Lecture 5 - DNA R,R,R pt 2

Question 1

1. what is crossing over and gene conversion and how do they compare?

Answer 1

1. They both Exchange of genetic material on homologous chromosomes
2. Crossing over thinks big and does large portions or regions of DNA
3. Gene conversion think small and is movement of single genes

Question 2

1. What notices a double strand break?

Answer 2

1. Spo11 in yeast
2. Mre11 in humans
   
   1. Identifies DNA damage and process ends

Question 3

1. What is a Holiday junction?

Answer 3

1. DNA intermediate containing four DNA strands from two different helices
2. Don’t last very long
3. RuvA and B form junction
4. Strands are cleaved by an endonuclease(RuvC)

Question 4

1. What do holiday junctions usually lead to?

Answer 4

1. Crossing over
   
   1. Rare only 2 or 3 crosses over events in genome
2. Gene conversion
   
   1. 90% of holiday junctions go this route

Question 5

1. what cuts are made to a holiday junction to create gene conversions

Answer 5

1. If cuts made in same direction it is gene conversion
2. If cuts made in opposite direction its cross over junction

Question 6

1. What is branch migration

Answer 6

1. The point at which strand invasion occurs move through DNA
2. Some are spontaneous
   
   1. b/c no helicase
3. Some require proteins and ATP
   
   1. Includes helicase

Question 7

1. Since meiosis leads to even split of DNA from Paternal and Maternal counterparts, How do we get chromosomes that are 3/4 paternal and 1/4 maternal?

Answer 7

1. Mistach pair is identified
2. Gene Conversion!
3. DNA syntheses during HR
4. Repair of mismatch repair in regions of heteroduplex dna
   
   1. Since there is no actually damaged DNA both strands are correct. One sequence is removed RANDOMLY. Thereby creating a strand that maybe more paternal or more maternal

Question 8

What amount of DNA is swapped in DNA crossover and gene conversion?

Answer 8

look up this answer

Question 9

What is the structure and transposition mechanism of the 3 classes of transposons?

Answer 9

1. What are the three different types of Transposition Recombination?
   
   1. DNA- only transposons
   2. Retroviral-like retrotransposon
   3. Non-retroviral retrotransposon

Question 10

1. What is a transposable element

Answer 10

1. Selfish Dna; Jumping genes; Interspersed dna?
2. Specialized segments of DNA that move from one position in the genome to another
3. Range from 100s to 10000s of nucleotides
4. Unique set of genes and encode their own enzymes need for jumping
5. Since no sequence homology it can jump anywhere in the cell.
6. Move infrequently 10^5

Question 11

1. Why do transposable elements even exist?

Answer 11

1. Create antibody resistance in bacteria
2. Produces genetic variation
   
   1. Induce spontaneous

mutations

Question 12

1. What important gene is encoded in Transposon

Answer 12

1. Transposase enzyme encoded by the transposon it self
   
   1. Only sequence specificity to the transposon not to the rest of DNA

Question 13

1. What is a DNA only transposon?

Answer 13

1. Exist only as DNA in their movement
2. Occur largely in bacteria
   
   1. Spreading of antibody resistance
3. Cut and paste Transposition or replicative transposition
4. Encodes transposase
   
   1. Recognizes seq and is used to jump around

Question 14

1. How does cut and paste transposition work?

Answer 14

1. External sites are recognized by transposase
2. Loops together a complex by bind them together = transpososome
3. Cut sequence out = central intermediate
   
   1. DS break seal/repaired by poly and ligase
4. Central immediate makes a cut in target DNA and inserts itself

Question 15

1. How can you recognize where transposons have been?

Answer 15

1. They leave un even cuts that are often repaired using these small repeated sequences can tell you if a transposon has/has not/ or Is (been) there

Question 16

1. How can you recognize where transposons have been?

Answer 16

1. They leave un even cuts that are often repaired using these small repeated sequences can tell you if a transposon has/has not/ or Is (been) there
2. They leave un even cuts that are often repaired using these small repeated sequences can tell you if a transposon has/has not/ or Is (been) there
3. Ds break repair (if right after replication and there’s identically copy)
   
   1. Transposon is RESTORED
4. Homologous recombination using homologous chromosome
   
   1. Transposon is NOT RESTORED
5. Non-Homologous end joining?
   
   1. Will produce a mutation at the break site

Question 17

1. What does reverse transcriptase do?

Answer 17

1. Make DNA/RNA and then DNA/DNA double helix from the viral RNA

Question 18

1. How does a retrovirus like transposon compare to retrovirus?

Answer 18

1. Resembe retroviruses but lack a coat
2. Steps of jumping
3. DNA is transcribed into RNA
4. RNA - DNA is formed by retrotranscriptase
5. DNA-DNA is formed by again retrotranscriptase
6. Intrgrase then attacks each 3’OH phosphodiester end and inserts transposon

Question 19

1. How does a retrovirus like transposon compare to retrovirus?

Answer 19

1. Similar to retrovirus but lack coat and cannot leave resident cell
2. Both are entirely made by host

Question 20

1. How is integration of retrovirus-like transposon done?

Answer 20

1. Via Integrase double stranded DNA in placed on a site in the chromosome.
2. Encoded in transposon
3. Cuts each strand at different ends of viral sequence
4. Exposed 3’OH attacks a phosphodiester bond of target DNA
5. Viral DNA inserted leaving gaps to be filled
6. Leaving short repeated seq on each side on integrated DNA segment

Question 21

1. How much of the human genome do nonretroviral transposons make up?

Answer 21

40%

Question 22

1. What is nonretroviral transposon?

Answer 22

1. Endonuclease and reverse transcriptase bind to L1 RNA
2. Endonuclease nicks the target DNa at insertion point
3. Release 3’OH to serves as primer in reverse transcription step
4. Most immobile but few retain ability to move
5. Require endo nuclease and reverse transcriptase to move but it doesn’t code for it
6. Single stranded DNA copy of L1 “nonretroviral transposon” directly linked to target DNA
7. Insertion of DS double stranded DNA copy of L1 at target site

Question 23

1. What is conservative site-specific recombination?

Answer 23

1. Unlike the three mention before this type of rearrangement requires a specific sequence on each DNA that serves as recognition sites
2. Form high energy covalent bonds and use this energy to complete DNA arrangement
3. No protein/DNA intermediate
4. Gaps filled by Dna poly/ligase

Question 24

1. What takes advantage of this mechanism

Answer 24

1. Bacteria viruses move in and out of host
2. Ex: bacteriophage lambda

Question 25

1. How does orientation of DNa sites effect Conservative specific recombination?

Answer 25

1. If sites are in the same orientation, DNa sequence can be integrated or excised
2. IF sites are inverted in orientation, DNa sequence is inverted instead of Excised

Question 26

1. What are three examples of conservative site-specific recombination

Answer 26

1. Bacteriophage lambda
   
   1. Essential the lambda virus attaches to host cell
2. Injects its DNA
3. It is integrated via Conservative site-specific recombination
4. Enters two paths ways
   
   1. Prophage
      
      1. Exists passively in DNA and is replicated alongside with it
      2. Wont lysis until induction event
   2. Lytic pathway where it will rapidly replicate until cell lysis
5. Salmonella
   
   1. Uses process to control flagellin expression
   2. Site specific reaction can occur can switch the promotor (H2) from on to off
6. Mice

Tool for knocking out gene function in specific tissues in mice