Enzymes involved in homologous recombination

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.

13. SPO 11MECHANISM

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

18. ENZYME
MEDIATED
RECOMBINATION
IN
EUKARYOTES