homologous recombination in bacteria is a central phenomenon underlying not only the DNA repair mechanism but also the horizontal gene transfer

1. DR. VIBHA KHANNA
ASSO. PROF. (BOTANY)
S.P.C. GOVERNMENT COLLEGE
AJMER (RAJASTHAN)

2. CYTOGENETICS
• BLOCK 2:
• PRESENTATION 3:
BIOLOGY OF HOMOLOGOUS RECOMBINATION IN
BACTERIA

3. Homologous recombination and its
Significance
• Homologous recombination is a type of genetic recombination in
which nucleotide sequences are exchanged between two similar or
identical molecules of DNA.
• Homologous recombination is conserved across all three domains
of life (Archaea, Bacteria, and Eukarya) as well as viruses.
• It is most widely used by cells to accurately repair the double-strand
breaks.
• Homologous recombination also produces new combinations of
DNA sequences during meiosis.
• These new combinations of DNA represent genetic variation in
offspring, which lead to evolution.
• Homologous recombination is also used in horizontal gene transfer
to exchange genetic material between different strains and species
of bacteria and viruses
• Homologous recombination is also used in gene targeting, a
technique for introducing genetic changes into target organisms.

4. Repair Of DNA Strand Gaps Generated
By Horizontal Gene Transfer
• Horizontal gene transfer create Double Stranded
Breaks and Single Stranded Gaps
• The end of this linear DNA molecule, acquired by
horizontal gene transfer is seen as a Double Stranded
Break, and the recombinational repair of the Double
Stranded Break is initiated
• In wild-type Escherichia coli, two distinct pathways are
responsible for the repair of DNA by recombination:
the RecBCD- and the RecF-pathways.
• The RecBCD-pathway is specific to recombination
initiated at double-strand DNA breaks, whereas the
RecF-pathway is primarily responsible for
recombination initiated at single-strand DNA gaps,
although it can also act at double-strand breaks.

5. Homologous Recombination in
Escherichia coli.
• The concept of recombination pathways was initially
postulated by Clark.
• Homologous recombination has been most studied and
is best understood for Escherichia coli.
• The process of finding DNA sequence homology and
exchanging DNA strands occurs in three defined stages:
– (i) presynapsis, during which the RecA nucleoprotein is
assembled;
– (ii) synapsis, during which the homology search and
exchange of DNA strands occur; and
– (iii) postsynapsis, during which branch migration can occur.
• Afterward, resolution of the resulting recombination
intermediate produces a recombinant molecule.

6. Recombination Pathways
• In wild-type E. coli, the processing of a broken
DNA molecule, and the subsequent delivery of
RecA protein to this ssDNA, occurs by either of
two pathways:
– RecBCD- and
– RecF-pathways.
• The pathway are named on the basis of the
critical and unique enzymes acting in each of the
two pathways.
• The RecBCD-pathway is used primarily to initiate
recombination at a Double Strand Break, whereas
the RecF-pathway is used for recombinational
repair at Single Strand Gaps.

7. Recombination Pathways
• Both the RecBCD and RecF pathways include a
series of reactions known as branch migration, in
which single DNA strands are exchanged between
two intercrossed molecules of duplex DNA, and
resolution, in which those two intercrossed
molecules of DNA are cut apart and restored to
their normal double-stranded state.
• In both pathways, the Holliday junctions that
result are resolved by the RuvABC enzyme
complex into the recombinant progeny

8. RecBCD Pathway
• The RecBCD pathway is the main recombination pathway
used in bacteria to repair double-strand breaks in DNA.
• In this pathway, a three-subunit enzyme complex called
RecBCD initiates recombination by binding to a blunt or
nearly blunt end of a break in double-strand DNA.
• After RecBCD binds the DNA end, the RecB and RecD
subunits begin unzipping the DNA duplex through helicase
activity.
• The RecB subunit also has a nuclease domain, which cuts
the single strand of DNA that emerges from the unzipping
process.
• This unzipping continues until RecBCD encounters a specific
nucleotide sequence (5′-GCTGGTGG-3′) known as a Chi site.

9. RecBCD Pathway
• Upon encountering a Chi site, the activity of the
RecBCD enzyme changes drastically.
• DNA unwinding pauses for a few seconds and then
resumes at roughly half the initial speed.
• This is likely because the slower RecB helicase unwinds
the DNA after Chi, rather than the faster RecD helicase,
which unwinds the DNA before Chi.
• Recognition of the Chi site also changes the RecBCD
enzyme so that it cuts the DNA strand with Chi and
begins loading multiple RecA proteins onto the single-
stranded DNA with the newly generated 3′ end.

10. Formation of Holliday Junction
• The resulting RecA-coated nucleoprotein filament
then searches out similar sequences of DNA on a
homologous chromosome.
• The search process induces stretching of the DNA
duplex, which enhances homology recognition (a
mechanism termed conformational
proofreading).
• Upon finding such a sequence, the single-
stranded nucleoprotein filament moves into the
homologous recipient DNA duplex in a process
called strand invasion.

11. Formation of Holliday Junction
• The invading 3′ overhang causes one of the strands of the
recipient DNA duplex to be displaced, to form a D-loop. If
the D-loop is cut, another swapping of strands forms a
cross-shaped structure called a Holliday junction.
• Resolution of the Holliday junction by some combination of
RuvABC or RecG can produce two recombinant DNA
molecules with reciprocal genetic types, if the two
interacting DNA molecules differ genetically.
• Alternatively, the invading 3′ end near Chi can prime DNA
synthesis and form a replication fork.
• This type of resolution produces only one type of
recombinant (non-reciprocal).

12. RecBCD Pathway
Beginning of the RecBCD pathway. This model
is based on reactions of DNA and RecBCD
with Mg2+ ions in excess over ATP.
• Step 1: RecBCD binds to a DNA double strand
break.
• Step 2: RecBCD initiates unwinding of the
DNA duplex through ATP-dependent helicase
activity.
• Step 3: RecBCD continues its unwinding and
moves down the DNA duplex, cleaving the 3′
strand much more frequently than the 5′
strand.
• Step 4: RecBCD encounters a Chi sequence
and stops digesting the 3′ strand; cleavage of
the 5′ strand is significantly increased.
• Step 5: RecBCD loads RecA onto the 3′ strand.
• Step 6: RecBCD unbinds from the DNA
duplex, leaving a RecA nucleoprotein filament
on the 3′ tail.

13. RecBCD enzyme
• The RecBCD enzyme is a
heterotrimer consisting
of the three non-
identical polypeptides,
• Catalytic activities,
which include
– DNA-dependent ATPase,
DNA helicase,
– ssDNA endo- and
exonuclease, and
– dsDNA exonuclease.

14. Secondary Pathway: RecF Pathway
• This pathway represents a unique combination of DNA
metabolic processes, with major elements of the DNA
replication, recombination, and repair enzymatic
machinery presumably interacting in a highly regulated
sequence.
• When a replication fork is slowed down at the site of
an unrepaired DNA lesion, the lesion is left in a single-
strand gap in the DNA.
• Recombinational DNA repair (postreplication repair) of
such lesions relies upon RecA protein as well as a
number of proteins generally assigned to the RecF
recombination pathway.

15. Need for the RecF Pathway
There are several problems with repairing of the Single Strand Gaps.
• First, the ssDNA gap may be coated with the single-strand DNA
binding-protein (SSB), which greatly inhibits the nucleation step in
RecA filament assembly.
• Second, RecA filaments undergo an end-dependent disassembly
reaction in the presence of SSB, proceeding from the end opposite
to that at which RecA monomers are added in filament extension, a
process that could eliminate RecA filaments before they could
initiate DNA strand exchange.
• Third, extension of RecA filaments into the adjoining dsDNA has no
apparent limit other than the availability of free RecA protein.
These considerations strongly suggest a need for the regulation of
RecA filament assembly and disassembly. This regulation is
achieved by the RecF, RecO, and RecR proteins.

16. Secondary Pathway: RecF Pathway
• A model for postreplication repair was proposed
by Howard-Flanders and colleagues.
• RecA protein forms a filament in the exposed
DNA gap.
• The bound ssDNA is then paired with
homologous dsDNA from the opposite side of the
replication fork.
• Unidirectional DNA strand exchange ensues,
converting the lesion-containing strand into
duplex DNA.
• Recombination is terminated by resolving the
DNA crossover, the lesion is repaired, and
replication is restarted.

17. Secondary Pathway: RecF Pathway

18. Secondary Pathway: RecF Pathway
• The RecFOR proteins function at an early stage of
recombination and recombinational repair
• RecF and RecR proteins in replication restart at disrupted
replication forks
• RecF protein binds to ssDNA and dsDNA and exhibits a
weak ATPase activity.
• RecO protein binds to both ssDNA and dsDNA.
• RecO also promotes an ATP-independent renaturation of
complementary DNA strands and a weak D-loop formation
activity.
• There is clear evidence for interactions between the RecO
and RecR proteins and between the RecF and RecR
proteins.

19. RecF Pathway
• At sufficiently high concentrations, RecF protein alone
inhibits RecA binding to DNA and RecA protein-mediated
DNA strand exchange
• In contrast, RecO protein interacts with SSB, and the RecO
and RecR proteins stimulate RecA protein binding to ssDNA
coated with SSB proteins.
• This RecO and RecR complex has the additional effect of
preventing the end-dependent disassembly of filaments.
• Thus, the RecO and RecR proteins overcome the inhibition
of RecA filament nucleation by SSB and the possible
premature end-dependent disassembly of those same RecA
filaments. In both cases, the RecO and RecR proteins act
together, with no effects seen with either protein alone.

20. Steps in the pre-synapsis phase of homologous
recombination in bacteria: RecF Pathway

21. Resolution Of Holliday Intermediates by
the RuvABC Complex
• The RuvABC proteins of Escherichia coli play an important role in the processing of
Holliday junctions during homologous recombination and recombinational repair.
• Homologous pairing and strand exchange reactions catalyzed by RecA lead to the
formation of a Holliday junction, a recombination intermediate that consists of two
homologous DNA duplexes linked by a single-stranded crossover.
• During the final stage of recombination, Holliday junctions are processed into
mature recombinant molecules.
• Three Ruv proteins involved in this process, RuvA, RuvB, and RuvC, have been
characterized .
• RuvA specifically recognizes Holliday junctions and allows the RuvB helicase to
bind.
• Together, RuvA and RuvB catalyze branch migration of recombination
intermediates, leading to the extension of heteroduplex DNA.
• Additional binding of the RuvC endonuclease allows the RuvABC complex to
resolve Holliday junctions by nicking strands of like polarity
• The final stage of this process (i.e., the resolution of Holliday intermediates by the
RuvABC complex) is common to both pathways