Genetic Recombination

Evolution could not happen without genetic recombination

 When mutations occurred, it would not be possible to separate favorable

from unfavorable changes. Ultimately, the chromosome would
accumulate so many deleterious mutations that it would fail to function

 By shuffling genes, recombination allows favorable and unfavorable

mutations to be separated and tested as individual units in new
assortments. It provides a means of escape and spreading for favorable
alleles, and a means to eliminate an unfavorable allele without bringing
down all the other genes with which it is associated. This is the basis of
natural selection.
Lewin, Genes VIII, p. 419 L0-_BBT000_000010
 Types of Recombination
 Homologous (or generalized): in all forms of life,
it occurs at meiosis in males and in females in
the formation of the gametes

 Site-specific: phage integration; inversion of

specific regions of the bacterial chromosome

 Transposition: does not depend on sequence

homology; specific enzymes involved

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Breakage and Joining of DNA

Each variation is characterized

by a crossover event that joins
DNA segments that were
previously separated.

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 Homologous Recombination
 Recombination is a breakage-and-reunion process
 Recombination due to crossing over between homologous pieces of DNA
 Identical or very similar sequences in the crossover region
 Complementary base pairing between double-stranded DNA
molecules
►Only conceivable mechanism for alignment of similar sequences
►Can only be envisioned if two double-stranded DNA molecules are
nicked
 Heteroduplex formation if similar complementary sequences base pair,
mismatches must exist, unless the sequences are identical
 Recombination enzymes must catalyze the various covalent and non-
covalent rearrangements
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 The Holliday model of recombination
A. Nicks occur in the same places in homologous chromosomes
(blue and red).

B. Strands of the two chromosomes exchange.

C. Nicks are sealed, permanently joining the two chromosomes

through two of their four strands and yielding a Holliday junction.

D. Branch migration occurs by breaking some base pairs and re-

forming others.

E. E and F These are simply bends and twists introduced into this
illustration of the Holliday junction to make the subsequent
events easier to understand.
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 The Holliday double-strand invasion model
 1964, Robin Holliday
 Has withstood the test of time

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The Holliday double-strand invasion model

2nd nick on 2nd nick on

same strand as different strand;
1st; patch splice
NonCrossover Crossover

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L0-_BBT000_000010
L0-_BBT000_000010
 The Meselson-Radding Model
1975, Matthew Meselson
and Charles Radding

Note: D-loop
digested
away

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The RecBCD Pathway

RecBCD protein, helicase

Chi site, 5′-GCTGGTGG-3′
RecA & SSB, D-loopL0-_BBT000_000010
RecBCD
Pathway

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Chi Sites Control RecBCD

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 RecA
 1st The RecA protein intertwines the ss and
duplex DNA in a sequence-independent manner.
 2nd the DNA ss "searches" the duplex for
homologous sequence (may involve transient base pairs formed
between the single strand and bases that flip out from the duplex DNA)

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 RecA Protein Assembles on Single-Stranded DNA
and Promotes Strand Invasion

Three possible structure combinations that participate in DNA pairing and strand
exchange. The brackets in parts a and c show the location of a gap in one of the
strands.

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 Three stages of participation of RecA in strand
exchange
 Presynapsis (Slow), in which RecA, (and SSB)
coat the single stranded DNA.
 Synapsis (Fast), or alignment of complementary
sequences in the single-stranded and double-
stranded DNAs that will participate in strand
exchange.
 Postsynapsis (Slow), or strand exchange, in
which the single stranded DNA replaces the (+)
strand in the double stranded DNA to form a new
double helix.

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Newly Base-Paired Partners Are Established within the
RecA Filament

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 Once a homologous sequence has been located,
a strand invasion occurs: the single strand
displaces one strand of the duplex as it forms
conventional base pairs with the other strand

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Role of RecA in assimilation of invading strands
 1- aggregates into long filaments with ss- or ds-DNA: 6 RecA monomers
per turn of filament which has a deep groove containing the DNA. 3
nucleotides per RecA monomer
 2- DNA held in form that is 1.5 times extended from normal B form (one
turn per 18.6 nt). dsDNA contacts RecA along minor groove leaving its
major groove accessible for reaction with 2nd DNA molecule
 3- First step is for RecA to bind the ssDNA. Then the dsDNA is
incorporated into the structure. the dsDNA is incorporated into the
structure.
 4- Interaction of the two DNAs takes place in the filament groove before
actual strand exchange (which requires a nick-3’ end). At end of the
reaction, RecA is bound to dsDNA.
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 Role of RecA in assimilation of invading strands

 5- Large amounts of ATP are hydrolyzed during the release

of RecA from the duplex DNA. ATP may act in an allosteric
fashion changing the affinity of RecA for DNA: high affinity
when ATP is present, low affinity when absent.
 6 The strands of the “invaded” dsDNA are displaced and can
then pair with respective strands of the “invading” DNA 
Holliday Junction.

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 RuvAB and Branch
Migration
 RuvA
 Holliday Junction Specific DNA binding
Protein
 Recognizes the structure (independent of DNA
sequence)
 Recruits RuvB to the site
 RuvB
 Hexamaric helicase
 The RuvB ATPase provides the energy to
drive the exchange of base pairs that move
the DNA branch.

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L0-_BBT000_000010
 RuvAB and Branch Migration
 RuvA binds to all four strands of DNA at the crossover point and
forms two tetramers that sandwich the DNA.
 RuvB is a hexameric helicase with an ATPase activity that provides
the motor for branch migration. Hexameric rings of RuvB bind around
each duplex of DNA upstream of the crossover point.
 The RuvAB complex can cause the branch to migrate as fast as 10 to
20 bp/sec. A similar activity is provided by another helicase, RecG.
 RuvAB displaces RecA from DNA during its action.

 The RuvAB and RecG activities both can act on Holliday junctions,
but if both are mutant, E. coli is completely defective in recombination
activity.

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 RuvC- End of Recombination
RuvC cleaves DNA with modest sequence
specificity 5'A/T-│-T-T-G/C
Occurs every 64 nucleotides
Major Holliday junction resolving
endonuclease
Functions in concert with RuvA and RuvB
nicks two of the homologous DNA strands
that have the same polarity
 Cleavage results in DNA
ends that terminate with 5'-
phosphates and 3'-OH
groups that can be directly
joined by DNA ligase

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Recombination
in eukaryotic
organism

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 Eucaryotes: Recombination is initiated at double-
strand breaks
 Comparison with single strand break model
 rad50 mutation

4 7

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Proteins involved EUC Recombn

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Prokaryotic and Eukaryotic Factors That Catalyze
Recombination Steps

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L0-_BBT000_000010
 Proteins involved EUC Recombn
 Rad51 is widely expressed in cells
dividing mitotically and meiotically, Dmcl
is expressed only as cells enter
Mre11,
Rad50
meiosis.
Xrs2  Dmc1-dependent recombination is
preferentially between the nonsister
homologous chromatids, rather than
between the sisters

1kb long

 The strands terminating with 3' ends

are not degraded
 SP011, introduces double-strand
breaks at a very specific time in
chromosomal regions that are not
tightly packed with nucleosomes, to
initiate meiotic recombination L0-_BBT000_000010
Recombination in eukaryotic organism

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Recombination in eukaryotic organism

Patch+Patch Patch+Splice
Splice+Splice

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Gene conversion
 A model for gene conversion without mismatchrepair.
We begin in the middle of the DSB recombination
scheme just after strand invasion.
 (a) This time, the invading strand is partially resected,
which causes partial collapse of the D loop.

 (b) DNA repair synthesis is more extensive because of

the resection, which produces a region (indicated at top and
bottom) in which all four DNA strands are allele a (red).

 (c) and (d) Branch migration and resolution do not change

the nature of the four DNA strands in the region in which
alleles A and a differ: All are allele a. Thus, this process
has converted a DNA duplex that was allele A to allele a.

When two similar but not identical DNA sequences

interact, the possibility exists for gene conversion—
the conversion of one DNA sequence to that of the
other. The sequences participating in gene
conversion can be alleles, as in meiosis, on
nonallelic genes, such as the MAT genes that
determine mating type in yeast.
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Resolutions of Holliday Junction

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 RECOMBINATION
 Conservative site-specific recombination (CSSR)
and transpositional recombination (generally
called transposition)
Recombination between two
defined sequence elements

Recombinase

Recombination between
specific sequences and
nonspecific DNA sites

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 CONSERVATIVE SITE SPECIFIC RECOMBINATION
 The segment of DNA that will be moved carries specific
short sequence elements, called recombination sites
 sequences specifically bound by the recombinases
►recombinase recognition sequences positioned symmetrically
 sequences where DNA cleavage and rejoining occur
►short asymmetric sequence (crossover region)
As the crossover
region is asymmetric,
a given recombination
site always has a
defined polarity

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 Recombinase recognition sequences
 Crossover region

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 Site-Specific Recombinases
 Serine recombinases
 Tyrosine recombinases
 Every DNA bond that is broken during the reaction is resealed
by the recombinase
 No external energy, is needed for DNA cleavage and joining by
these proteins
 DNA topoisomerases and Spo11 use the same mechanism

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Recombinases by Family and by Function

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 Serine Recombinase
 CSSR always occurs between two recombination sites
 Same DNA molecule (inversion or deletion), different
(integration)
 The serine recombinases cleave all four strands prior to
strand exchange

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Tyrosine Recombinases
 Tyrosine Recombinases Break
and Rejoin One Pair of DNA
Strands at a Time.
 In contrast to the serine
recombinases, the tyrosine
recombinases cleave and rejoin
two DNA strands first, and only
then cleave and rejoin the other
two strands .
 This cleavage occurs at the first
nucleotide of the crossover
region
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 Mechanism of DNA Exchange
 Cre recombinase & lox site

Cre-lox system belongs to

tyrosine recombinase family
 Only Cre protein and the
lox sites are needed for
complete recombination
 Green colour represents
active conformation of cre
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Application of Site-Specific Recombination to
Genetic Engineering

Recombination requires four

subunits of Cre, with each
molecule bound to one
binding site on the substrate
DNA molecules.

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 Biological Role
1. Many phage insert their DNA into the host
2. To alter gene expression
3. Maintain the structural integrity of circular DNA

Recall
 Assembly of the recombinase protein on the
DNA
 The arrangement of the two recombination sites
 Accessory Proteins
 organize DNA into a specific shape
 control the direction of a recombination reaction

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 Phage λ Integration
 Lysogenic state/ Lytic cycle

 λ Integrase (Tyr Recombinase)

 Similar to Cre in action

 att (attachment) sites

 attP (240 bp) & attB

 Central core (30bp)

 Similar to Cre in action, λ integration

requires accessory proteins

 Highly asymmetric organization of
the attP and attB sites
 Architectural protein called
integration host factor (IHF)
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 Phage λ Excision
 Xis (for excise), binds to specific DNA sequences
and introduces bends in the DNA.
 Two sequence motifs present in one arm of attR
(and also present in attP)
 DNA binding by Xis also inhibits integration

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 Salmonella Hin recombinase
 The Salmonella Hin recombinase inverts a segment of the
bacterial chromosome to allow expression of two
alternative sets of genes
 Hin recombinase is an example of programmed
rearrangements in bacteria
 In the case of Hin inversion,recombination is used to help
the bacteria evade the host immune system
 Hin is a serine recombinase which promotes inversion

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The Hin Recombinase

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 Hin Recombination Requires a DNA Enhancer
 Hin recombination requires a DNA enhancer in addition to
the hix sites
 Enhancer function requires the bacterial Fis (factor for
inversion stimulation) protein
 The enhancer-Fis complex activates the catalytic steps of
recombination
 Hin-catalyzed inversion is
not highly regulated,
rather, inversion
occurs stochastically

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 Transposable elements or transposons
 Transposition is a specific form of genetic recombination that
moves certain genetic elements from one DNA site to another.
 Recombination between the DNA sequences at the very ends
of the transposable element and a sequence in the DNA of the
host cell
 Often show little sequence selectivity in their choice of
insertion sites
 Gene disruption, changed expression

 The most common source of new mutations in many organisms

 50% of human genome has transposon related sequence.

L0-_BBT000_000010
L0-_BBT000_000010
 Classification
 Based on structure and recombination
mechanism transposable elements are devided
into three major families

LTR retrotransposons.

Nonviral retrotranposon
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 DNA Transposition by a
Cut-and-Paste Mechanism
1. The transposase binds to the terminal inverted
repeats
2. Excision of the transposon DNA from its original
location in the genome.
3. The 3'OH ends of the transposon DNA attack the
DNA phosphodiester bonds at the target DNA

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 Viral-like Retrotransposons
 A cycle of transposition starts with transcription
of the retrotransposon (or retroviral) DNA
sequence into RNA by cellular RNA
polymerase.
 Transcription initiates at a promoter sequence
within one of the LTRs.
 The RNA is then reverse transcribed to
generate the cDNA
 The cDNA is recognized by Integrase and
recombinate with a new target DNA site
 Integrase assembles on the ends of this cDNA
and cleaves a few nucleotides off the 3’ ends
of each strand
 Integrase catalyzes the insertion of cleaved 3’
ends into a DNA target site in the host cell
genome using the DNA strand transfer
reaction.
 Gap repair reaction generates target-site
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 Transposase & Integrase
 Many different tranposases and integrases carry a catalytic
domain that has a common three-dimensional shape
 This domain contains two D and a E
 The tranposase/integrase proteins use this same site to
catalyze both the DNA cleavage and the DNA strand transfer

Tranposases and integrases

are only active when
assembled into a synaptic
complex, also called a
transpososome, on DNA
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Poly-A Retrotransposition and Reverse Splicing
The Poly-A Retrotransposons use an
RNA intermediate but use a mechanism
different than that used by the viral-like
elements. This mechanism is called target
site primed reverse transcription

1. First, the DNA of an integrated element is

transcripted by a cellular RNA polymerase
2. Then, newly synthesized RNA is exported
to cytoplasm to produce ORF1 and ORF2
proteins
3.The protein-RNA complex then reenters the
nuclease and associates with the cellular
DNA
4.The endonuclease initiates the intergration
reaction by introducing a nick in the
chromosomal DNA
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 Poly-A Retrotransposition and Reverse Splicing

5. The 3’OH DNA end generated

by the nicking action then serves
as the primer for reverse
transcription of the element RNA

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L0-_BBT000_000010
 REGULATION OF TRANSPOSABLE ELEMENTS
 Two types of regulation
Transposons control the number of their copies
present in a given cell to limit their deleterious
impact on the genome of the host cell.
 Transposons control target site choice.
Preference for the regions of the chromosome
where insertion of transposon is not harmful to the
host cell.
Transposition target immunity: avoid transposing
into their own DNA.

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 Bacterial transposon Tn I0.
 Tn1O is a well-characterized representative of the
IS4 family, Tn5
 Tn10 transposes via the cut-and-paste
mechanism, also has a IHF binding site
 3 functional modules

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 Antisense
regulation of Tn10
expression
 Tn10 limits its copy number in any
given cell by strategies that restrict
its transposition frequency.

 One mechanism is the use of an

antisense RNA to control The
expression of the transposase
gene

 By this mechanism, cells that carry

more copes of Tn10 will transcribe
more of the antisense RNA, which
in turn will limit expression of the
transposase gene.

 The transposition frequency will,

therefore, be very low in such a
strain. L0-_BBT000_000010
Transposable Elements: Ds and Ac of Maize
 Barbara McClintock discovered the first
transposable elements in a study of maize (corn)
in the late 1940s
 The original mutation resulted Wild-type
from an insertion of a active C locus
transposable element, called Ds
(dissociation) into the C gene
 A transposable element, Ac
(activator) could induce Ds to
transpose out of C, causing C locus
reversion, autonomous mutated
transposon

C locus
reverted

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Ac & Ds

4500 bp

Ds must have transposed

into C before it (Ds) became
defective, or else it had help
from an Ac element.
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 Transposable Elements (pea gene)

 The first pea gene described by Mendel (R or r),

governs round versus wrinkled seeds,
 The R locus encodes an enzyme (starch
branching enzyme)
 The wrinkled phenotype results from a
malfunction sbe gene;
 Insertion of an 800-bp piece of DNA that seems
to be a member of the Ac/Ds family.
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 SINEs and LINEs: a parts list and
SINEs and LINEs owner's manual

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