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Genome Research
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INTRODUCTION |
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 Introduction
 References
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Recombination between homologous DNA sequences occurs in all organisms, and the resultant exchange of information is critical for the survival of species. Recombination is an essential cellular process catalyzed by proteins explicitly expressed for this purpose. It provides an effective means of generating genetic diversity that is important for evolution. The proteins involved in recombination allow cells to retrieve sequences lost when DNA is damaged by radiation or chemicals, by replacing the damaged section with an undamaged strand from a homologous chromosome. The process of homologous recombination has also been used to study gene function by way of gene knockouts. However, recombination and factors involved in recombination may also be a source of harmful mutations and disease.
Specific DNA sequences are known to mediate or enhance the rate of
recombination in the genomes of many organisms. Attempts to identify
and decipher recombination hot spots have focused on determining the
influence of various DNA sequences on the rate and type of DNA
rearrangements. The human nuclear genome contains a large number of
highly repeated DNA sequence families (Jelinek and Schmid 1982
; Hardman
1986; Vogt 1990), broadly classified as tandemly repeated DNA or
interspersed repetitive DNA. Because of their role in mediating
disease-causing recombination errors, a brief overview of the various
repeats is presented.
Tandemly repeated DNA is characterized by blocks or arrays of tandemly
repeated DNA sequences. They are subclassified based on the size of the
blocks or arrays of tandem repeats into satellite (0.1 to >2 Mb),
minisatellite (0.1-2.0 kb), and microsatellite (~150 bp) DNA.
Satellite DNA is further sub-classified based on the size of the repeat
unit within these blocks into types 1 (25-48 bp), 2 and 3 (5 bp),
(alphoid DNA; 171 bp), and 
(Sau3A family; 68 bp).
Minisatellite DNA consists of telomeric DNA that has a 6-bp repeat
unit, and the polymorphic VNTRs (variable
number tandem repeats) or
hypervariable minisatellite DNA, where the size of the repeat unit
ranges from 9 to 24 bp. VNTRs have been shown to be hot spots for
homologous recombination in human cells (Wahls et al. 1990).
Microsatellite DNA consists of small arrays of tandem repeats (usually
1-4 bp units) that are interspersed throughout the genome, in blocks
consisting of <150 bp.
In contrast to tandemly repeated DNA, interspersed repetitive DNA
consists of repeat units dispersed throughout the genome. Based on the
repeat unit length, two major classes are recognized: short
interspersed nuclear elements [(SINES) e.g., the Alu repeat family] and long interspersed nuclear elements [(LINES) e.g., the
LINE-1 or L1 element (Singer 1982)]. The Alu repeat
containing a 280-bp repeat unit occurs approximately once every 4 kb in
the human genome. Mispairing between such repeats has been shown to be
a frequent cause of deletions and duplications. Breakpoints of
disease-causing deletions have been clustered within Alu
sequences in genes for the low density lipoprotein receptor
(LDLR) (Lehrman et al. 1985, 1987), and the complement
component 1 inhibitor (C1I) (Stoppa-Lyonnet et al. 1991). Such
observations have suggested a general role for Alu sequences
in promoting recombination and recombination-like events. Alu
repeats or other dispersed repetitive elements are also thought to have
played a role in the evolution of clustered multigene families by
mediating unequal crossover events that lead to gene duplications. The
average length of the L1 element repeat unit is 1.4 kb. In addition,
there are smaller repeat sequence families belonging to this class,
including the THE-1 (transposable human
elements), MER (medium
reiteration), HERV (human
endogenous retroviruses), and
RTLV (retrovirus-like elements) repeats. Members of many of the
interspersed repeat families are considered retrotransposable elements,
that is, unstable DNA elements that can migrate to different regions of
the genome by transposition via an RNA intermediate. These endogenous
retroposons are thought to have played an important role in shaping the
genomes of vertebrates by intracellular transposition events and by
generating hot spots of recombination (Leib-Mosch and Seifarth 1995).
The various families of repetitive elements are highly relevant to a number of different mechanisms of mutagenesis in human genes. As discussed in the following sections, recombination between such sequences can lead to rearrangements, including deletions, duplications, inversions, and fusion genes.
Deletions and Duplications Caused by Homologous Recombination
Hot spots of homologous recombination between misaligned
repetitive elements have been observed at the duplication and deletion breakpoints in a number of human genetic diseases. In Escherichia coli, direct repeats in close proximity can mediate efficient RecA-independent intramolecular recombination. A replicational model
for DNA recombination between direct repeats was suggested by Bi and
Liu (1996). They proposed that misalignment of repeats at the
replication fork creates a recombinogenic intermediate that can be
differentially processed and that the proposed sister-strand recombination mediated by direct repeats might be a general mechanism of deletion or duplication of repeated sequences in prokaryotic and
eukaryotic genomes.
Large-scale deletions and duplications may be generated by the pairing
of nonallelic interspersed or tandem repeats, followed by breakage and
rejoining of chromatid fragments. Repeat DNA sequences may predispose
to abnormal chromosome pairing and unequal crossing-over, with
deletions and duplications representing the reciprocal products of such
events. Large deletions within duplicated regions may occur either
interchromosomally because of misalignment of non-sister chromatids
during meiosis (Pentao et al. 1992; Chance et al. 1994) or
intrachromosomally because of either sister chromatid exchange during mitosis or DNA slippage during replication (Krawczak and Cooper
1991).
Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy
with liability to pressure palsies (HNPP) are two autosomal dominant
peripheral neuropathies resulting from DNA rearrangements that are
reciprocal products of an unequal crossing-over event between
misaligned flanking CMT1A-REP (repeat) elements on
chromosome 17p (Chance et al. 1994; Patel and Lupski 1994) (Fig.
1). The proximal and distal CMT1A-REP elements are
~30 kb in length, extremely AT rich (64% A + T), and display
98% sequence identity (Reiter et al. 1996). Given the high degree of
homology between the proximal and distal repeats, a recombination event
can potentially occur anywhere within the 30-kb region. However,
through the detection of novel junction fragments from the recombinant
CMT1A-REP elements in both CMT1A and HNPP patients, a 1.7-kb
recombination hot spot within the ~30-kb CMT1A-REP was identified
in 75% of CMT1A duplication patients and 84% of HNPP deletion
patients examined (Reiter et al. 1996). Sequence analysis showed there
was no particular increase in the degree of sequence identity over this
1.7-kb region, and, interestingly, a mariner transposon-like element
(MITE) was identified in the vicinity of the hot spot. Three exons were
identified in the repeat, one of which showed homology at the amino
acid level to the conserved region of several insect transposases.
Kiyosawa and Chance (1996) further investigated the MITE and found that it is probably nonfunctional, as several stop codons were found in its open reading frame. However, it is possible that this
nonfunctional mariner sequence may be a target for a functional form of
the transposase protein transcribed from a gene located elsewhere.
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Northern blot analysis with the distal CMT1A-REP, which encompasses
the putative transposase, identified a low-abundance transcript expressed in testes but not in ovaries (Reiter et al. 1996). This finding was interesting, because until recently it was considered that
the unequal crossing-over resulting in CMT1A and HNPP occurred solely
during male meiosis. Investigation of the origin of de novo
duplications revealed a paternal origin for the mutation in 13 sporadic
cases (Raeymaekers et al. 1991; Palau et al. 1993; Wise et al. 1993;
Hertz et al. 1994). Palau et al. (1993) proposed that male-specific
factors may operate during spermatogenesis to help form the duplication
and/or stabilize the duplicated chromosome. However, Blair et al.
(1996) on analysis of de novo duplications in eight families, found
seven to be paternal and one, the first reported, to be maternal in
origin, indicating that this was not a phenomenon associated solely
with male meiosis. Two loci within the duplication region (D17S122 and
D17S61) that were physically mapped within 1.0 Mb of each other were
found to span an average genetic distance of 4 cM in males and 14 cM in
females (Pentao et al. 1992). This large discrepancy between the
genetic and physical distances of these duplicated markers suggests
that this region appears to be extremely prone to meiotic
recombination. Because recombination fractions for the duplicated
region in CMT1A are larger in females than in males, oogenesis may
afford greater protection from misalignment during synapsis, and/or
there may be lower activity of those factors or mechanisms that lead to unequal crossing-over at the CMT1A locus (Blair et al. 1996).
Allelic variants of the human cytochrome P450 CYP2D6 gene have
also been shown to arise from homologous unequal crossing-over involving a 2.8-kb direct repeat (Steen et al. 1995). The cytochrome P450 enzyme debrisoquine 4-hydroxylase metabolizes many different classes of commonly used pharmacological drugs. Among Caucasians, 5%-10% are classified as poor metabolizers (PM) because of autosomal recessive inheritance of two CYP2D6 deletion alleles. In these individuals, administration of average therapeutic doses results in
toxic plasma concentrations and adverse drug reactions. In contrast, up
to 5% demonstrate ultrarapid metabolism (UM) caused by an inherited
duplication of functional CYP2D6 genes, thus requiring higher
doses of drugs to maintain desired plasma levels. A 2.8-kb repeated
sequence (CYP-REP), containing an Alu element and a tandem 10-bp direct repeat flanks the active CYP2D6 gene in the
wild-type allele. It is thought that the CYP2D6 deletion and
duplication alleles are reciprocal products, generated by homologous
recombination between nonallelic CYP-REP elements.
Large-scale rearrangements are detected in 80% of familial and 65% of
sporadic cases of juvenile nephronophthisis (NPH), representing the
most frequent inherited cause of chronic renal failure in children
(Konrad et al. 1996). Large homozygous deletions (250 kb) involving a
100-kb inverted duplication were found at the NPH1 locus on
chromosome 2q13. Further characterization of this region revealed the
presence of low-copy repeats.
X-linked icthyosis is a disease characterized by an extremely high
frequency of submicroscopic deletions involving the steroid sulfatase
(STS) gene (Bonifas et al. 1987; Conary et al. 1987; Ballabio
et al. 1989a,b). Eighty-four percent of patients with steroid sulfatase
deficiency possess deletions of their STS genes (10 exons
spanning 146 kb of genomic DNA). The deletions have breakpoints
clustered around or within a number of low-copy repetitive sequences
called S232 type repeats, flanking the STS gene (Yen et al.
1990). These repeats resemble VNTRs (Li et al. 1992) and indicate that
the high frequency of deletions at this locus may be attributable to
recombination involving these repetitive sequences.
Deletion of both growth hormone (GH1) genes causes the autosomal
recessive disease familial growth hormone deficiency type 1A. In 9 of
10 patients with GHI gene deletions, the crossovers occur
within two 99% homologous, 594-bp regions flanking the GH1 gene (Vnencak-Jones et al. 1988; Vnencak-Jones and Phillips 1990). The
presence of these highly homologous DNA sequences flanking the
GH1 gene predisposes recurrent unequal recombination events.
Fascioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant
neuromuscular disease that has been linked to deletions within a tandem
array of 3.2-kb repeats (D4Z4) adjacent to the telomere on chromosome
4q35 (van Deutekom et al. 1993). The majority of sporadic cases of FSHD
are associated with de novo deletions at the D4Z4 locus. The D4Z4
sequence contains two homeoboxes and two previously described
repetitive sequences, LSau and a GC-rich low-copy repeat designated
hhspm3 (Hewitt et al. 1994; Lee et al. 1995). D4Z4 is a member of a
dispersed family of homeobox-containing repeats, subsets of which are
clustered on the short arms of acrocentric chromosomes (Lyle et al.
1995). Analysis of the evolutionary distribution and structural
organization of D4Z4 showed that tandem arrays closely related to D4Z4
were conserved at loci syntenic to human 4q35-qter in apes and lower
primates, suggesting a functionally important role for these sequences
(Clark et al. 1996; Winokur et al. 1996). However, no single gene has
yet been associated with FSHD, and the etiopathogenesis of FSHD is not
yet known. It has been proposed that the deletions mediated by these
repeat sequences may invoke a position effect on a nearby gene.
The most common molecular defect underlying thalassemia involves
a deletion of one or both of the duplicated -globin genes (Dozy et
al. 1979; Higgs et al. 1979). The mechanism by which the thalassemia deletions occur is related to the underlying molecular
structure of the -globin complex (Embury et al. 1980; Lauer et al.
1980). Each gene is located within a region of homology, ~4 kb
long (thought to have resulted from an ancient duplication event), and
interrupted by short nonhomologous regions. During evolution, these
homologous segments were divided by insertions and deletions to give
rise to three homologous subsegments referred to as X, Y, and Z. The
duplicated Z boxes are 3.7 kb apart, and the duplicated X boxes are 4.2 kb apart. Misalignment and reciprocal crossover between these segments
at meiosis give rise to chromosomes with either single (Embury et al.
1980) or triplicated -globin genes (Goossens et al. 1980).
Recombination between the homologous X or Z boxes (4.2 or 3.7 kb apart)
gives rise to chromosomes with a 4.2- or 3.7-kb deletion with one
-globin gene and the reciprocal chromosome with three -globin
genes. Although the breakpoints of these deletions vary within these
regions of homology, the mechanism of homologous unequal recombination
appears to be the same. These recombination events have been reported
in several ethnic groups (Higgs et al. 1989).
Inversions Caused by Homologous Recombination
Occasionally, highly similar inverted repeats may be located
within or close to a gene. The high degree of sequence similarity between inverted repeats may predispose to pairing of the repeats by a
mechanism that involves a chromatid bending back on itself. Subsequent
chromatid breakage at the mispaired repeats and rejoining can result in
an inversion, thus disrupting a functional gene. An example of
pathogenic inversions caused by such a mechanism is seen in hemophilia
A. Forty-five percent of patients with severe hemophilia A have an
inversion and disruption of the coagulation factor VIII gene mediated
by an unequal crossing-over event (Lakich et al. 1993). Within intron
22 of the factor VIII gene lies another gene, F8A, which has
two additional copies situated 500 kb upstream (telomeric) of the
factor VIII gene on Xq but in the opposite orientation. Evidence
suggests that the tip of Xq flips back on itself, aligning the
homologous intragenic and extragenic F8A sequences in meiosis.
Unequal crossing-over results in recombination between one of the
upstream genes and the intragenic F8A gene, generating an
inversion of the intervening factor VIII gene sequence (Fig.
2). Such inversions result in disruption of the
factor VIII gene, separating exons 1-22 from exons 23-26 by 200-500
kb. As the single X chromosome in males remains largely unpaired in
meiosis, the tip of the chromosome is free to flip up on itself
a
phenomenon that does not occur in female meiosis, where both X
chromosomes pair along their length like autosomes, apparently
restricting the movement of Xqter. Rossiter et al. (1994) hypothesized
that pairing of Xq with its homolog inhibits the inversion process and,
therefore, the event should originate predominantly in male germ cells.
They examined 20 informative cases in which the inversion originated in
a maternal grandparent, and by analysis of DNA polymorphisms determined
that it occurred exclusively in the male germ line. In addition, they
showed that all but one of the 50 mothers of sporadic cases resulting
from an inversion were carriers. These data supported their hypothesis
that factor VIII gene inversions leading to severe hemophilia A
occurred exclusively in male germ cells.
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Another example of an inversion caused by homologous recombination is
that involving the iduronate-2-sulfatase (IDS) gene. Deficiency of the enzyme IDS results in Hunter syndrome, an X-linked recessive disorder also known as mucopolysaccharidosis II. A linked IDS pseudogene-like sequence (IDS2) was detected
~90 kb downstream of the IDS gene (Bondeson et al. 1995;
Rathmann et al. 1995). This region is involved in a homologous
recombination event with the IDS gene in 20% of patients with
Hunter syndrome (Bondeson et al. 1995). The intrachromosomal
recombination between homologous sequences present in the IDS
gene and the IDS2 locus results in disruption of the
IDS gene in intron 7 with an inversion of the intervening DNA.
No detectable deletions or insertions are observed as a result of this
inversion event.
Unequal exchange between large homologous repeats leading to genome
variation in the absence of disease-causing mutations was reported
recently in the Xq28 region, which harbors the neighboring genes for
filamin (FLN1) and emerin (Small et al. 1997). Flanking the 48-kb
FLN1/emerin region are two large inverted repeats, 11.3 kb in
length, that exhibit 99% sequence identity (Chen et al. 1996). During
the characterization of a rare mutation involving a complete deletion
of the emerin gene and a partial duplication of the FLN1
gene in a patient with Emery-Dreifuss muscular dystrophy (EMD), a
common rearrangement resulting from mispairing of these large repeats
was identified (Small et al. 1997). Recombination betweeen these
inverted repeats leads to a complete inversion of the 48-kb
FLN1/emerin region without any sequence alteration in either
gene. This inversion was detected in the heterozygous state in 33% of
normal females and helps explain the discrepancies between the genetic
and physical map distances in this region of the X chromosome (Small
et al. 1997). These investigators speculate that regional variation in
the genetic map of humans may reflect the influence of similar, as yet
uncharacterized, inversions mediated by large inverted repeats.
Fusion Genes Generated by Recombination
Besides deletions, duplications, inversions, or other rearrangements, recombination between homologous sequences also results in the creation of fusion genes. Sequence homology between members of gene families or pseudogenes results in such rearrangements.
Several hemoglobin variants containing fused or hybrid globin chains
have been described. Hemoglobin Lepore (Hb Lepore) was the first to be
reported and is an example of gene fusion resulting from the deletional
removal of intervening DNA sequence (Gerald and Diamond 1958). This
hemoglobin, which is synthesized in reduced amounts, is an abnormal
molecule with the amino-terminal 50-80 amino acids of 
-globin and
the carboxy-terminal 60-90 residues of -globin. The reduction in
globin synthesis is caused by the reduced synthesis of mRNA encoding
the fusion product to a level intermediate between those of the
-globin and -globin genes. Thus, Hb Lepore contains normal
chains, and its non- chain is a - fusion chain.
Three different varieties of Hb Lepore have been described (Hb Lepore
Hollandia, Hb Lepore Baltimore, and Hb Lepore Boston), in which the
transition from to occurs at different positions (Baglioni
1962; Barnabus and Muller 1962; Ostertag and Smith 1969). Misalignment
of chromosome pairing during meiosis results in pairing of the
-chain gene with the -chain gene instead of its homologous
partner. The fusion chains arise by nonhomologous crossing-over between
part of the locus on one chromosome and part of the locus
on the complementary chromosome (Fig. 3). This
mechanism gives rise to two abnormal chromosomes: the Lepore
chromosome, which has no normal or loci but has a
- fusion gene, and the anti-Lepore chromosome, with a
- fusion gene (Hb anti-Lepore) and normal or loci. A variety of anti-Lepore hemoglobins have been described,
including Hb Miyada, Hb P-Congo, Hb Lincoln Park, and Hb Nilotic
(Lehmann and Charlesworth 1970; Ohta et al. 1970; Badr et al. 1973;
Honig et al. 1978). Hemoglobin Kenya is analogous to Hb Lepore, except
that the abnormal hybrid chain is a 
- fusion chain (Huisman
et al. 1972). The anti-Kenya chromosome contains intact
A
-, -, and -globin loci. The Lepore variants
result in the clinical phenotype of or thalassemia.
The anti-Lepore variants and Hb Kenya are not associated with any
significant hematolgical changes.
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Unequal crossover or unequal sister chromatid exchange between a
functional gene and a related pseudogene can result in deletion of the
functional gene or formation of fusion genes containing a segment
derived from the pseudogene. Alternatively, the pseudogene can act as a
donor sequence in gene conversion events and introduce deleterious
mutations into the functional gene. In steroid 21-hydroxylase deficiency, virtually all pathological mutations arise as a result of
sequence exchanges between the functional 21-hydroxylase gene (CYP21B), and a very closely related pseudogene
(CYP21A) (Fig. 4). The two genes occur on
tandem 30-kb repeats that show ~97% sequence identity. The repeated
segments also contain other duplicated genesnamely, the complement
C4A and C4B genes. About 25% of pathological mutations at the 21-hydroxylase locus are large deletions resulting in
removal of 30 kb of DNA resulting from unequal crossover or unequal
sister chromatid exchange (Sinnott et al. 1990). The
remaining 75% of mutations are point mutations where small-scale gene
conversions of the CYP21B gene are thought to occur. A small
segment of the CYP21A gene containing deleterious mutations is
inserted into the CYP21B gene replacing a short segment of the
original sequence. Analysis of one such mutation that arose de novo
showed that the conversion tract was 390 bp in length (Collier et al.
1993).
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Glucocorticoid-suppressible hyperaldosteronism (GSH) is an
autosomal dominant form of hypertension caused by oversecretion of aldosterone. Gene fusion between the cytochrome P450 genes CYP11B1 and CYP11B2 has been shown to cause GSH
(Lifton et al. 1992). CYP11B1 and CYP11B2 are two
highly homologous genes closely linked on chromosome 8q22 that
encode steroid biosynthetic enzymes catalyzing cortisol production
and aldosterone production under the control of corticotrophin
(ACTH) and angiotensin, respectively. Production of a hybrid gene
due to unequal meiotic crossing-over between CYP11B1 and
CYP11B2 results in a new gene that contains the promoter of
CYP11B1 and the coding region of CYP11B2. This fusion
gene produces an enzyme that catalyzes the formation of aldosterone but
is sensitive to ACTH. As a result, normal levels of ACTH, which
normally maintain low levels of cortisol, lead to excessive production
of aldosterone with consequent hypertension and hypokalemia.
The genes involved in visual dichromacy or red/green color blindness
are those encoding the red and green visual pigments. These are highly
homologous (98% sequence identity in exons, introns, and 3
flanking regions) and are linked in tandem on chromosome Xq28. The
red/green gene arrays are composed of a single red pigment gene and one
or more green pigment genes located downstream (3) of the red
pigment gene. Gene expression studies indicate that when several green
pigment genes are present, only the most proximal is expressed in the
retina. Deficiencies in red/green color vision arise from unequal
recombination of these normal X-linked genes (Nathans et al. 1986).
Such events lead to deletions of the green pigment genes or the
formation of full-length hybrid genes consisting of portions of both
red and green pigment genes. (Fig. 5). With a few
exceptions, deletion of the green pigment genes leaves a single red
pigment gene and is associated with deuteranopia (G
R+). Affected
individuals are dichromatic, as they completely lack green cones.
Individuals with 5-green-red-3 fusion genes have deuteranomaly (GR+), a milder type of color vision defect, with a slightly red-shifted absorption maximum for the green pigment. Individuals with 5-red-green-3 fusion genes are associated
with protan abnormalities (R or R). Those who have a hybrid
gene only are always protanopic (R) and are therefore, dichromats. Those who have normal green genes in addition are either protanopic (R) or protanomalous (R) and have a milder defect with
slightly green-shifted absorption maximum of the red pigment.
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Hybrid SMN (survival motor
neuron) genes have been identified in patients with
autosomal recessive spinal muscular atrophy (SMA) (Hahnen et al. 1996).
The SMN gene is a strong candidate for SMA and is
present as two highly homologous copies, telomeric SMN and
centromeric SMN (TELSMN and CENSMN,
respectively) within the SMA region. A large percentage of SMA patients
(90%-98%) carry homozygous deletions in TELSMN, affecting
either exon 7 or both exons 7 and 8. Hybrid SMN genes were
identified in 42 patients with SMA, who showed homozygous deletions of
exon 7 but not of exon 8 of the TELSMN copy (Fig.
6). Besides the SMN gene, which is present
in at least two copies per chromosome, all other genes and markers
present in the SMA region are also present in several copies, and these
regions are prone to unequal crossing-over resulting in deletions,
duplications, and gene conversion events. A putative recombination hot
spot represented by recombination-stimulating sequence elements (TGGGG
and TGAGGT) was identified in exon 8 of the SMN gene. These
sequences are homologous to polymerase arrest sites (Weaver and
DePamphilis 1982) and the deletion hot spot consensus sequences in the
immunoglobulin switch region (Gritzmacher 1989) and the -globin
gene cluster (Nicholls et al. 1987).
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Sex-Specific Meiotic Recombination Hot Spots
Meiotic recombination in the genome does not occur randomly but
tends to be concentrated in hot spots, regions with relatively high
recombination rates separated by stretches of diminished recombination.
There is a striking sexual dimorphism in cytogenetic chromosome length,
with human female pachytene chromosomes being 50% longer than those in
males (Wallace and Hulten 1985). It seems likely that the higher rate
of recombination seen in human females versus males is a reflection of
the more condensed state of male chromosomes. Chromatin conformation
may influence meiotic recombination in humans. This is suggested by the
observation that during spermatogenesis, the X and Y chromosomes are
transcriptionally inactive and experience restriction of recombination,
whereas the X chromosomes, which are transcriptionally active during
oogenesis, participate in unrestricted recombination (Handel and Hunt
1992). Recombination is probably prevented during meiosis in males by
specific heterochromatization of the sex chromosomes (McKee and Handel
1993). There is virtual absence of crossing-over in constitutive
heterochromatin, which is highly condensed and devoid of transcribed
genes; this may be further evidence for an influence of chromatin
structure on recombination.
In an effort to determine the pattern of chromatin condensation and
recombination at meiosis in an imprinted region, Robinson and Lalande
(1995) carried out fine-scale genetic mapping in the 4-Mb Angelman
Syndrome/Prader-Willi Syndrome (AS/PWS) region. Their results
indicated that the male/female recombination ratio varies significantly
over short regions. A male recombination hot spot was localized to a
region that is adjacent to, but outside, the putative AS/PWS-imprinted
regions. In females, a region of relatively high recombination was
observed that spans a domain of paternal allele-specific transcription,
implicated in the Prader-Willi syndrome.
The preponderance of CMT1A duplication and factor VIII gene inversion in the male germ line has been discussed in the previous sections.
Proteins Involved in Homologous Recombination and their Role in Cancer
Human cancer is commonly associated with rearrangements of DNA that result in deletions of tumor suppressors and altered expression or amplification of proto-oncogenes. Multiple mutations are present in most human tumors, and these genetic modifications appear to be necessary to produce and select premalignant, malignant, and metastatic cells. Destabilization of genes in cancer could be explained by pre-existing hot spots or by creation of de novo hot spots by the rearrangements themselves. In this section proteins involved in homologous recombination, and "recombination hotspot-binding" proteins and their role in carcinogenesis are discussed.
Homologous recombination is a fundamental biological process, the
biochemical understanding of which is most advanced in E. coli. The proteins involved in promoting genetic exchange include RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, SSB proteins, DNA
polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA
helicases. Collectively, they define biochemical events essential for
efficient recombination (Kowalczykowski et al. 1994). In addition to
these proteins, a cis-acting recombination hot spot sequence
chi (5-GCTGGTGG-3), is also known to play an important role
(Smith 1989). The central events in homologous recombination are the
pairing of homologous molecules and the initiation of strand exchange.
The E. coli RecA protein is the prototype of proteins that can
catalyze these reactions by promoting interaction between homologous
DNA molecules (Kowalczykowski and Eggleston 1994). Evidence is
accumulating that all organisms have a protein that shares significant
functional homology to this bacterial protein, suggesting that the
fundamental mechanisms of recombination are conserved in all species
(Heyer 1994). Homologs of RecA are known in yeast (Saccharomyces
cerevisiae; ScRad51) (Aboussekhra et al. 1992; Basile et al. 1992;
Shinohara et al. 1992); mouse (MmRad51) (Morita et al. 1993); and human
(HsRad51) (Shinohara et al. 1993; Yoshimura et al. 1993).
Genes involved in recombination in the yeast S. cerevisiae
include those of the RAD52 epistasis group
(RAD50-RAD57), involved in meiotic and mitotic recombination
(Shinohara et al. 1993; Bishop 1994) and double strand break (DSB)
repair (Game 1993). Accurate repair of these genotoxic lesions is
essential for the prevention of chromosomal fragmentation,
translocations, and deletions, which can lead to carcinogenesis through
activation of oncogenes and/or inactivation of tumor suppressor genes.
Key genes in the RAD52 DNA repair pathway include
RAD54 and RAD51. The primary sequence of many
RAD52 group genes is conserved from yeast to human. Analysis of the phenotype of mouse MmRad54 / cells by Essers et
al. (1997) demonstrated that homologous recombination in these cells
was reduced compared to wild-type cells, implying that homologous recombination contributes to the repair of DSBs in mammalian cells.
ScRad51 mutants also show defects in genetic recombination and repair
of damaged DNA (Game 1993). A role of ScRad51 in meiosis is shown by
its presence in meiotic nuclei along with ScDMC1, another homolog of
RecA specific to meiosis (Bishop et al. 1992). Evidence for the role of
HsRad51 protein in meiosis comes from the findings that antibody to
HsRad51 stained murine synaptonemal complexes early in meiosis, and
stained numerous foci in nuclei of human cells exposed to DNA-damaging
agents (Haaf et al. 1995; Plug et al. 1996). The role of the
MmRad51 gene in mitosis and meiosis was shown by the finding
of high levels of transcription of homologs of the Rad51 gene
in lymphoid and reproductive organs (Shinohara et al. 1993). A
homozygous MmRad51 mutation (MmRad51 /) was
lethal early in murine embryogenesis (Tsuzuki et al. 1996). Lim and
Hasty (1996) showed that the embryonic lethal phenotype of
MmRad51 / is suppressed by a mutation in the p53
oncogene. Sturzbecher et al. (1996) reported that p53 interacts with
HsRad51 and RecA. These observations suggest that functional wild-type p53 may select directly the appropriate pathway for DNA repair and
control the extent and timing of the production of genetic variation
via homologous recombination. Therefore, rearrangements may occur as a
direct consequence of a defect in p53-mediated control of homologous
recombination processes attributable to mutations in the p53
gene.
Individuals with mutations in either the BRCA1 or
BRCA2 tumor suppressor genes have a dominant predisposition to
breast and ovarian cancer (Smith et al. 1992; Easton et al. 1993;
Wooster et al. 1994; Gayther et al. 1997). Colocalization and
coimmunoprecipitation experiments have shown that human BRCA1 protein
associates with HsRad51 in mitotic and meiotic cells (Scully et al.
1997). Thus, there appears to be a role for BRCA1 in nuclear processes
that leads to normal chromosomal recombination and control of genome integrity. Embryonic lethality and radiation hypersensitivity mediated
by Rad51 was shown in mice lacking Brca2 (Sharan et al. 1997).
Using a yeast two-hybrid assay, Sharan et al. 1997 also identified an
interaction between Brca2 and MmRad51. The homozygous mutant phenotypes
of Brca1, Brca2, and Mmrad51 are similar, indicating that these genes function in similar pathways (Hakem et al. 1996; Lim
and Hasty 1996; Sharan et at 1997). The association of Rad51 with Brca1
and Brca2 and the resultant sensitivity of MmRad51 /
and Brca2 / cells to irradiation may explain the high
penetrance of early onset cancer phenotypes exhibited by patients with
either BRCA1 or BRCA2 mutations. In mammary
epithelial cells that have lost BRCA1 or BRCA2 activity, the
HsRad51-mediated DNA repair mechanisms may be compromised, thus
destabilizing the genome. Thus, Rad51 probably suppresses tumor
formation through its interaction with both Brca1 and Brca2, all three
of which may be involved in detecting and repairing DSBs, thereby
controlling cell cycle progression (Sharan et al. 1997).
A recombination hotspot-binding protein, translin, which is associated
with chromosome translocations and binds to consensus sequences at
breakpoint junctions of chromosomal translocations in many lymphoid
malignancies, was reported by Aoki et al. (1995). ReHF-1, a
recombination hot spot-associated factor specifically recognizes novel
target sequences at the sites of interchromosomal rearrangements in
T-cell acute lymphoblastic leukemia (T-ALL) (Kasai et al. 1994).
Nuclear proteins have been identified that bind to target sequences
within the recombination hot spot regions of the Bcl-2
oncogene that is involved in rearrangements associated with follicular
lymphomas (Aoki et al. 1994). These proteins appear to be similar to
ReHF-1. Nuclear proteins were also shown to bind to the recombination
hot spot region of the retinoic acid receptor gene on chromosome
17 (Tashiro et al. 1994, 1995), which along with the PML gene
on chromosome 15 is involved in the 15;17 translocations found in acute
promyelocytic leukemia. The interaction of these proteins with
conserved target sequences at chromosomal breakpoint junctions suggests
that they may be involved in enzymatic mechanisms reminiscent of the
general features of DNA recombination or replication events in E. coli or S. cerevisiae.
Recombination is of eminent importance in germ cells to generate genetic diversity during meiosis and to safeguard DNA from genotoxic damage in somatic cells. Identification of human homologs of genes encoding components of the recombination and replication pathways of lower organisms will yield insight into mechanisms of disruption of these pathways that lead to human disease. Further characterization of the human genome has allowed identification of previously unknown sequence elements that may be involved in the recombination process. Understanding the complex mutational mechanisms in disease genes will allow us to discover new hot spots and mechanisms of recombination resulting in human disease.
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FOOTNOTES |
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4 Corresponding author.
E-MAIL pragna{at}bcm.tmc.edu; FAX (713) 798-8526.
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