Molecular basis of
recombination, the importance
of recombination in genetics
1
DNA recombination
 exchange of segments of
DNA molecules between
chromosomes
 often occurs during meiosis
in sexual reproduction replacing
parts of
homologous chromosomes
 increase of genetic
diversity in the offsprings an
evolutionary advantage
for offsprings
 It exists in prokaryotic cells
(after transfer of foreign
DNA by transformation,
transduction or
conjugation)
2
Homologous and non-homologous recombination
 homologous
recombination
 allows the exchange of
genetic material between
chromosomes that are so
similar that can lead to
base pairing between
them
 common between the two
copies of the same
chromosome (in meiosis)
 non-homologous
recombination
 rarer, does not require
sequence homology
 requires specific proteins
3
Molecular basis of
homologous recombination
 reciprocal recognition
of homologous
segments of doublestranded
DNA
 interruption of one
strand of each helix
 replacement of strands
 reuniting to form
Holliday structure
4
5
Holliday structure
can isomerize
separation of
recombinant
molecules by
resolvase
Recombination during meiosis
6
Repair of
double-
strand
breaks
MRE11
RAD50
BRCA1
BRCA2
7
Site-specific
recombination
 recombination between non-homologous
sequences
 mechanism whereby the genome moves mobile
genetic elements
 controlled by enzymes which recognize short
sequences at the ends of the mobile elements,
does not require extensive DNA homology
8
Transposons, mechanisms of
transposition, retroelements
9
Transposons = mobile
genetic elements
 Cause changes in genetic information (insertional
inactivation, activation, modulation of gene
expression, mutation)
 Significantly involved in the architecture of
genome - most of repetitive DNA in the genome
of the plant consists of transposon sequences
 In plants, transposons do not carry genes which
directly increase the fitness (unlike many bacterial
transposons)
10
Transposons = mobile
genetic elements
 segments of DNA capable of transfer
to another site of the genome
(transposition)
 in all prokaryotes and eukaryotes yet
analyzed (with the exception of the
parasite Plasmodium falciparum)
 Transposase enzyme
 They do not exist as a separate
independent form like plasmids or
phages
 important source of genomic
instablity
 - In plants - thousands of families
(80% of the genome)
 - animals 3-45%,
 - fungi 2-20%
 - human 40%
11
The discovery of transposons
 Barbara McClintock (1902-1992)
 The Nobel Prize in Physiology and Medicine in 1983 for
discovering (knowledge of the nature) of mobile genetic
elements in maize
 Study of chromosomal breakage in maize
 increased incidence of breaks in a certain area (= a marker
called "dissociation" Ds)
 position of marker was not stable after crossing with some lines,
and shifted to other spots (= line carrying the "activator" Ac)
Demerec (1937) described the unstable mutations in the D. melanogaster
B. McClintock ( Nobel Prize 1983) showed in the 40s and 50s while studying
chromosomal breaks in maize that its genome contains many mobile
elements causing somatic mutations (ac / ds)
Molecular analysis of these elements may be implemented up roughly from
the late 70s ,the first cloned elements are elements of D. melanogaster
(1978), which are now known as "Copia-like" elements
Transposons are found in all organisms, which have been searched, (except parasite Plasmodium
falciparum) in plants up to 80% of the genome, in animals 3-45%, fungi 2-20%
Transposons are segments of DNA capable of transfer to another place of the genome
(transposition), either autonomously or with the help of related elements.
12
 Barbara McClintock discovered the first TEs in maize (Zea mays) at the Cold Spring
Harbor Laboratory in New York. McClintock was experimenting with maize plants
that had broken chromosomes.[5]
 In the winter of 1944–1945, McClintock planted corn kernels that were selfpollinated,
meaning that the silk (style) of the flower received pollen from its own
anther.[5] These kernels came from a long line of plants that had been selfpollinated,
causing broken arms on the end of their ninth chromosomes.[5] As the
maize plants began to grow, McClintock noted unusual color patterns on the
leaves.[5] For example, one leaf had two albino patches of almost identical size,
located side by side on the leaf.[5] McClintock hypothesized that during cell
division certain cells lost genetic material, while others gained what they had
lost.[6] However, when comparing the chromosomes of the current generation of
plants with the parent generation, she found certain parts of the chromosome had
switched position.[6] This refuted the popular genetic theory of the time that genes
were fixed in their position on a chromosome. McClintock found that genes could
not only move, but they could also be turned on or off due to certain
environmental conditions or during different stages of cell development.[6]
 McClintock also showed that gene mutations could be reversed.[7] She presented
her report on her findings in 1951, and published an article on her discoveries in
Genetics in November 1953 entitled "Induction of Instability at Selected Loci in
Maize."[8]
 Her work would be largely dismissed and ignored until the late 1960s-1970s when it
would be rediscovered after TEs were found in bacteria.[9] She was awarded a
Nobel Prize in Physiology or Medicine in 1983 for her discovery of TEs, more than
thirty years after her initial research.[10]
 Approximately 90% of the maize genome is made up of TEs,[11] as is 44% of the
human genome.[12]
13
Types of transposition
 Class I -conservative transposition - „CUT and PASTE“
 transposon excision and transfer to another
place of the genome
 Only transfer, without multiplication
 Class II-replicative transposition - „COPY and PASTE“
 replication, the copy is placed in a new location
 original element remains, the number of copies ~
number of replications
 copying through RNA intermediate or direct insertion
of copied DNA
14
Conservative x duplicative transposition
15
Conservative x duplicative transposition
16
Conservative x duplicative transposition
17
Classification of transposons
Nature Rev. Genet. 2008
1. Class: Depending on whether or not with RNA intermediate
• DNA transposons
• Retrotransposons
2. Subclass: According to the mechanism of replication (for
DNA transposons)
3. Order: According to the basic structural features
4. Superfamily: By sequence homology
18
Basic types of TE
Lisch 2013, Nature Rev. Genet.
Rolling
circle
replication
???
Retrotransposons DNA transposons
19
Types of transposition 20
Types of transposable
elements (Not all transposons encode
the necessary enzymatic activity)
 autonomous elements
– encoding the gene whose product ensures
transposition / replication
 non-autonomous elements
– derived from autonomous
– lost the genes required for the transposition,
but can be mobilized by other related
autonomous elements
– have cis sequences necessary to mobilize
21
A) Transposable elements in bacteria
 First TE studied at the molecular level
 relocated within the bacterial chromosome or the
chromosome and plasmid
 The main types:
 IS-elements (only include genes coding for
proteins providing transposition)
 composite transposons and Tn3-elements
(additionally contain genes which encode
products functionally unrelated to the process of
transposition)
22
IS-elements
 usually less than 2500 bp
 framed by short identical sequences - inverted
terminal repeats
 mutations in terminal repeats eliminates
transposition capability
 They contain only genes for ensuring and
controlling transposition
 encode transposase enzyme: binds to the ends of
the element, cleaves both DNA strands - thus the
element is released from the original site
23
Transposon
Tn3
 It contains genes that are not necessary for
transposition
 the ends are formed by simple inverted repeats
 at the target site duplication occurs
 Structure:
 transposase/resolvase gene and their repressor
 gene for beta-lactamase (Amp resistance)
24
Importance of bacterial
transposons in medicine
 often they contain genes for resistance to
antibiotics
 that these genes can spread easily and thereby
increase the resistance of pathogenic bacteria to
antibiotics
 today it is difficult to treat a variety of infectious
diseases (diabetes, gonorrhea, tuberculosis, etc.).
 spread of resistance is promoted by the
widespread use of antibiotics
 transposons (transfer between the molecules of
DNA within the bacterial cells) and conjugative
plasmids (transfer between different bacterial
strains)
25
Bacterial transposons:
summary
 Insertion sequences - IS-elements, "cut and paste"
transposon, part of bacterial chromosomes and
plasmids
 Composite transposons generated by 2x ISelements,
flank area for one or more genes for
resistance to antibiotics
 Tn3-type replicative transposon, temporarily
connects the molecules to form co-integrate, when
unfolded, each molecule contains 1xTn3
 Bacterial transposons bounded by inverted repeats
are duplicated after incorporation
 Conjugative plasmids - carrying a transposon
containing the resistance genes from one bacterium
to another
26
B) Transposons in eukaryotes
 mainly types of "cut and paste" and retrotransposons
 P-elements in Drosophila
 Ty-elements in yeast
 human retrotransposons LINE constitute about 15% of the
genome (mostly immobile due to mutations - incapable of
transposition)
 some can maintain mobility and can cause diseases (e.g.
transposition into the gene for a factor required for blood
clotting - hemophilia)
27
DNA transposons subclass
I:
 encode transposase, the
edges are inverted repeats
 transposition - complex
process – binding of IR,
cleavage (transposase),
cleavage of the target
sequence, DNA synthesis,
ligation
 duplication of short
sequence (2-8 bp) in
incorporation site =
footprint after re-excision
28
DNA transposons subclass
I:
 usually integration in the vicinity of the original insertion
 usually a few to a few hundred copies in the genome
 Ac, Spm, Mu (maize), Tam (Antirrhinum), TphI (petunia)
Tags (Arabidopsis), Stowaway, Tourist> 10,000 copies
every 30 kbp (maize, insertion into the TA-rich sequences)
 MULE (Mutator-like elements) with rice - over 1,000 gene
fragments mobilized - 5% is expressed - evolution of new
genes
 mutated non-autonomous forms Ac/Ds (Ds1, Ds2),
Spm/dSpm
29
Movements and propagation of DNA transposons
30
break repair after TE excision by homologous second chromatid section
(possibility of reconstructing the original sequence with TE = amplification)
The activation mechanism during replication? Hemimethylated status?
DNA transposons subclass
II:
 order: Helitron - a
single-stranded
break, DNA
migration, insertion
 in maize 4-10
thousands of mobile
genetic elements
31
32
RETROTRANSPOZONS
Class I TEs are copied in two stages: first, they are transcribed from
DNA to RNA, and the RNA produced is then reverse transcribed to
DNA. This copied DNA is then inserted back into the genome at a
new position.
The reverse transcription step is catalyzed by a reverse transcriptase,
which is often encoded by the TE itself. The characteristics of
retrotransposons are similar to retroviruses, such as HIV.
Retrotransposons are commonly grouped into three main orders:
•TEs with long terminal repeats (LTRs), which encode reverse
transcriptase, similar to retroviruses
•Long interspersed nuclear elements (LINEs, LINE-1s, or L1s), which
encode reverse transcriptase but lack LTRs, and are transcribed by
RNA polymerase II
•Short interspersed nuclear elements (SINE) do not encode reverse
transcriptase and are transcribed by RNA polymerase III
[Note : Retroviruses can also be considered TEs. For example, after conversion of
retroviral RNA into DNA inside a host cell, the newly produced retroviral DNA is
integrated into the genome of the host cell. These integrated DNAs are termed
proviruses. The provirus is a specialized form of eukaryotic retrotransposon, which can
produce RNA intermediates that may leave the host cell and infect other cells. The
transposition cycle of retroviruses has similarities to that of prokaryotic TEs, suggesting a
distant relationship between the two].
Retrotransposons
33
Retrotransposons
- replication through RNA intermediate
- 1-13 kbp size (apart form SINE), millions of copies (up to 40-80% of
the genome)
 often in heterochromatic regions, in euchromatin especially among
genes - possibly as a result of selection pressure
34
Retrotransposons –
elements similar to
retroviruses
Retrotranspozons
 Odrer: LTR –významnější TE u rostlin
 LTR (long terminal repeat): promoter, terminator,
direct repeat
 short duplication of the target sequence
 - protease, reverse transcriptase, RNase H,
integrase, nucleocapsid protein
35
LTR retrotransposons
 Ty1- copia group
 BARE-1 barley, 12.1 kbp> 50,000 copies of transcript in
leaves and callus
 Opie -1, maize, 8.7 kbp,> 30,000 copies, roots, leaves,
integration into the LTR
 PREM-2, maize, 9.5 kbp,> 10,000 copies in microspore
 TNT1, tobacco, 5.3 kbp,> 100, protoplasts, roots, activation
after injury, pathogen attack, integration into
euchromatin
 Ty3 – gypsy group
 potential ancestors of animal retroviruses, sometimes envlike
sequences
 Athila, A.t., 10,5 kbp, >10000, paracentromeric regions
 Athila-1-1, A.t., 12 kbp, 730, env-like sequences
 Cinful-1, maize, 8,6 kbp, 20000, leavse, env-like seq.
36
LTR retrotransposons
- replication
 - replication analogous
to retroviruses - LTR (U3,
R, U5)
 - PBS (primer binding site):
tRNA primer
 - jumps between
templates
 (direct repeat - R)
37
Transposable elements in humans
 44% of human DNA is derived from transposable elements
 many different types:
 long dispersed nuclear elements (LINE) - size about 6 kb
 short dispersed nuclear elements (SINE) - less than 400 pb
 use of reverse transcription
38
Retrotransposons w/o LTR
 LINE (long interspersed nuclear elements)
 - apparently phylogenetically oldest predecessor of
transposons with LTR
 - 5´end – promoter; 3´end - terminator
 Cin4, maize, 1-6,8kbp, 50-100, variously truncated
forms
 SINE (short interspersed nuclear elements)
 - using RT apparatus of other transposons (non-
autonomous)
 derived from RNA polymerase III products (tRNA
7SLRNA, rRNA)
 < 500 nt
39
Regulation of the activity of
transposons
 Retrotransposons
 enormous potential to change gene function and
genome structure
 regulation by own control mechanisms and host
(mostly inactive - methylation, controlled activation
developmentally, external conditions)
 coevolution of mechanisms regulating transposition,
insertion specificity, mutagenic potential
 functions: changes in gene regulation, role in DNA
repair, centromeres
 DNA transposons
 regulation of activity by environmental conditions:
 Tam1 in snapdragon (1000 * at 15 ° C)
 Methylation
40
Methylation of transposons
 Inactivation - temporary, permanent, the possible
cause of methylation mechanisms
 For retrotransposons - similarities with the silencing
of multiple-copy genes
 The activity of Ac and Spm is different depending
on the type of gametes (changes during
gametogenesis)
 The increase of Spm and Mu methylation with
development (leaves), demethylation in the early
stages of development
 Methylation is needed especially during meiosis –
safeguarding of integrity (x illegitimate crossover)
41
Significance of transposons
 inducing mutations = increasing variability
 modulating the expression (activation, repression, during
development, during stress)
 creation of new genes
 induction of chromosomal rearrangements induced by
recombination between transposons
- in plants transposons do not carry genes which directly
increase the fitness (resistance etc.).
- increasing fitness by randomly induced mutations (e.g.
activation by stress conditions) - very low probability ...
- great importance in the domestication (breeding) of plants
42
Mutations caused by transposons
 place of incorporation (different preferences: GC, AT)
 character of carried regulatory sequences
 Modulation of expression (time and place) - promoter,
enhancer
 changes in the stability of the transcript and
posttranscriptional editing (splicing) - UTR, introns,
terminator
 change in the sequence of the resulting protein,
premature termination of translation, creation of
chimeric genes, ... - exons, introns
43
Regulation of gene
expression by transposons
 prevention or reduction of transcription
 modulation of time and site specific expression
 changes in the stability of the transcript and
posttranscriptional editing (splicing)
 change in the structure of the resulting protein
 e.g. Maize- inactivation of the gene CCT
(response to photoperiod length) by inserting a
cacti-like element (TE DNA) in the promoter region
- expansion of cultivation in temperate zones
(flowering during a long day)
44
Significance in the evolution of genes
 insertional mutagenesis (premature termination)
 possible participation in the multiplication of genes
- directly or indirectly via homologous recombination
- advantageous to have a gene family of different
regulations, respectively backup copies of genes
 creation of intron-free copies of genes (reverse transcription)
 may participate in the creation of entirely new genes - eg.
fusion of transmitted fragments of existing genes (helitrons,
MULE)
 genes that were originally of transposon origin were
"domesticated" by many eukaryotic organisms for new
features (eg. telomerase, syncitin, ...)
 natural genetic engineering tools
 they spread and thus can provide a selective advantage for
the host
 others are genetic parasites
45
Changes on the level of the
genome
 possible participation in the multiplication of
genes
 creation of intron-free copies of genes
 chromosomal rearrangements (repetitive
sequences)
 breaks, inversions, deletions, duplications,
translocations, ...
46
TE in disease
 TEs are mutagens and their movements are often the causes of genetic disease. They can
damage the genome of their host cell in different ways:[27]
 a transposon or a retrotransposon that inserts itself into a functional gene will most likely
disable that gene;
 after a DNA transposon leaves a gene, the resulting gap will probably not be repaired
correctly;
 multiple copies of the same sequence, such as Alu sequences, can hinder precise
chromosomal pairing during mitosis and meiosis, resulting in unequal crossovers, one of the
main reasons for chromosome duplication.
 Diseases often caused by TEs include hemophilia A and B, severe
combined immunodeficiency, porphyria, predisposition to cancer,
and Duchenne muscular dystrophy.[28][29] LINE1 (L1) TEs that land on the
human Factor VIII have been shown to cause haemophilia[30] and
insertion of L1 into the APC gene causes colon cancer, confirming that
TEs play an important role in disease development.[31]
 Additionally, many TEs contain promoters which drive transcription of their own
transposase. These promoters can cause aberrant expression of linked genes, causing
disease or mutant phenotypes.
47
Discovery of transposons
 Barbara McClintock (1902-1992)
 The Nobel Prize in Physiology and Medicine
in 1983 for discovering (knowledge of the
nature) the mobile genetic elements in
maize
 Study of chromosomal breakage in maize
 increased incidence of breaks in a certain
area (= a marker called "dissociation" Ds)
 position of marker was not stable after
crossing with some lines, and shifted to other
spots (= line carrying the "activator" Ac)
48
Discovery of transposons
 in one line a Ds marker shift caused a loss of
purple discoloration of the caryopses
 light color caryopses (c) caused by the insertion
of the Ds element were not stable in a crossing
with lines carrying Ac - appearance of caryopses
with purple spots
 triploid endosperm
•c/c/c = light color
•C/c/c or C/C/c pr C/C/C = purple color
49
Transposition and
coloration of the caryopses
 If the c is reversed to C, red pigment
begins to form in the cell thus forming a
spot on a light background
 the earlier in the development of the
caryopsis reversion occurs, the greater
the stain
 B. McClintock concluded that „c" allele
was created by integrating the nonautonomous
transposon "Ds" to "C" allele
(Ds = dissociation)
 reversion of c to C is due to the
transposition of the Ds element from the
C allele which is mediated by
autonomous transposable element
 "Ac" (Ac = activator)
50