Recombination Models are discussed

1. MOLECULAR GENETICS
Unit 2: Sex Determination and Dosage Compensation 05 hrs
Sex determination in mammals and Drosophila; Secondary sex determination in humans; Dosage
compensation in mammals and Drosophila.
Unit 3: Genetic Recombination 14 hrs
Mechanism of recombination – Holliday, White House, Messelson and Radding models; Enzymes
involved in homologous and site-specific recombination; Breakage and reunion of DNA at specific
sites; Synapsis of homologous duplexes; Role of Rec A in recombination; Genetic recombination in
Bacteria - Transformation, natural transformation systems, mechanism, gene mapping by
transformation; Conjugation – discovery, interrupted mating and temporal mapping, Hfr; Transduction
– Generalized and specialized transduction; Gene mapping by specialized transduction, abortive
transduction; Identification and selection of mutants.
Unit 4: Transposons 10 hrs
Bacterial and yeast transposons; Replicative and non-replicative transpositions; Common intermediates
for transpositions – role of transposase and resolvase; Controlling elements in Bacteria; Ac Ds system
in maize; P elements in Drosophila; Transposons in humans; Retrotransposons.

2. GENETIC
RECOMBINATION

3. Recombination is the production of new DNA molecule(s)
from two parental DNA molecules or different segments of
the same DNA molecule.
Types of recombination:
General or homologous recombination occurs between
DNA molecules of very similar sequence, such as
homologous chromosomes in diploid organisms (meiotic
recombination).
Illegitimate or nonhomologous recombination occurs in
regions where no large scale sequence similarity is
apparent,
e.g. translocations between different chromosomes or
deletions that remove several genes along a
chromosome.

4. • Site-specific recombination systems mediate DNA
rearrangements by breaking and joining DNA molecules at
two specific sites, termed recombination targets (RTs).
• Site-specific recombination requires a special enzymatic
machinery, basically one enzyme or enzyme system for
each particular site.
• Good examples are the systems for integration of some
bacteriophage, such as λ, into a bacterial chromosome
and the rearrangement of immunoglobulin genes in
vertebrate animals.
• Replicative recombination generates a new copy of a
segment of DNA.
• Many transposable elements use a process of replicative
recombination to generate a new copy of the transposable
element at a new location.

5. TYPES OF RECOMBINATION

6. HOMOLOGOUS
RECOMBINATION
Functions served:
Meiotic crossing over leading to reassortment
of alleles on a chromosome
DNA repair
Resuming a stalled replication fork

7. MODELS FOR HOMOLOGOUS RECOMBINATION
1.White House Model-1963
(Polaron-Hybrid DNA Exchange Model)
Proposed and developed by Whitehouse and Hastings
Defines a ‘polaron’ as a segment of the
Chromosome where Hybrid DNA is formed and
cross over takes place.
Steps:
Two homologous double stranded DNA align
together
An endonuclease nicks the DNA in one strand of
each DNA molecule at the start of a polaron
(depicted as solid circles in the image)

8. • The nicks are made in strands of opposite polarity (unlike holiday
model)
• The nicked strands starts unwinding from their sister strands.
• New DNA strands are synthesized along the single stranded regions
using the intact strand as the template.
• The newly synthesized DNA separatesform their templates
• The newly synthesized strands base pair with their non-sister strands
and form double stranded regions
• The intact strands which are single stranded are degraded and the
remaining nicks are sealed. This completes recombination.
• The mismatches in the DNA hybrids may be repaired later to give non-
reciprocal recombination (due to gene conversion)
• If it is not repaired it can lead to post meiotic segregation

10. 2. HOLLIDAY MODEL (1964)
This model was proposed by Robin Holliday
The model illustrates the strand invasion, branch migration
and junction resolution in a simple and effective way.
Explained reciprocal exchange of DNA segments, gene
conversion and post meiotic segregation
But this model is insufficient to explain the mechanism of
crossing over because
It involves only single stranded breaks (Meiotic
recombination is initiated by Double stranded breaks)
No DNA synthesis takesplace
(all recombination events involve some new DNA synthesis)

11. STEPS
1. Alignment of two homologous DNA molecules
2. Introduction of breaks in both strands of DNA
3. Strand Invasion: Formation of initial short regions of
base pairing between two recombining DNA
molecules
Occurs when a single stranded region from one
parent DNA pairs with a strand of the other homolog
Strand invasion results in the connection
between two DNA molecules by crossing DNA
strands. This structure is called Holliday
Junction

12. Branchmigration: the holliday junction moves
along the DNA by repeated melting and formation
of base pairs.
Resolution: this involves the cleavage of the holliday
junction.
This steps finishes the process of genetic exchange
depending on the type of resolution both cross over
DNA and non cross over DNA can be produced

17. 3. MESELSON-RADDING MODEL
(1975)
The model proposed by Meselson and Radding generates the
Holliday structure with one single-strand cut in only one
chromosome in contrast with the Holliday model, in which a
nick is made in one strand in each of the two homologous
chromatids.
Steps involved
(a) A duplex is cut on one chain.
(b) DNA polymerase displaces one chain.
(c) The resulting single chain displaces its counterpart in the
homolog.
(d) This displaced chain is enzymatically digested.
(e) Ligation completes the formation of a Holliday junction,
which is genetically asymmetric in that only one of the two
duplexes has a region of potentially heteroduplex DNA. If the
junction migrates, heteroduplex DNA can arise on both
duplexes.
(f) Resolution of the junction occurs as in the Holliday model.

19. 4.DOUBLE STRAND BREAK REPAIR PATHWAY
FOR HOMOLOGOUS RECOMBINATION (1983)
Involves double stranded DNA breaks instead of single stranded
breaks.
The break is introduced only in one DNA duplex involved in
recombination
The DNA adjacent to the break site is then degraded to produce
single stranded tails with a free 3’ end.
One of the ssDNA tails then invade the other duplex.
The strand displaced from the second duplex invades the first duplex.
New DNA synthesis takes place at the 3’ ends of the invading strand
and the non-invading broken strand.
The process of branch migration results in formation of two Holliday
junctions which can be resolved in many alternative forms
Based on the type of resolution either a splice/cross-over
product or a patch/non cross over product is obtained

22. RESOLUTION OF DOUBLE–
HOLLIDAY JUNCTION

23. 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

24. 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

25. 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)

26. 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

27. 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

28. 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

29. RUVAB IN A HOLLIDAY
JUNCTION

30. 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

31. RUV C IN A HOLLIDAY
JUNCTION

32. 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

33. 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.

34. SPO 11MECHANISM

35. 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.

36. 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

37. 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

38. MUS 81
Essential formeiosis
Isan endonuclease
Probably involved in holiday junction
resolution

39. ENZYME
MEDIATED
RECOMBINATION
IN
EUKARYOTES