The document explains the molecular mechanisms of recombination, detailing the processes of homologous recombination, including crossing over, gene conversion, and various steps involved in forming Holliday junctions. It discusses different models of recombination such as the double-strand break model and site-specific recombination, highlighting the roles of specific proteins and enzymes. Additionally, it describes types of site-specific recombination, including conservative and transpositional types, emphasizing their applications in genetic engineering.

1. UNIT 3
MOLECULAR
MECHANISM OF
RECOMBINATION

2. INTRODUCTION
• Gene is a polynucleotide chain that can control a
specific trait
• Can be unit of function- cistron
• Unit of mutation-mutan
• Unit of recombination – recon
• Recombination- process of formation of new
recombinant chromosomes by combining genetic
materials from 2 organisms.

3. • Steps involved:
 Crossing over
 Gene conversion
 Exchange between sister chromatids
 Repair of DNA damage

4. HOMOLOGOUS RECOMBINATION
• Between complementary strands of 2 homologous
DNA molecules
• Crossing over: genetic exchange between
chromosomes as a result of HR
• Frequency of crossing over is proportional to the
physical distance between genes
• Helps to get new combination and repair the prior
damages

5. • Steps:
 Allignment of 2 DNA molecules
 Introduction of breaks in DNA( single or double strand
break)
 Strand invasion – formation of initial short region of base
pairing
 Causes the formation of a junction ie:- 2 DNA connected
through crossing over – Holliday junction
 Movement of holliday juction by continuous melting and
formation of base pairs – branch migration
 Cleavage of holliday juction - resolution

6. HOLLIDAY MODEL

7. • Pairing: align homologous duplexes
• Single strand invasion:
– Endonuclease nicks at corresponding regions of the
same strands of homologous chromosomes
– Ends generated by the nicks invade the other,
homologous duplex
– Ligase seals nicks to form a joint molecule.
– (“Holliday intermediate” or “Chi structure”)
• Branch migration expands heteroduplex region.

8. • Can occur in one of two ways
• The Holliday junction can be nicked in the same strands
that were initially nicked = “horizontal resolution.” This
results in NO recombination of flanking markers.
• The Holliday junction can be nicked in the strands that
were not initially nicked = “vertical resolution.” This
results in RECOMBINATION of flanking markers.
• Heteroduplex formation
•Resolution of joint molecules

9. Vertical & horizontal resolution
A+
B+
B-
A-
H
V
2. Vertically V
B-
A+
A- B+
This leaves a region of heteroduplex, and
the flanking markers have recombined.
1. Horizontally H
A+ B+
B-
A-
A region of heteroduplex is left, but the
flanking markers are not recombined.
or

10. Enzymes involved
• rec A protein and recBCD endonuclease are purified
from bacterial systems
• Topoisomerases
• int proteins, xis protein and IHF Protein in λ phage
• rec A and rec BCD promotes basepairing in single
strand of DNA

11. Double strand break model
• This model provides a better explanation for recombination
events in yeast
• A double strand break precedes recombination.
• HR in bacteria helps in repair of DSBs.
• One DNA molecule is used preferentially as the donor of genetic
information.
• A double-strand break is expanded to a gap, which is then
repaired by copying a homologous sequence
• commonly results in crossover
• 2 holliday junctions will form
http://web.mit.edu/engelward-
lab/animations/DSBR.html

13. Steps
• DSB is introduced into one of the DNA
• the DSB is processed by either a helicase, a nuclease, or a
combination of both, to yield a free 3’-ssDNA overhang(s)
• free 3’-terminal end is homologously paired with its homologue
by a RecA-like protein to form an intermediate known as a joint
molecule
• pairing of the displaced complementary strands results in the
formation of characteristic intermediates known as Holliday
junctions
• Holliday junction(s) is extended unidirectionally by branch
migration proteins
• the Holliday junction is cleaved by a resolvase to yield
recombinant products (e.g. spliced or patched)

14. Site specific recombination
• Homologous recombination is to ensure that the
genome of organism is nearly identical from
generation to generation
• some recombinations alter the relative position of
nucleotide sequence in chromosome- site specific
recombination
• It can be of 2 types
 Conservative site specific recombination (CSSR)
 Transpositional site specific recombination (TSSR)

15. Conservative Site Specific
Recombination
• GENERAL FEATURES:
 Recombinase recognize specific sequences where
recombination occurs
 It brings these specific sequences together to form a
protein- DNA complex(synaptic complex)
 Within synaptic complex recombinase catalyse
cleavage and rejoing of DNA molecule either to invert
or to move a segment to a new site
 One recombinase enzyme will do all these steps
 Controlled process to minimise the DNA damage

16. Recombinases
• Cleaves all 4 strands prior to exchange
• For each strand 4 molecules of reombinase is needed
Recombinase covalently linked to DNA through
phosphotyrosine/phosphoserine bond based on type of
recombinase(Serine recombinase &Tyrosine recombinase(cre-lox
recombination))
• Energy released by cleavage will be compensated when the
recombinase join the molecule forming Protein-DNA
intermediate
• Hence called conservative site specific recombination
• Then cleaved strands will be rejoined with new molecules and
hence form a holliday intermediate.
• In some cases no holliday junction is formed

17. Eg :CSSR is integration of phage λ genome in bacterial
chromosome:
 During λ integration recombination occurs at exactly same
nucleotide sequence on phage and host DNA.
 Recombination sites are ~20bp long
 It have sites for the attachment of recombinase enzyme
 3 different arrangements occur due to CSSR based on the
organisation of recombination sites
i. Insertion
ii. Deletion
iii. Inversion

18. • Prophage in lysogeny
• Independent in lytic
• Transition between the two involve site specific recombination

19. Because integrase remained bound with DNA
just like topoisomerase, the action of lambda
integrase does not require ATP.

21. excisionase

22. Application of CSSR
• To delete unwanted transgenes like markers
• To activate transgene expression/ to switch between alternate
transgenes
• To fascilitate precise transgene integration
• Chromosome engineering

23. Transpositional recombination
• Does not produce heteroduplex
• Require no specific sequence
• Mobile genes are involved
• Jumping genes
• Can move from 1 position to another on same or different
chromosome
• Transposible elements encode integrase enzyme
• These enzymes will recognize specific DNA
Steps:
• Integrase makes a cut at 1 strand at each end of DNA-results in
3’ OH
• These OH groups will invade phospho diester bond on opposite
strands of randomly selectd site on target chromosome
• DNA sequences insert to target and a short gap will be left
• Another repair process will occur to fill the gap and complete
the process
• Process result in short repeats of sequences( hall mark of
TSSR)