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ENZYMES
INVOLVED IN
HOMOLOGOUS
RECOMBINATION
Dr. M. Sonia Angeline
Assistant Professor
Kristu Jayanti College
ENZYMES INVOLVED IN HOMOLOGOUS
RECOMBINATION IN E. COLI
Protein Function
RecBCD Processes DNA togenerate single
stranded regions
RecA Brings about strandexchange
Ruv A, RuvB Enables branch migration
Ruv C Holliday Junctionresolution
In addition to these dedicated proteins involved in
recombination DNA polymerases, Single stranded DNA binding
proteins, topoisomerases and ligases also take part in the process
In bacteria there is no enzyme known to introduce breaks in
DNA.
Breaks arise by UV rays, or by replication errors
RECBCD
Processes broken DNA molecules to generate regions of ssDNA.
Helps load the recA strand exchange protein to the ssDNA ends.
Helps a cell to choose whether to recombine with or destroy
DNA molecules that enter the cell
It contains three subunits, the B, C & D subunits. (total size-330
KDa)
Ithas both helicase ( B& D subunits) and nuclease activities
Itreleases energy by hydrolysis of ATP to fuel its activities
The activity of the enzyme complex is under the control of
Chi (crossover hotspot instigator)
Chi sites are regions in the bacterial genome near to which
recombination occurs at a higher frequency than expected
RECA
Rec A is the most important protein in homologous recombination
Itbelongs to a family of proteins called strand –exchange proteins
These proteins catalyse the pairing of homologous DNA molecules by
Search for sequence matches between strands of DNA
Generate regions of complementary base pairing between the
DNA
In order to perform its function, multiple (many to hundreds)
subunits of RecA protein bind to single stranded DNA with 3’
overhangs to produce a protein-DNAfilament.
The protein DNA filament is extended in length with a distance of
around 5 A between bases as against the normal 3.4 A
The RecA filament has a primary binding site that is bound to the
single stranded DNA and a secondary binding site that can
accommodate double stranded DNA
Homologs of RecA are found in Archae (Rad A) & Eukaryotes (Rad
51, Dmc1)
STAGES OF RECA CATALYSED
STRAND EXCHANEGE
1. RecA assembles on one of the participating DNA
molecule containing a region of ssDNA to form a recA-ss
DNA complex that searches for regions of
complimentarity in the other strand.
The secondary binding site samples large stretches
of DNA for sequence complimentarity
A sequence homology of around 15 bp triggers strand
exchange
STAGES OF REC A CATALYZED
STRAND EXCHANGE
2. Once a region of base pair complementarity is located
Rec A promotes the formation of a stable three
stranded complex called Joint molecule.
3. Then the single stranded DNA in the primary site binds
to its compliment in the duplex bound by the
secondary site.
This process of strand exchange involves breakage and
formation of bonds
RUV AB
Ruv A protein (a tetramer) recognises and binds
specifically to Hollidayjunctions
Itrecruits Ruv B protein to the site.
Ruv B is a hexameric protein which acts as a
ATPase
The energy derived by hydrolysis of ATP by RuvB
enables the exchange of base pairs that result in
branch migration
RUVAB IN A HOLLIDAY
JUNCTION
RUV C
Ruv C is the major endonuclease that resoves holliday
junction in bacteria
Itfunctions in concert withRuvAB
RuvC is recognises the junction in complex with RuvAB
and nicks two DNA strands of the same polarity
Depending on the strands which are cut either patch
products or splice products are formed
The protein exhibits some level of sepecificity
It recognises the sequence 5”-A/T-T-T-T -G/C-3” which is
on an average present once every 64 nucleotides in the
genome
This specificity ensures that at least some branch
migration has happened before the resolution of the
junction
RUV C IN A HOLLIDAY
JUNCTION
ENZYMES INVOLVED IN EUKARYOTIC
RECOMBINATION (MEIOTIC RECOMBINATION)
Homologous recombination events that occur during
meiosis are called meiotic recombination.
Many proteins associate together to form large
recombination factories to bring about the process
Few well characterised enzymes are listed below
SPO11
• SPO11 gene encodes a protein that introduces DSBs in
chromosomal DNA to initiate meiotic recombination.
• The Spo11 protein cuts the DNA at many chromosomal locations,
with little sequence selectivity, but at a very specific time during
meiosis.
• Spo11- mediated DNA cleavage occurs right around the time
when the replicated homologous chromosomes start to pair.
• Spo11 cut sites, although frequent, are not randomly distributed
along the DNA.
• The mechanism of Spo11 DNA cleavage is as follows:
• A specific tyrosine side chain in the Spo11 protein attacks the
phosphodiester backbone to cut the DNA and generate a covalent
complex between the protein and the severed DNA strand.
• Two subunits of Spo11 cleave the DNA two nucleotides apart on
the two DNA strands to make a staggered DSB.
• Spo11 shares this DNA cleavage mechanism with the DNA
topoisomerases and the site-specific recombinases.
SPO 11MECHANISM
MRX–ENZYME COMPLEX
• During meiotic recombination, the MRX–enzyme complex is
responsible for this DNA-processing event.
• MRX is composed of protein subunits called Mre11, Rad50,
and Xrs2; the first letters of these subunits give the complex
its name.
• Processing of the DNA at the break site occurs exclusively
on the DNA strand that terminates with a 50 end—that is,
the strands covalently attached to the Spo11 protein (as
described above).
• The strands terminating with 3’ends are not degraded.
• This DNA-processing reaction is therefore called 5’-to-3’
resection.
• The MRX-dependent 5’-to-3’ resection generates the long
ssDNA tails with 30 ends that are often 1 kb or longer.
• The MRX complex is also thought to remove the DNA-linked
Spo11.
RAD51 AND DMC1
• Eukaryotes encode two well-characterized homologs
of the bacterial RecA protein: Rad51 and Dmc1.
• Both proteins function in meiotic recombination.
• Whereas Rad51 is widely expressed in cells dividing
mitotically and meiotically, Dmc1 is expressed only as
cells enter meiosis.
• Dmc1- dependent recombination is preferentially
between the non-sister homologous chromatids,
rather than between the sisters.
• They promote strand invasion & exchange between
non sister chromatids
• Rad 51 associate with single stranded DNA to form
DNA-protein filaments
• The assembly of these filaments are promoted by
another protein called Rad52
RAD52
• Rad52 is another essential recombination protein
that interacts with Rad51.
• Rad52 functions to promote assembly of Rad51
DNA filaments, the active form of Rad51.
• It does this by antagonizing the action of RPA, the
major ssDNA-binding protein present in eukaryotic
cells.
• In this respect, Rad52 shares an activity with the E.
coli RecBCD protein.
• Rad52 protein also promotes the annealing and
base pairing of complementary ssDNA molecules,
and this activity may also play a role in the strand-
pairing reactions that occur during initiation
of recombination
MUS 81
Essential formeiosis
Isan endonuclease
Probably involved in holiday junction
resolution
ENZYME
MEDIATED
RECOMBINATION
IN
EUKARYOTES

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Enzymes involved in homologous recombination.pdf

  • 1. ENZYMES INVOLVED IN HOMOLOGOUS RECOMBINATION Dr. M. Sonia Angeline Assistant Professor Kristu Jayanti College
  • 2. ENZYMES INVOLVED IN HOMOLOGOUS RECOMBINATION IN E. COLI Protein Function RecBCD Processes DNA togenerate single stranded regions RecA Brings about strandexchange Ruv A, RuvB Enables branch migration Ruv C Holliday Junctionresolution In addition to these dedicated proteins involved in recombination DNA polymerases, Single stranded DNA binding proteins, topoisomerases and ligases also take part in the process In bacteria there is no enzyme known to introduce breaks in DNA. Breaks arise by UV rays, or by replication errors
  • 3. RECBCD Processes broken DNA molecules to generate regions of ssDNA. Helps load the recA strand exchange protein to the ssDNA ends. Helps a cell to choose whether to recombine with or destroy DNA molecules that enter the cell It contains three subunits, the B, C & D subunits. (total size-330 KDa) Ithas both helicase ( B& D subunits) and nuclease activities Itreleases energy by hydrolysis of ATP to fuel its activities The activity of the enzyme complex is under the control of Chi (crossover hotspot instigator) Chi sites are regions in the bacterial genome near to which recombination occurs at a higher frequency than expected
  • 4. RECA Rec A is the most important protein in homologous recombination Itbelongs to a family of proteins called strand –exchange proteins These proteins catalyse the pairing of homologous DNA molecules by Search for sequence matches between strands of DNA Generate regions of complementary base pairing between the DNA In order to perform its function, multiple (many to hundreds) subunits of RecA protein bind to single stranded DNA with 3’ overhangs to produce a protein-DNAfilament. The protein DNA filament is extended in length with a distance of around 5 A between bases as against the normal 3.4 A The RecA filament has a primary binding site that is bound to the single stranded DNA and a secondary binding site that can accommodate double stranded DNA Homologs of RecA are found in Archae (Rad A) & Eukaryotes (Rad 51, Dmc1)
  • 5. STAGES OF RECA CATALYSED STRAND EXCHANEGE 1. RecA assembles on one of the participating DNA molecule containing a region of ssDNA to form a recA-ss DNA complex that searches for regions of complimentarity in the other strand. The secondary binding site samples large stretches of DNA for sequence complimentarity A sequence homology of around 15 bp triggers strand exchange
  • 6. STAGES OF REC A CATALYZED STRAND EXCHANGE 2. Once a region of base pair complementarity is located Rec A promotes the formation of a stable three stranded complex called Joint molecule. 3. Then the single stranded DNA in the primary site binds to its compliment in the duplex bound by the secondary site. This process of strand exchange involves breakage and formation of bonds
  • 7. RUV AB Ruv A protein (a tetramer) recognises and binds specifically to Hollidayjunctions Itrecruits Ruv B protein to the site. Ruv B is a hexameric protein which acts as a ATPase The energy derived by hydrolysis of ATP by RuvB enables the exchange of base pairs that result in branch migration
  • 8. RUVAB IN A HOLLIDAY JUNCTION
  • 9. RUV C Ruv C is the major endonuclease that resoves holliday junction in bacteria Itfunctions in concert withRuvAB RuvC is recognises the junction in complex with RuvAB and nicks two DNA strands of the same polarity Depending on the strands which are cut either patch products or splice products are formed The protein exhibits some level of sepecificity It recognises the sequence 5”-A/T-T-T-T -G/C-3” which is on an average present once every 64 nucleotides in the genome This specificity ensures that at least some branch migration has happened before the resolution of the junction
  • 10. RUV C IN A HOLLIDAY JUNCTION
  • 11. ENZYMES INVOLVED IN EUKARYOTIC RECOMBINATION (MEIOTIC RECOMBINATION) Homologous recombination events that occur during meiosis are called meiotic recombination. Many proteins associate together to form large recombination factories to bring about the process Few well characterised enzymes are listed below
  • 12. SPO11 • SPO11 gene encodes a protein that introduces DSBs in chromosomal DNA to initiate meiotic recombination. • The Spo11 protein cuts the DNA at many chromosomal locations, with little sequence selectivity, but at a very specific time during meiosis. • Spo11- mediated DNA cleavage occurs right around the time when the replicated homologous chromosomes start to pair. • Spo11 cut sites, although frequent, are not randomly distributed along the DNA. • The mechanism of Spo11 DNA cleavage is as follows: • A specific tyrosine side chain in the Spo11 protein attacks the phosphodiester backbone to cut the DNA and generate a covalent complex between the protein and the severed DNA strand. • Two subunits of Spo11 cleave the DNA two nucleotides apart on the two DNA strands to make a staggered DSB. • Spo11 shares this DNA cleavage mechanism with the DNA topoisomerases and the site-specific recombinases.
  • 14. MRX–ENZYME COMPLEX • During meiotic recombination, the MRX–enzyme complex is responsible for this DNA-processing event. • MRX is composed of protein subunits called Mre11, Rad50, and Xrs2; the first letters of these subunits give the complex its name. • Processing of the DNA at the break site occurs exclusively on the DNA strand that terminates with a 50 end—that is, the strands covalently attached to the Spo11 protein (as described above). • The strands terminating with 3’ends are not degraded. • This DNA-processing reaction is therefore called 5’-to-3’ resection. • The MRX-dependent 5’-to-3’ resection generates the long ssDNA tails with 30 ends that are often 1 kb or longer. • The MRX complex is also thought to remove the DNA-linked Spo11.
  • 15. RAD51 AND DMC1 • Eukaryotes encode two well-characterized homologs of the bacterial RecA protein: Rad51 and Dmc1. • Both proteins function in meiotic recombination. • Whereas Rad51 is widely expressed in cells dividing mitotically and meiotically, Dmc1 is expressed only as cells enter meiosis. • Dmc1- dependent recombination is preferentially between the non-sister homologous chromatids, rather than between the sisters. • They promote strand invasion & exchange between non sister chromatids • Rad 51 associate with single stranded DNA to form DNA-protein filaments • The assembly of these filaments are promoted by another protein called Rad52
  • 16. RAD52 • Rad52 is another essential recombination protein that interacts with Rad51. • Rad52 functions to promote assembly of Rad51 DNA filaments, the active form of Rad51. • It does this by antagonizing the action of RPA, the major ssDNA-binding protein present in eukaryotic cells. • In this respect, Rad52 shares an activity with the E. coli RecBCD protein. • Rad52 protein also promotes the annealing and base pairing of complementary ssDNA molecules, and this activity may also play a role in the strand- pairing reactions that occur during initiation of recombination
  • 17. MUS 81 Essential formeiosis Isan endonuclease Probably involved in holiday junction resolution