Dale Ramsden
Recombination
RECOMBINATION AND GENOME
REARRANGEMENTS
Date:
Time:
Room:
Lecturer:
August 19, 2005 *
1:00pam- 1:50 pm *
G-202 Biomolecular Building
Dale Ramsden
32-044 Lineberger
dale_ramsden@med.unc.edu
966-9839
*Please consult the online schedule for this course for the definitive date and time for this
lecture.
Office Hours: by appointment
Assigned Reading: This syllabus.
Important terms are bolded. Illustrative and supplementary information (rare) is
italicised.
Basic Principles: For those of you with little background in molecular biology, a review of
basic principles of nucleic acid structure and metabolism will be presented (GUTS, or
Get Up to Speed session) immediately before this lecture, on August 15 between 9:00
and 9:50 PM
Overall objectives for “The genome, and genomic instability”
Genome Maintenance I:
Genes and Chromosomes:
August 15th
Genome Maintenance II:
Genome Replication:
August 17th
Genomic Instability I:
DNA damage and repair:
August 18th
Genomic Instability II:
Recombination:
August 19th
This section consists of four lectures: In the the first two you will learn how to define the
genome, and describe how genomes are maintained. In the second two lectures, you will learn
why genomes are unstable, the consequences of genomic instability, and how cells cells mitigate
the risk of genomic instability.
Lecture objectives: At the end of this lecture, you should have an
idea
a) How double strand breaks are repaired, and the consequences of repairing double strand
breaks different ways.
b) How retroviruses and other mobile elements work
c) Why you do V(D)J recombination, and how V(D)J recombination relates to both mobile
DNA elements and double strand break repair.
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I.
Recombination
GENERAL CONSIDERATIONS
Recombination and rearrangements represent a gross change in the organization of your genome.
For example, recall you have one maternal and one paternal copy (homologous copies) of each
chromosome; During meiotic recombination, paternal and maternal information is mixed up (see
e.g. crossover in figure below). However, there also more severe rearrangements, including
exchange of material between chromosomes that are NOT homologous (see e.g. translocation
between Ch 14 and Ch18 in figure below). Duplications/amplifications, insertions, deletions,
and fusions can also occur. Such rearrangements are typically the consequence of DNA double
strand break (DSB) repair.
Figure 1. Examples of genome rearrangements
Sources of DSBs that generate genome rearrangements
1) DSBs can be intentionally made by the cell, as the resulting recombination is required for
certain NORMAL developmental programs; meiosis, V(D)J recombination
(rearrangement is a good thing)
2) DSBs from exogenous DNA damaging agents (e.g. ionizing radiation, ROS,
chemotherapy) are typically repaired so that rearrangements do NOT occur. However,
aberrant repair of these DSBs can lead to rearrangements that cause cell death, or cancer
(rearrangement is a bad thing)
3) Integrations and excisions of mobile DNA elements (including transposition, retrovirus
integrations; see example below) are another source of genome rearrangements.
Certain rearrangements (translocations/deletions/insertions) often cause cell death and
developmental problems, and are seen in many (most?) cancers
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Recombination
II. DSB REPAIR
A. Homologous recombination
REVIEW Chromosomes are found in pairs as
homologues (one homologue inherited from
mom, one homologue from pop), both of which
are duplicated prior to each cell division. The
recently replicated copy is termed a sister
chromatid.
Homologous chromosomes are similar, but
because they are derived from different parents,
they are not identical. Sister chromatids are
made by replication in S phase of every cell
cycle, and are identical (with the exception of
errors made during replication).
How it works
Figure 2. Homologous recombination
1) make a double strand break
2) At the region of the break, align the equivalent unbroken region from either the intact
homologous chromosome, or the intact sister chromatid
3) use the intact homologue or sister chromatid as a template for repair
(yes, its true; the term homologous recombination isnt great).
The choice of homologue or sister is made according to cell type.
In Meiotic cells: Programmed rearrangement for genetic variability
Genetic variability is derived from recombination during meiosis. Meiosis is the process
required to make the haploid (one copy of each chromosome) cell (germ cell in multicellular
eukaryotes; sperm or oocyte) that is the precursor to sexual reproduction. A diploid progenitor
(parent cell) undergoes a round of replication and produces sister chromatids, then undergoes
homologous recombination, then divides twice without replication, producing four daughter cells
with a haploid DNA content. Each of the four haploid germ cells now contains half the number
of chromosomes as the parent cell; also, each chromosome in a given haploid cell is now a
unique mixture of the maternal and paternal version of that chromosome.
What does meiotic recombination have to do with double strand break repair? Recombination
between two homologous chromosomes requires one of the chromosomes to be broken. Meiotic
cells are programmed to do introduce a double strand break approximately at random in each
chromosome. They have an enzyme to do this (expressed only in meiotic cells, called spo11) that
looks like a type II topoisomerase, except after making the double strand break, it doesn’t reseal
it. The intact homologue is then used as a template for repair.
Recombination occurs between homologues
In meiosis “un-productive” recombination with sister chromatids is blocked; such
recombinations would be un-productive because sisters are identical, thus no variability would
result
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Dale Ramsden
Recombination
Recombination between homologous
chromosomes generates genetic
variability in two ways.
Gene conversion. Short patch of
information around break is “converted”
to be like the unbroken. i.e. if paternal
chromosome has double strand break, the
region around break is converted to
maternal information after repair is
finished. Non-reciprocal (I.e maternal
copy stays the same).
Cross-over. Two chromosomes exchange
arms as a consequence of repair.
Figure 3. Meiotic recombination
WHY homologous recombination can result in gene conversion or crossover is a consequence of
how the mechanism works; for details regarding this, see…
http://sekelsky.bio.unc.edu/Research/Meiotic_rec.html
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CrossingOver.html
In Mitotic cells: DNA Repair
In addition to its role in meiosis, homologous recombination is a critical pathway for repair of
double strand breaks made accidentially (e.g. replication through unrepaired strand breaks, or
exogenous agents like ionizing radiation). Homologous recombination may also be used for
repair of other types of DNA damage.
Hereditary breast cancer is caused in part by defects in a gene required for recombinational
repair (BRCA2).
In mitotic cells, Recombination occurs between sister chromatids
When recombination is not recombination. Recombination in cells undergoing mitosis is
skewed such that the intact sister chromatid is used as a template for repair, rather than a
homologuous chromosome (opposite to meiotic recombination). This is significant, as sister
chromatids are identical (not homologous). Exchange of material between sister chromatids is
thus normally conservative, i.e. will not result in a rearrangement or “recombination” from a
genetic standpoint. This ensures the genetic makeup of daughter cells is the same as the parent
before recombination. This is why it makes for a good repair pathway in mitotic cells, and why
you avoid repair by the sister chromatid in meiotic cells. Nevertheless, the mechanism by which
sister chromatids exchange material is essentially the same as homologous recombination, so the
term homologous recombination is used to refer to the proccess in general terms, identifying the
paired molecules as sisters or homologues when necessary.
When recombination is still recombination. Alignment of chromatids is based on
homology/identity. Therefore, chromatids can be misaligned when there is homology between
two regions on the same chromosome. This is not uncommon, due to frequent repetitive DNA
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Dale Ramsden
Recombination
elements in the genome (see Genes and chromosomes, and also below) (e.g. Alu elements).
Recombination and crossover between misaligned chromatids can result in unequal sister
chromatid exchange; the products are a duplication of the region between repetitive elements in
one sister chromatid, and deletion in the other.
Figure 4. Unequal sister chromatid exchange
B. End joining
Figure 5. End joining.
End-joining refers to a pathway for repair of double strand breaks in which broken DNA ends
are simply joined back together.
How it works
Pretty simple, really. Get rid of damage at ends (using nucleases), make the ends compatable
(polymerases) then stick whatever is left back together with a ligase.
Note that repair junctions almost always involve loss of sequence, thus this pathway is
innaccurate.
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Recombination
Given that end joining is less accurate, why would you choose to do it over homologous
recombination?
Essentially, End joining is more flexible and easier to do.
Accuracy
Efficiency
Flexibility
Homologous recombination
Conservative
Requires lots of synthesis,
chromatid/chromosome synapsis
Restricted to S phase (cells must be
actively cycling)
End joining
Loss of information at ends, prone to
translocation
Easy, efficient
Anytime (can occur in resting or
differentiated cells)
III. MOBILE ELEMENTS
A. Transposition
Structure
Figure 6. A transposon
Transposons are mobile elements within a host genome that are able to directly transport
themselves to other locations within the genome, or sometimes to another host’s genome. The
DNA excises from one location, and then integrates into another location. Transposition is a
common way for bacteria to acquire resistance to antibiotics. Each transposon contains a gene
(or genes) encoding a transposase, the enzyme involved in insertion of the transposon to a new
site. Transposons have two signals, which usually flank the genes necessary for transposition.
Mobility
Excision is targeted by signals.
However, the target site for
integration is typically chosen
with much less specificity.
Excision from the host genome is
initiated by transposase-mediated
strand breaks at the border
between signal and host DNA,
resulting in a “free” linear
transposon DNA segment, and a
double strand break in the host
genome at the site of excision.
These breaks are dangerous, and
unless accurately repaired by the
host’s double strand break repair
machinery can lead to death or
recombination.
Figure 7. Transposon mobility
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Recombination
Integration occurs by the introduction of a double strand break at a new “target” site in the host
genome. The linear transposon DNA is inserted by joining the ends of the transposon to the ends
of the broken target site.
For these reasons, transposons are frequently harmful to the host. After excision, the double
strand breaks left behind can lead to cell death, or cancer-causing recombinations. They also can
integrate such that they disrupt nearby genes or the expression of nearby genes.
B. Retro elements
Retro-element “life” cycle
Retro elements are a general term for mobile elements that have an obligatory RNA
intermediate. They integrate into host genomes by the same mechanism as transposons do.
However, the integrated retro element is NOT excised. Instead, the retro element’s genome is
transcribed by host transcription
Pro ces s
Repl ic ation Tran script ion Rever se
machinery as directed by DNA
transc ri ption
Enyz
m
e
DNA
RN
A
Rever se
sequences at the 5’ end of the
po
lym
erase
po
lym
erase
transc ri ptas e
element’s genome. This RNA
Tem plate DNA
DNA
RNA
copy of the genome is then
Produ
ct
DNA
RN
A
DNA
converted into a DNA copy by
the process of reverse
transcription. The new DNA copy can now integrate elsewhere. Reverse transcription
represents an important reversal of the normal flow of information in molecular biology (see
table).
Retro elements can be further divided into
1) Retroviral elements: their genome can
be packaged into infectious virus, and thus
have an extracellular phase in their life
cycle, and 2) Retroposons, whose activity
are limited to intracellular events.
Like most higher eukaryotes, we have a
REDICULOUS number of MOSTLY
inactive retroposons already resident in our
genome; about 1/4 of our genomic DNA
comes from retroposons (remember only
about 1/20th actually codes for genes).
They do move occasionally….
Insertional mutagenesis
As previously mentioned for transposons,
insertion of retroelements can disrupt
genes. A significant number of congenital,
disease-causing mutations are due to
insertion of a retroposon into an important
gene in germ cells. With respect to
retroviruses, the risk of disruption is even
Figure 8. Retroviruses vs. Retrotransposons
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Recombination
greater, as they have a strong preference for integration into or near active genes (likely to a
preference for euchromatin). Moreover, the integrated viral genome has strong transcriptional
enhancers, thus even the insertion of a retrovirus nearby a gene can result in over expression or
anomalous expression of that gene.(see also case conference I)
Pathogenic Retroviruses
In addition to “accidental” interference with host cell growth due to its integration, some
retroviruses specifically carry genes to disrupt normal cell growth.
Retroviral transduction of host sequences and non-autonomous viruses
The retrovirus can also acquire growth disrupting capability by recombination between the
integrated retrovirus (provirus) and the host genome, or aberrant splicing of the viral RNA to a
host gene. This produces a hybrid viral genome, with host sequence partly substituted for part of
the viral sequence. The hybrid RNA can contain information necessary for packaging and
integration, as well as the host gene, but it usually cannot produce infectious virus on its own
because it lacks all the necessary viral genes. This partially defective virus is not active on its
own (non-autonomous). However, missing
functions can be provided “in trans”, by an intact
“helper” virus present in the same cell.
In some cases the encoded host/viral hybrid
protein alone is sufficient to cause cancer. For
example, v-src was is an aberrant version of a
normal host gene carried by the Rous sarcoma
retrovirus, and causes cancer in chickens.
In a similar manner, retroviruses can be
engineered for use in gene therapy, to deliver
genes to cells that are deficient in that gene (see
case conference I).
Figure 9. Non-autonomous retroelements.
IV. V(D)J RECOMBINATION
A. Generation of diversity
Vertebrates (including us) possess an “adaptive” immune system. We are able to mobilize
subsets of cells that can specifically respond to any one of a virtually infinite variety of different
foreign agents, and protect us against them. This ability rests in the diversity present in a class of
receptor proteins (e.g. in B cells, the antibody or immunoglobulin) found on the surface of the
lymphocytes (white blood cells). As lymphocytes mature, they each generate a different receptor,
each with a different specificity. Lymphocytes shown to be useful in recognizing foreign
pathogen are activated, and now help clear the body of pathogen. In the spleen and lymph nodes,
lymphocytes with specific receptors that have proven useful in this way are then “saved” for
future use (immunological memory, the reason why vaccines work), while the rest are
eliminated.This process goes on for the life of the organism.
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Recombination
Figure 10. Generation of receptor diversity
Diversity in antibodies is primarily derived through V(D)J recombination. Multiple versions of
coding segments (V, D, or J segments) are distributed at a specific locus in the chromosome, as
shown above. One each of a V, D, and J segment is then assembled to code for the complete
variable domain of the mature receptor. The variable domain provides specificity for different
foreign agents. The remaining domains (constant domains) mediate the immune response to the
foreign agent, and are thus common to all the different antibodies. The assembly process, termed
V(D)J recombination, occurs at the DNA level and only in developing lymphocytes.
One of the loci coding for human antibodies has an estimated 300 different Vs, 12 different Ds,
and 4 different Js. This leads to a potential 300X12X4 or over 14,000 different combinations.
The junctions of these recombinations are also imprecise, further increasing diversity. Estimates
suggest over 100 million different receptors are possible, a number that likely exceeds the pool
of lymphocytes we have available at any given time.
B. How it works
V(D)J recombination occurs in two
steps: a lymphocyte specific
initiation step (cleavage), and a
resolution step (joining) that can
occur in any cell type.
Initiation: cleavage makes a pair
of double strand breaks
RAG1 and RAG2 (Recombination
Activating Genes 1 and 2)
recognize a pair of recombination
signals and introduce double strand
breaks precisely at the borders
between signals and coding
segments. RAG1/RAG2 are
expressed only in developing B/T
cells.
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Figure 11. V(D)J recombination.
Dale Ramsden
Recombination
Resolution: End joining
Resolution of these double strand breaks does not require lymphocyte specific genes; rather,
resolution requires genes encoding end-joining double strand break repair factors, which are
expressed in all cell types. Mutations in factors required for end joining repair leads to both
immunodeficiency (because lymphocytes cannot resolve double strand break intermediates in
V(D)J recombination) as well as general cellular sensitivity to other sources of double strand
breaks (e.g. ionizing radiation sensitivity, because they cannot efficiently repair other sources of
double strand breaks either).
The ends of coding segment DNA are joined together by the end joining pathway for repair of
double strand breaks, forming the variable domain of the antibody. Signal ends are typically also
joined together to make a signal junction product. This circular DNA has been removed from its
normal chromosomal context, and, under most circumstances, is lost in subsequent cell divisions.
An adapted transposon
V(D)J recombination possesses many mechanistic similarities to transposition. 1) a pair of
signals are used to target a pair of double strand breaks, resulting in the excision of the
intervening DNA from the host genome. 2) After excision, the break in the flanking “host” DNA
must be rejoined by host repair machinery.
However, we appear to have mostly re-trained this transposon so that the excised DNA is formed
into a circular deletion product, rather than integrating elsewhere.
C. V(D)J recombination and disease?
Severe combined immunodeficiency (scid)
A failure in the cleavage step (RAGs) results in complete deficiency in both B and T cells (scid)
because they cannot initiate V(D)J recombination (no cleavage).
However, some people have radio-sensitive scid, or rs-scid; in addition to no B and T cells, these
patients have a general cellular sensitivity to double strand break inducing agents (e.g. ionizing
radiation). These people have a defect in a gene required for end joining.
Lymphoid malignancy
Lymphoid malignancies (leukemia, lymphoma, thymoma) are common. They are often due to
translocations, and these translocations frequently have breakpoints in antibody T cell receptor
loci near where double strand break intermediates in V(D)J recombination occur.
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Dale Ramsden
Recombination
Figure 8 reproduced with permission from Genes VII, by B. Lewin, Oxford University Press, 2000, p. 486.
Figure 9 reproduced with permission from Genes VII, by B. Lewin, Oxford University Press, 2000, p. 495.
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