Mole Bio Exam 3

What is homologous Recombination

the “crossing over” event during meiosis

essential for diversity, segregation, and repair during mitosis

What is Somatic Recombination

recombination in non-sex cells

mainly in Immune cells

Double Strand Break Repair Pathway

1. Recruitment of MRN and RAD proteins

2. Resection by exonucleases

3. Strand invasion

Types of Double Stranded Break Repair

1. Double Holliday Junction

2. Synthesis-dependent Single-strand Annealing

What types of Strand-Transfer proteins are there

1. Generic : promote exchange between DNA strands

2. Controlled : sequence specific DNA transfers (site-specific recombinases)

How does Phage lambda integrate into plasmids

Recombination between attP(phage plasmide) and attB(bacterial plasmid) sites

What do RAD and MRN do in DNA repair

• RAD: recombination-repair

• MRN single-stranded overhang in DNA ends

Non-Homologous End Joining

• joining of double stranded breaks when homologous sequence is not available

• Ligates blunt DNA ends

Break Induced Replication

1. break in chromosome at end

2. Uses non-homologous chromosome

3. Can cause Non-Reciprocal translocation (unbalance translocations of chromosome segments)

LoxP site and Cre recombinase

• LoxP site is two inverted 13bp repeats sandwiching an 8bp core

• Cre recombinase can

1. Excise LoxP + DNA outside of site

2. Inverts DNA outside of LoxP

3. Translocate DNA outside of LoxP

FLP recombinase and FRT sites

• FLP: Flippase that recognizes FRT site

• FRT: like LoxP

• Mechanism: FLP excises DNA between two FLP sites facing same direction

What is Recombination signal sequence (RSS)

• The V+D+J recombination machinery uses sequences of 7bp separated by nonamers of 12-23bp (RSS)

• signals VDJ recombination

VDJ recombination events in blood cells

1. Rag 1 or 2 recognizes RSS flanking VDJ

2. Rag complexes with HMGB1 and hairpin the DNA

3. Double stranded break

4. non-homologous repair and end joining (NHEJ)

5. random nucleotides are added at junctions

Transcription direction

moves 3’→5’ on DNA

Upstream vs Downstream of RNA polymerase

• Up: polymerase has already passed over it

• Down: yet to be transcribed

Primary Transcript

the newly synthesized RNA

Prokaryotic RNA Polymerase holoenzyme

• 5 subunits + sigma factor

• C Terminal Domain contacts regulatory proteins

Prokaryote Transcription steps

1. Polymerase binds to promoter forming closed complex

2. Transcription is initiated forming bubble (open complex)

3. elongation moving 3’→5’ on DNA

4. termination

what does Sigma Factor do

• Required for initiation

• increases affinity for DNA promoter in RNA pol.

• recognizes consensus sequences like TATA

• can compete with other sigmas to bind at different promoters

• displaced when transcription starts

Ternary Complex in Prokaryotic Transcription

• Formed at initiation

• RNA pol. + DNA + Dinucleotide primer

UP element

sequence upstream to promoter that enhances transcription

binds to CTD on RNA pol

What is promoter strength

frequency of RNA pol. transcription initiation

Down mutation vs up mutation in promoters

• down : decrease promoter efficiency specificity to consensus sequence

• up : opposite

Footprinting

1. DNA is amplified with PCR

2. radio labeled at ends

3. reacted with DNA binding proteins

4. treated with DNAase1 (cant endonuclease where protein is bound)

5. Creates different lengths of protein

6. analyzed with gel electrophoresis

7. no band is part of DNA with protein bound to it

Termination of Transcription

• intrinsic : RNA polymerase recognizes terminator sequence in DNA→ makes hairpin in new RNA → cleavage

• rho-dependent: needs rho proteins that bind to new RNA and cleaves RNA at rut site sequence on RNA