Replication, Maintenance and Rearrangement of Genomic DNA part 3 Recombination

Question 1

Explain Homologous Recombination by complementary base pairing

Answer 1

How can parental DNA molecules be broken at the same point?’
Alignment by base pairing of complementary DNA strands during meiosis (four stranded stage
Staggered breakage and crosswise rejoing can occur at any point
The two resulting strands are derived from different parental molecules
Genetic differences between the 2 strands can be corrected by DNA repair machinery
1. homologous recombination is based on alignment of the parent DNA molecules during the four-strand stage of meiosis
2. DNA breakage occurs (can be staggered) and can be exchanged crosswise resulting in recombination of the genetic material

Question 2

Explain the Holiday model for homologous recombination

Answer 2

1. formation and repair of the previouly described structure lead to the holiday model
2. Original model: recombination initiated by the introduction of nicks at the same position on both strands, followed by strand exchange and ligation of the hybrid molecules
3. a crossed strand intermediate also referred to as holiday junction was predicted

Question 3

Isomerization and resolution of holliday juntion

Answer 3

1. Initial Holliday junction
   nick and rejoin crossed strands
2. rotate bottom portion 180 degrees
   rotate right hand portion 180 degrees results in isomerized Holliday junction
   nick and rejoin crossed strands
3. resolution of the four-stranded structure requires a rotation of the heteroduplex followed by nicking and re-ligation
   Nonrecombinant only exchange one strand

Question 4

Initiation of recombination by double strand breaks

Answer 4

1. only one DNA duplex cleaved
2. 5’-3’ nuclease digestion of the donor strand
3. New DNA synthesized to fill the gap
4. Generation of recombinant DNA (branch migration)-both DNA molecules are unwound to allow strand invasion DNA synthesis (branch migration)
5. second parental DNA molecule no breakage

Question 5

What is the function of the RecA Protein (E. coli)

Answer 5

1. first protein characterized in E coli was RecA a SSBP
2. mutation studies helped to indentify the proteins required for efficient recombination
3. Screens indentified RecA, DNA pol ligase, then RuvA, B, C (no homolog in Eukaryotes)
4. RecA promotes strand exchange

often referred to as Rec for recombination. Best characterized is RecA in E coli. SSBP promotes strand exchange

Question 6

Branch migration and resolution of holliday junctions

Answer 6

1. RuvA bids first and recruits RuvB
2. RuvB catalyzes the branch migration
3. RuvC resolves the Holliday junction by cleaving the crossed strands
4. strands are re-ligated

Question 7

Yeast enzymes

Answer 7

1. RecA-related protein: RAD51 required for recombination and repair of double strand breaks
   higher eukaryotes posses RAD51 homologues
   most of yeast counterparts are RAD proteins also involved in DNA repair

Question 8

In DNA rearrangements lytic and lysogenic pathways of bateriophage gamma

Answer 8

1. site specific recombination
2. lambda phage can replicate or intergrate its DNA into bacterial chromosome
3. lambda DNA can eventually be excised
4. Recombination occurs at specific sites call att sites
5. it requires a lambda protein called integrase
   the phage can choose between two cycles:
   a. lytic: the phage gets its DNA replicated using bacterial proteins and the DNA is packaged into new virus particles. Bacterial will be lyzed and new viruses will be released to go and infect other bacteria
   b. lysogenic: DNA from bacteriophage lambda gets recombined with the bacterial DNA (integration) and the event occurs at specific sites called att sites. it requries the phage encoded enzyme integrase

Question 9

Integration of Lambda DNA by site specific recombination

Answer 9

Both phage and bacteria posses att sites
attP for the phage and attB for the bacterium
Once the integranse has been expressed the phage can integrate its DNA into the bacterial chromosome at the attB site

Question 10

What is the mechanism of lambda site specific recombination

Answer 10

1. homology between attP and attB the att sites are -15 nucleotides
2. integrase cleaves at specific sites. staggered cuts by 7 bp in both attP and attB
3. strand exchange followed by ligation
4. ends are joined cross wise
5. integrase protein can also be involved in excision of the lambda DNA from the bacterial chromosome