Double-strand breaks (DSBs),
their repair and misrepair
How double-strand breaks are generated
DSBs are caused by several factors:
• arrest of replication and restart of DNA synthesis (replication forks tend to stall in
regions of repeat elements - e.g. tRNA genes, retroposons, and telomeres); major source
of DSBs!
• transposon excision
• during meiotic recombination (needed for recombination)
• mechanical pulling (e.g. in dicentric chromosomes)
• experimentally (radiation by X-rays, DSBs inducing chemicals, rare cutting restriction
endonucleases, DNA transposons)
 in vegetative (mitosis) and generative cells (meiosis)
 DSBs have to be repaired before genomes are replicated (S phase)
 in plants, errors in DSB repair (DSBs misrepair) can have the evolutionary
significance because changes in meristematic cells can be transferred to the
offspring >>> chromosome rearrangements
DSBs in meiosis
 necessary for homologous recombination (cross-overs)
 induced by the Spo11 topoisomerase
DSBs in in somatic plant cells and their repair
homologous recombination (HR)
• after replication: uses sister chromatids
as a template to rejoin DSBs (most
frequently)
• minor pathway
• error-free repair
• in late S-G2 phase
non-homologous end joining (NHEJ)
• also known as homology-independent
recombination or “illegitimate
recombination”
• main mode of DSB repair in higher
eukaryotes (somatic plant cells)
• the broken ends are re-ligated directly
• often an error-prone process
• throughout the cell cycle (mainly G1
phase)
DSBs repair
complementary nucleotides
The two main pathways of non-homologous end joining
(NHEJ) in plant cells
DNA lost but some DNA can be inserted (filler DNA)
deletion inversion reciprocal translocation multiple translocations
NHEJ-mediated rearrangements
NHEJ in plant somatic cells
• NHEJ seems to be the main mode of DSB repair in higher eukaryotes
• NHEJ might lead, in some cases, to genomic changes (deletions, insertions or various kinds of
genomic rearrangements)
• genomic alterations in meristematic cells can be transferred to the offspring
• alternative NHEJ can mediate genome size loss
Arabidopsis vs. tobacco (genome size larger in tobacco)
- tobacco: almost every second deletion event is accompanied by the insertion of filler sequence
- Arabidopsis: no insertions
- overall length of the deletions is about one-third shorter in tobacco than in Arabidopsis
>>> inverse correlation between genome size and the medium length of deletions
>>> ??? species-specific differences in DSB repair pathways can contribute to the
evolution of eukaryotic genome size ???
1C = 4.5 Gb
1C = 157 Mb
- A. thaliana (157 Mb) has lost 6 more introns than Arabidopsis lyrata (210 Mb) since the
divergence of the two species but gained very few introns
Homologous recombination
The two main pathways of homologous recombination
in somatic plant cells
single-strand annealing
synthesis-dependent
strand annealing
all sequence
information between
the repeats is lost in
the rejoined molecule
a restored dsDNA
molecule
without changing
sequence of the
donor locus
DNA loss
(evidence: solo
long terminal
repeats of LTR
retrotransposons) genome size decrease
(template needed)
relevant term:
gene conversion
1C = 157 Mb 1C = 5 500 Mb
Vu et al. (2017) New Phytol 214
A. thaliana: deletion size and frequency considerably larger (than in barley)
HR via SSA (and NHEJ) can contribute to evolutionary genome shrinking.
~70% of retrotransposon sequences in the A. thaliana genome are no longer autonomous
= probably as the consequence of SSA (truncated retroelements and solo LTRs)
= these elements cannot contribute to genome expansion
HR: single-strand annealing (SSA)
The role in genome size increase/decrease.
DSBs experimentally
induced
>>> DSB repair
Templates for homologous recombination (HR)
via SDSA (needs a template)
sister chromatid
(S and G2 phase)
homologous
chromosome
(allelic sequence)
ectopic sequences
(heterologous chromosomes)
Intra-
chromosomal
homology
repair using a sister
chromatid or homologue
repair using a sister
chromatid or homologue
AND
ectopic recombination
(generating chromosome
abberations)
ectopic DSB repair competes
with repair using the sister
chromatid or homologue
DSB repair and misrepair can lead to
chromosome rearrangements
Argueso et al. (2008) PNAS 105
• in somatic plant cells, DSBs are mainly repaired by NHEJ
• the NHEJ repair can be associated with deletions, but also insertions due to copying
genomic sequences from elsewhere into the break; chr. rearrangements can be generated
• inverse correlation of deletion size to genome size (Arabidopsis vs. tobacco) >>> NHEJ
might contribute significantly to evolution of genome size
• DSB repair by HR might also influence genome organization
• intra-chromosomal HR is frequent (sequences in close proximity to the break), interchromosomal
HR (allelic and ectopic) is hardly used for repair
• a ‘single-strand annealing’ (SSA) mechanism of HR that
leads to sequence deletions between direct repeats is
particularly efficient >> might explain the accumulation of
single LTRs of retroelements in some plant genomes
(?evolution of genome size?)
NHEJ vs. HR (short summary)
Vu et al. (2017) New Phytol 214
HR: synthesis-dependent strand annealing (SDSA)
The role in genome size increase/decrease.
deletion(1019bp),insertion(170bp)
Insertion size and frequency
larger in barley
(than in A. thaliana)
1C = 5 500 Mb