. Transposon is cut from DNA and directly inserted into new site

replicative transposition

Copy and past. Transposon is duplicated and then put into a new site.

rRNA

Ribosomal RNA. Structural components of Ribosome

mRNA

Messenger RNA. Protein coding RNA

tRNA

Transfer RNA. Amino acid carrier RNA

Transcription occurs

on one strand of DNA, conveteed to RNA. If both strands are both being transcribed then an RNA hybrid forms, and can inhibit translation

Cis-acting elements

A seqence of DNA.

1. Promoter sequence. Upstream of the open reading frame

2. Terminator sequence: downstream

Trans-acting elements

Proteins that will act on the DNA

1. RNA polymerase holoenzyme: recognizes promoter and starts transcription

2. elongation factors. Help RNA polymerase continue making RNA,

3. termination factors: stps transcription

4. Gyrase/topoisomerase: helps relieve the coiling that occurs when DNA is being transcribed.

Positive (+) strand

coding strand. sense strand, NONTEMPLATE STRAND

• this is in the 5’-3’ direction. RNA polymerase wants to make a copy of something in the 5’-3’ direction, so it needs 3’-5’ to copy.

Negative (-) strand

non coding strand. antisense, TEMPLATE STRAND. runs in the 3’5’ direction so RNA polymerase can make mRNA easily. it will copy her. They are complementary and antiparralell yay.

A will turn into U

Promoter

a sequence of DNA where RNA polymerase will bind to initiate transcription. Upstream of transcription start site

Start point

+1

Operator sites

Where activator or repressor binds to, to regulate transcription

Terminator

a stop point for transcription

-35 region TTGACA

Region of the dna that is recognized by the sigma factor on the RNA polymerase. Inital recruitment of DNA polymerase onto DNA.

-10 region TATAAT

The pribnow box. Initiates DNA unwinding, allowing for transcription od DNA.

holoenzyme

The core enzyme+ the sigma factor.

• the core enzyme makes the RNA

• the Sigma factor, finds the starting point.

Prokaryotic transcription step 1: Initiation

sigma 70 recognizes promoter sequence -35 (TTGACA) and -10 (TATAAT) pribnow.

then RNa polymerase subunit beta’ unwinds DNA at -10 region to open the complex making a transcription buble which is 12-17 bp long.

RNA polymerase adds a few ribonucleotides at the +1 region after 9-10 bases the sigma factor gets dropped.

Prokaryotic transcription step 2: elongation

• once the sigma factor is dropped elongation factors NusA GreA GreB bind to the DNA polymerase complex.

• RNA polymerase reads the templete strand, negative (-) strand thats in the 3’-5’ direction so it transcribes in the 5’-3’ direction

• transcription bubble is maintained btw 12-17bp

• DNA ahead of the RNA polymerase is unwinded by grease, and the DNA behind the polymerase is rewound by topoisomerase 1.

• RNA continues until termination sequence is found.

Prokaryotic transcription step 3: termination

Termination can either be Rho independent or dependent.

1. Rho independent: No use of Rho Protein. GC rich portion gets transcribed, followed by UUUU which causes a hairpin loop, acts as a termination sequence

2. Rho dependent: Uses the Rho protein, which uses ATP.
   - Rho is an ATP dependent helicase. It binds to the rut sequence on the RNA and follows behind RNA polymerase until it cases up. It gets to the RNA polymerase and unwinds the RNA DNA complex releasing the RNA polymerase off of the sequence.
   - Rut is a C rich region

Bacterial gene regulators

OPERONS YAYY

Types of operons

1. Constitutive operons.
   - Always ON. nessecary for bacteria to have on cannot have it off.

2. Conditional operons

• inducible operons: Are always OFF but can be turned on (induced on) in response to specific environemental signals or stimuli

• Repressible operons: are always On but can be turned off (repressed) due to repressors binding to a repressor protein. This blocks transcription

rRNA size

120-5000 nucleotides

mRNA size

300- 10,000

tRNA

76-90 nucleotides

pre-mRNA processing

1. Transcription. Adds 5’ cap.

2. endonucleases, makes cleavage at poly (A) site. AAUAAA

3. polyadenylation PAP makes the 3’ A tail

4. Splicing of non coding introns, pushing exons together.