Low copy repeats (LCRs) are typically
chromosome-locus-specific sequences sharing greater than 95%
sequence identity over a range of 10 to 400 kilobases. These
repeats make up at least 5% of the human genome (1). Meiotic
recombination between these repeats results in
translocation, deletion, duplication and / or inversion of
the chromosomal regions flanked by the repeats. Several
human diseases are the direct result of this process
(reviewed in (2)) including Smith-Magenis syndrome (3),
Charcot-Marie-Tooth disease (4), DiGeorge / velocardiofacial
syndrome (5) and the inversion form of severe hemophilia A
(6, 7). In all disease cases the recombination event between
repeats necessarily involved crossing-over. Since homologous
recombination between repeated sequences is a major form of
mitotic double-strand break repair (8), crossing-over
between LCRs mitotically would have the potential for
significant genome destabilization.
The evidence that crossing-over can happen as the result
of mitotic repair is much less direct than for meiotic
repair, although a case report of somatic mosaicism in a
female hemophilia inversion carrier is highly suggestive
(9). In order to directly assay mitotic crossing-over as a
double-strand break repair mechanism, I created the XO-GFP
reporter gene (figure 1).
The reporter consists of two truncated coding sequences for
the green fluorescent protein (GFP), one truncated at the 3'
and the other at the 5' end (SceGFPD5'),
inversely oriented with respect to each other (figure
1a). Neither sequence is capable of producing a
functional protein. Additionally, the
sequence contains an 18 basepair recognition site for the
homing endonuclease I-SceI (10). An intervening
puromycin resistance gene allows selection for stable
chromosomal integration. Expression of the endonuclease
generates a double-strand break in SceGFPD5',
stimulating homologous recombination with the
sequence. If the recombination occurs without crossing-over,
the I-SceI recognition site is gene converted to GFP
sequences, using GFPD3'
as a template, changing SceGFPD5'
a 5' truncated, non-functional GFP gene without an
I-SceI recognition site (figure 1b). In
contrast, if recombination occurs with crossing-over, the
I-SceI recognition site in SceGFPD5'
is again gene converted to GFP sequences, but the cross-over
event also inverts all intervening sequences between the
repeats. This inversion brings the front half of
register with the back half of the SceGFPD5'
yielding a functional GFP gene (figure 1c).
Additionally a doubly-truncated internal GFP fragment
(iGFP) is created and the orientation of the drug
resistance marker is inverted. Cells in which this
crossing-over occurs then constitutively express GFP and are
easily detectable by flow cytometry.
Figure 1: XO-GFP crossing over
drug resistance gene
I have integrated the XO-GFP reporter stably into the
genome of mouse embryonic stem cells and detected GFP+ cells
in response to I-SceI transfection (11). Southern
blot analysis of these cells directly demonstrated inversion
of the drug resistance marker, confirming mitotic
crossing-over in response to a double-strand break.
Preliminary evidence indicates that the bias between
non-cross-over repair and cross-over repair is on the order
of 30 : 1 in favor of non-cross-overs. I will explore the
genetic factors responsible for this bias by co-transfecting
expression vectors of various DNA repair proteins such as
the Bloom's syndrome protein and by reducing expression of
these proteins by co-transfection of small inhibitory
double-stranded RNA sequences (12).
Of all classes of homologous recombination mediated
double-strand break repair, those involving crossing-over
have the most dire consequences for genome stability.
Crossing-over between either direct or inverted repeats on
different chromosomes results in reciprocal translocations.
Crossing-over between direct repeats on the same molecule
results in deletion of all intervening sequences as an
episomal circle, while crossing-over between inverted
repeats on the same molecule inverts all intervening
sequences as described above. Crossing over between direct
repeats on a sister chromatid results in deletions and / or
expansions. Finally, crossing over between inverted repeats
on sister chromatids leads to the obligate formation of both
an acentric and a dicentric chromatid. This latter
possibility is especially serious as it can result in
prolonged breakage-fusion-bridge cycles (13) which have been
identified as an important pathway for the generation of
tumor genetic heterogeneity (14). The XO-GFP reporter has
the potential to rapidly identify cells in which this
process is actively occurring, enabling study of possibly
very early events in the cellular progression to cancer.
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