Ďouble-strand breaks (ĎSBs), their repair and m\srepa\r
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DSBs in meiosis
♦♦♦ necessary for homologous recombination (cross-overs)
♦♦♦ induced by the Spoil topoisomerase
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How are generated double-strand breaks
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!
•  during meiotic recombination
•  mechanical pulling (e.g. in dicentric chromosomes)
Transposon excision	
Ionizing radiation	Endonuclease
^s.	y^
a     b     c     d     e     f	g     h     i    j     k     1
DSB           ~	
• 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
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ĎSBs in mitosis and their repair
(in somatic plant cells) DSBs repair
non-homologous end joining (NHEJ)
•  also known as "illegitimate recombination"
•  the broken ends are re-ligated directly
•  often an error-prone process
•  throughout the cell cycle (mainly G-, phase)
•  main mode of DSB repair in higher eukaryotes (somatic plant cells)
homologous recombination (HR)
•  uses sister chromatids as a template to rejoin DSBs
•  error-free repair
•  in late S-G2 phase
•  minor pathway
Models describing the pathways of ĎSB repair
in somatic plant cells
synthesis-dependent strand annealing (SDSA)
mechanism
single-strand annealing (SSA)
mechanism
for details see Puchta (2005) JEB 56: 1-14
Different pathways of HR
Homologous information for repair can be copied from:
elsewhere in the genome ('ectopic') the homologue as in meiosis ('allelic')
inter-chromosomal
the same chromatid (chromosome) the sister chromatid (after replication)
intra-chromosomal
Intra eft rout osomal
Allelic
Ectopic
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
•     different classes of NHEJ repair events were characterized:
a)   the repair of the break was accompanied by incorporation of filler DNAs,
b)  the break ends were rejoined with or without deletions
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 ???
NHEJ vs. HR
•  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
•  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
•  inter-chromosomal HR (allelic and ectopic) is hardly used for repair, intra-chromosomal HR is frequent (sequences in close proximity to the break)
Genomic locus
•  'single-strand annealing' mechanism of HR that leads to                     |     Integration of Retrotransposon
sequence deletions between direct repeats is particularly       ^^                    nno--.
efficient >> might explain the accumulation of single LTRs
r             ■                 i                          ■          •                                        i             ■                                                                                                                                                                                W               Induction of a DSB by an active elemf
of retroelements in some plant genomes
---------1    LTR    |--------------------      --------------------1    LTR    \---------
w              Repair by homologous recombination
I   LTR   r---------               Production of a single LTR
ĎSB repair and misrepair can lead to chromosome
rearrangements...
(next lecture)