Lecture 12-Recombination and Genome Rearrangement (Nakai)

Question 1

What’s the process of homologous recombination?

Answer 1

• 1 ds break
• exonucleases make a gap, shortening 3’ ends LESS
• 3’ end of one strand pairs with copmliment, displaces other strand
• DNA Pol extends broken strand until the gap has filled, branch migration meanwhile creating 2 Holliday structures
• special endonucleases cleave the Holliday intermediates
• 1 of 2 products formed

Question 2

nondisjunction

Answer 2

• one of the roles for homologous recombination

- when chromosomes don’t separate during meiosis

Question 3

What are the 3 roles of homologous recombination?

Answer 3

• proper segregation of chromosomes preventing nondisjunction
• ds break repair
• restart of replication forks

Question 4

How do you reestablish the replication fork if there is a lesion?

Answer 4

• fork regression using RecA and RecFOR
• synthesizing strands form a duplex (actually a holliday structure) and one is shorter because the other strand will have synthesized a little more before the Pol stops
• DNA Pol I synthesizes the shorter strand complementary to the longer one, and this should take the strand past the lesion
• reverse branch migration occurs and the fork proceeds and the newly synthesized strand will be past the lesion
• if NER machinery available then the lesion will be fixed

Question 5

How do you reestablish the replication fork if there is a nick in one strand?

Answer 5

• exonucleases will shorten one of the strands so the other will bp with its homologous strand that did not have a nick using RecA and Rec BCD
• This forms a D loop and the branch will migrate so that the shorter of the two strands with the nick will eventually base pair with the other correct strand forming a Holliday structure
• Holliday structure resolved using RuvAB and RuvC and now both strands have a complete template

Question 6

Other than the RecA/RecFOR, DNA Pol I process for repairing lesions, what is another way the cell can repair DNA lesions?

Answer 6

• Error-prone DNA Pol: loosely attached active site that will read right past the thymidine dimers and incorporate 2As that can later be fixed if nucleotides are incorrectly incorporated.
• This pol is essential for lesion repair

Question 7

How does RecA/Rad51 work?

Answer 7

REQUIRES ATP! RecA will bind ssDNA and help it to search for homologous dsDNA. Once it finds it it mediates exchange of the ssDNA, displacing another DNA strand so it can homologously bind with its complement.

Question 8

P53

Answer 8

may play a role in controlling homologous recombination by Rad51

Question 9

Explain eukaryotice ds break repair involving BRCA2 (6)

Answer 9

• high fidelity
• occurs in somatic cells after S phase when sister chromosomes are present and cell is in G2; a lot of breaks are made during S phase so a lot that needs to be fixed
• RPA (eukaryotic form of SSB) binds ssDNA
• BRCA2 is at the junction of ss and dsDNA and loads Rad51 onto the strands displacing RPA
• Rad51 mediates the normal mechanism of repair by finding homologous chromosomes
• instead of cleavage/resolution of the Holliday structures the strands disengage to avoid diversity taking place

Question 10

Describe non-homologous end joining (4)

Answer 10

• occurs during G1 when no sister chromosomes available
• LOW FIDELITY: information may be lost if chunks are missing between exposed ends
• Ku proteins bring the ends together
• other proteins recruited (xrcc4-LigIV) to seal the gap

Question 11

What are the 3 repair mechanisms of the cell? which ones does BRCA1 control? BRCA2?

Answer 11

• Homologous recombination (error-free)
• Single Strand Annealing (SSA–error-prone)
• Non-Homologous end Joining (error-prone)

BRCA1: HR, SSA
BRCA2: HR

Question 12

What are transposable elements? (7)

Answer 12

• parasitic DNA that can jump places throughout the genome.
• One common on is L1 that takes up 17% of our genome and usually doesn’t move but is often found to in cancer
• can insert anywhere
• all genomes have these
• can be mutagenic
• encode their own machinery that will catalyze their movement to the new site
• can be inverted repeats

Question 13

How do bacteria use transposable elements?

Answer 13

• bacteria have developed these due to antibiotic overprescription and have passed the element between cells
• with antibiotic resistance the non-pathogenic bacteria can become the predominate bacteria of the body

Question 14

Basic reactions of transposition use what enzymes?

Answer 14

• transposases

- integrases

Question 15

briefly describe direct transposition (not the process). (2)

Answer 15

• “cut and paste”

- no RNA intermediate

Question 16

Explain the process of direct transposition

Answer 16

• transposases cleave the transposon on both ends (ds) and remain bound until it finds target DNA
• transposases promote 3’OH to bind with the target DNA
• DNA Pol fills in the gap
• ligase seals

Question 17

How does recombination play a role in Ig formation? (5)

Answer 17

• There are many V (variable) regions, ~4 J regions (go between constant and V regions) and 1 C region (constant, and actually C terminal of the light chain)
• recombination deletes DNA between V and J segments so there are a couple of J segments and some V segments left–this is the “mature light chain gene”
• transcription to mRNA removing more V sequences
• the other potential J segment is removed by splicing
• translation into mature light chain

Question 18

Explain how Igs remove V and J segments to make the light chain protein

Answer 18

• RSS (recombination signal sequences) signal to RAG1a and RAG2 where to make DNA breaks (these are just like the signals found at the end of transposable elements)
• 3’ OHs act as nucleophiles causing transesterification reaction forming looped ends and release of the segment. Loops protect the ends
• same enzymes from NHEJ will join the ends of the V and J DNA that was removed
• ## if DNA is lost due to the sloppy activity of NHEJ machinery then this just leads to greater variation

Question 19

What is also made by site specific recombination?

Answer 19

• T cell ab receptors