Mutation Research 616 (2007) 1123

Mini review

Molecular mechanisms of sister-chromatid exchange

David M. Wilson III a, , Larry H. Thompson b
a

Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224, United States
b Lawrence Livermore National Laboratory, P.O. Box 808, Livermore, CA 94551-0808, United States
Available online 6 December 2006

Abstract
Sister-chromatid exchange (SCE) is the process whereby, during DNA replication, two sister chromatids break and rejoin with
one another, physically exchanging regions of the parental strands in the duplicated chromosomes. This process is considered to be
conservative and error-free, since no information is generally altered during reciprocal interchange by homologous recombination.
Upon the advent of non-radiolabel detection methods for SCE, such events were used as genetic indicators for potential genotoxins/mutagens in laboratory toxicology tests, since, as we now know, most forms of DNA damage induce chromatid exchange upon
replication fork collapse. Much of our present understanding of the mechanisms of SCE stems from studies involving nonhuman
vertebrate cell lines that are defective in processes of DNA repair and/or recombination. In this article, we present a historical
perspective of studies spearheaded by Dr. Anthony V. Carrano and colleagues focusing on SCE as a genetic outcome, and the role
of the single-strand break DNA repair protein XRCC1 in suppressing SCE. A more general overview of the cellular processes and
key protein effectors that regulate the manifestation of SCE is also presented.
2006 Elsevier B.V. All rights reserved.
Keywords: Sister-chromatid exchange; Single-strand break DNA repair; CHO EM9; XRCC1; Bloom syndrome; Homologous recombination; DNA
replication forks

1. Introduction

1.2. Tony Carranos inuence from the perspective

of David Wilson

1.1. Purpose of this review

In this review, we pay tribute to our previous colleague and mentor Dr. Anthony V. Carrano (who most
knew as Tony) by discussing one of his major scientific interests: cytogenetics, which was the area of his
training. Specifically, we highlight the leading role he
played in studies that helped develop our understanding of sister-chromatid exchange (SCE) and the role of
the XRCC1 (X-ray repair cross-complementing 1) repair
protein in this form of genetic recombination. The mechanistic aspects of SCE in mammalian cells have been
infrequently reviewed in recent years [1].
Corresponding author. Tel.: +1 410 558 8153;
fax: +1 410 558 8157.
E-mail address: wilsonda@grc.nia.nih.gov (D.M. Wilson III).

0027-5107/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrfmmm.2006.11.017

When asked to write for this special issue in honour of

Tony, I did not hesitate to accept. Along with the established group of experts in the field of DNA repair (headed
by Larry Thompson), Tony was a major reason why I
joined the staff at Lawrence Livermore National Laboratory (LLNL) and enjoyed my time within the Biology
and Biotechnology Research Program. Tony exhibited
supportive, yet firm, leadership, offering advice and
assistance in my development as an independent investigator. He was an inspiration and a strong role model.
I thank him for his time and efforts while at LLNL.
But most importantly, I express my gratitude for his
emphasis of and dedication to high impact and high
quality research science. I am convinced that it was
this trait that drove him during his long and illustrious
career.

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D.M. Wilson III, L.H. Thompson / Mutation Research 616 (2007) 1123

Searching PubMed with Carrano AV, Tonys interest in the processes that affect chromosome stability
becomes obvious. His contributions to our understanding
of the mechanisms that are involved in or that police the
formation of genetic alterations are considerable. These
accomplishments include the development of assays to
measure chromosomal aberrations, the characterization
of genetic alterations associated with exposure to laboratory or environmental genotoxins, the delineation of
the contributions of specific DNA repair factors to the
maintenance of genome integrity, and the identification
of genetic mutations that lead to disease manifestation.
Indeed, Tonys enormous efforts in the Human Genome
Project allowed him to participate in defining several
genetic disorders associated with human disease.
1.3. Tony Carranos impact on DNA repair from the
perspective of Larry Thompson
I first met Tony one winter evening in 1973 at the
Holiday Inn in Livermore when I came for my interview
with Mort Mendelsohn, who was recruiting new staff
for the program he was reorganizing. Tony was in the
restaurant (or bar) with Mike Bender, who was puffing
on a cigar (I learned this year that Mike has also recently
passed away). We chatted awhile to see what we might
have in common. They were both cytogeneticists, and
Tony later went to work at LLNL.
After several years at LLNL, Tony and I developed a
very natural and productive scientific collaboration; with
our first publication in Nature in 1978, we were off to a
good start (see photograph of us in Fig. 2). This paper,
with Tony as the first author, was about chromosomal
SCE induced by chemical mutagens. Tony and I went on
to publish more than 20 papers together. As I isolated new
DNA repair mutants of hamster cells, which required
extensive characterization, Tony enthusiastically applied
his expertise in chromosome analysis to greatly enhance
the discoveries with these mutant cell lines. My last publication with Tony was in 1995 not long before he left
the Program.
Early on, Tony became my group leader, and this
arrangement was quite beneficial to both of us. As my lab
began isolating human DNA repair genes, Tonys DNA
sequencing shop run by Jane Lamerdin was invaluable
in determining the sequence of some of the first cloned
human repair genes. This was an exciting time for us
as we compared the sequences of the human genes with
those in other organisms such as yeast and mice, and
began to develop ideas about what the gene products
were doing at the molecular level. It was highly gratifying for me when the work Tony and I did together led to

his choosing human chromosome 19 as the major focus

of the new Human Genome Project.
With the goal of expanding the DNA repair effort at
LLNL in 1997, Tony and I did a national search for a new
staff member, which lead to our hiring David Wilson. It
seems fitting to note that David and I are now working together on XRCC1 studies, which Tony helped to
pioneer more than 25 years ago.
2. SCE as a cytological manifestation of
homologous recombination
2.1. Methodology for SCE visualization
During the S-phase of the cell cycle, DNA is replicated, and each chromosome becomes duplicated into
two closely associated daughter chromatids, which are
joined tightly at the centromere. Sister chromatids are
distinctly visible cytologically in late prophase and early
metaphase of mitosis before segregating to the resulting daughter cells. SCE is the process whereby the sister
chromatids effectively break and rejoin with one another,
physically exchanging regions (see Fig. 1 and Section
2.2).
SCE was first visualized by Taylor in plant cells
using tritium and autoradiography, which provided poor
spatial resolution [2]. It was later discovered that incorporation of the DNA base analog 5 -bromodeoxyuridine
(BrdUrd), in combination with Hoechst dye 33258 staining, would differentiate sister chromatids and reveal
SCEs [3]. BrdUrd closely resembles thymidine, and
is efficiently incorporated into the elongating DNA
strands during replication. The standard FPG (fluorescence plus Giemsa) staining method for visualizing
exchanges relies on growing cells in medium containing
BrdUrd [4]. Because replication of DNA is semiconservative, all of the BrdUrd is incorporated into the nascent
daughter strand of each duplex. During a second round of
growth in BrdUrd medium, the two sister chromatids differ in the amount of BrdUrd present. The sister chromatid
which has the original template strand of DNA now has
one strand of normal DNA and one that contains BrdUrd.
The other sister chromatid has BrdUrd incorporated into
both strands. Alternatively, the BrdUrd can be removed
from the medium during the second S phase, such that
the second-division metaphase cells have one BrdUrdlabeled strand in one chromatid and none in either strand
of the sister chromatid. In either case, BrdUrd has the
effect of bleaching so that the chromatid with more
BrdUrd is lighter in appearance.
A convenient alternative to the FPG technique,
which requires exposure to ultraviolet (UV) radiation,

D.M. Wilson III, L.H. Thompson / Mutation Research 616 (2007) 1123

13

Fig. 1. (A) Mechanism for occurrence of SCE when the leading strand of a replication fork encounters a SSB or gap. Steps 1 and 2: fork approaches
a SSB. Step 3: fork breaks. Step 4: repair synthesis occurs at the gap in the unbroken chromatid. The curved black arrow signifies a conformational
change to facilitate visualization of subsequent events. Step 5: processing of the broken duplex creates a 3 single-strand tail. Step 6: Rad51 mediates
strand invasion. Step 7: resolution of the Holliday junction in the orientation shown by the green arrows results in SCE, as illustrated by the red/blue
color junctions in the new parental strands. Resolution in the orientation shown by the purple arrowheads would not produce an SCE. Step 8:
the replication fork is restored. (B) Extraordinarily high SCE in CHO EM9 metaphase stained with propidium iodide (red) and antibody against
BrdUrd-substituted single-stranded DNA (following denaturation). The cells were grown for one cycle in the presence of BrdUrd, followed by one
cycle in normal medium, and stained according to Ref. [5]. The arrow indicates the only chromosome not having at least one exchange event. (C)
Human hTERT-NHF1 fibroblast, after two cycles of growth in 25 M BrdUrd, stained for SCE using DAPI (which stains DNA; blue fluorescence)
and antibody against BrdUrd-substituted DNA (green fluorescence). Arrows mark sites of exchange.

utilizes an antibody that binds to BrdUrd-substituted

DNA following denaturation [5]. DNA counter-stained
with propidium iodide (red staining in Fig. 1B)
or with the fluorescent dye DAPI (4 ,6-diamidino2-phenylindole) (blue staining in Fig. 1C) generates
images that permit easy scoring of SCE events.
An advantage of the antibody detection method is
that relatively low levels of BrdUrd substitution
can be used, thereby minimizing BrdUrd-dependent
SCE.
2.2. Molecular mechanisms of SCE
SCEs occur naturally as events associated with normal DNA replication and upon natural fork collapse,
with estimates being 34 exchanges per cell per cycle
under conditions where the level of BrdUrd incorporation is very low or zero [58]. As alluded to above,
BrdUrd incorporation itself contributes to SCE [5,9],

likely by increasing the level of single-strand breaks

(SSBs) and alkali-labile sites [10]. In E. coli, biochemical and genetic studies suggest that incorporated BrdUrd
is dehalogenated to uracil, which is then removed by
uracilDNA glycosylase, followed by the appearance of
single-strand nicks produced either by an abasic endonuclease or further dehalogenation [11,12]. As discussed
below, SSBs are prominent DNA intermediates that promote SCE.
In experiments where biotindUTP was used for
labeling instead of BrdUrd, it was discovered that most
SCEs produced by ionizing radiation or UV-C were
attributable to the presence of BrdUrd [13]; conversely,
SCEs induced by mitomycin C (MMC) were not the
result of BrdUrd incorporation [8]. Crosslinking agents
are in general potent SCE inducers, presumably because
homologous recombination (HR) is required to repair
the resulting broken replication forks that arise during
crosslink unhooking [14].

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D.M. Wilson III, L.H. Thompson / Mutation Research 616 (2007) 1123

Conditions that increase the cellular burden of SSBs

generally induce SCE efficiently: XRCC1 deficiency
[15], inhibition or knockout of poly(ADP-ribose) polymerase 1 (PARP-1) [16,17], exposure to hydrogen
peroxide [18], and inhibition of DNA synthesis by
hydroxyurea, aphidicolin, or camptothecin [1921],
which are drugs that produce broken replication forks
that behave as DSBs during non-denaturing pulsed-field
gel electrophoresis [22,23]. Thus, the simplest pathway
by which SCE likely occurs is through HR-mediated
restart of a broken DNA replication fork when it encounters a nick or gap in one parental strand as shown in
Fig. 1A [2325]. XRCC1-deficient cells, which carry an
elevated level of breaks (see Section 4), have a markedly
elevated level of Rad51 foci [23], which are indicative of
sites of HR. It is likely that HR similarly processes broken forks produced by genotoxins, including replication
inhibitors such as camptothecin [26].
Although certain models have invoked nonhomologous end joining (NHEJ) in mediating SCE [19,27,28],
most evidence points toward HR being the main pathway. In chicken DT40 cells, HR defective mutants,
including mutants of Rad51, Rad54, and the Rad51 paralogs, consistently have reduced SCE [29]. However, in
mammalian cells, the data are more complex and confusing. Some Rad51 paralog mutants show modest, not
extreme, reductions in SCE (see discussion in Ref. [30]),
but notably, isogenic rad51d mutant lines prepared by
gene targeting in both Chinese hamster ovary (CHO)
and mouse fibroblasts show no decrease in spontaneous
SCE [30,31]. Rad54 knockout mouse cells also show little or no reduction in spontaneous SCE, but like with
rad51d mouse cells, there is a noticeable deficiency in
MMC-induced SCE [31,32]. This deviation in phenotype between spontaneous and induced SCE suggests
that there may be more than one molecular pathway
responsible for SCE. However, since an SCE only arises
when the Holliday junction intermediate is resolved in
one of the two orientations (Fig. 1A), changes in the
bias of resolution, for example in mutant cells, might
account for some of these apparent inconsistencies (see
discussion in Ref. [30]).
3. Tonys chromosomal studies in DNA repair
and genomics
3.1. SCE as an endpoint for characterizing
repair-decient cells
SCE quickly became a popular cytogenetic endpoint
when it became possible to identify this manifestation
of chromosomal recombination using non-radioisotope

Fig. 2. Photograph of Tony Carrano and Larry Thompson taken in 1978

after the publication in Nature of a paper entitled Sister chromatid
exchange as an indicator of mutagenesis.

methods (e.g. FPG) to distinguish the DNA strands [4].

It also became clear that SCEs arose during DNA replication as cells passed through S phase [33]. As reported
in Nature in 1975 [34], The (SCE) test gives a very
sensitive and rapid method for detecting chromosome
mutagenicity of chemical agents and provides a powerful new method for detecting environmental mutagens.
As this cytogenetic phenomenon gained momentum, Tonys first studies dealing with chromosome
exchange addressed the influence of BrdUrd incorporation and chromatin compaction on SCE frequency
[35,36].
Since the biological significance of SCE was poorly
understood, Tony and Larry decided to address this
issue in a doseresponse study in which induced SCE
and hprt mutation were compared for several prototype
mutagens (see photograph in Fig. 2 associated with this
publication). Over the same dose range, each genotoxin
linearly induced both SCE and single-gene mutations,
leading to the conclusion that the two forms of genetic
alteration were closely and linearly related [37]. Thus,
although SCEs arise in close association with mutations,
these exchange events themselves were likely to serve as
markers of DNA damage and to occur (as we now under-

D.M. Wilson III, L.H. Thompson / Mutation Research 616 (2007) 1123

stand much better) through an error-free HR mechanism,

analogous to repair events measured by unscheduled
DNA synthesis performed by nucleotide excision repair
(NER).
Subsequent studies focused on characterizing the
newly isolated CHO repair-deficient mutants and their
hypersensitivity for induced SCE and single-gene mutation. These mutant cell lines were isolated with the
goal of eventually elucidating the molecular steps
and specific proteins involved in various DNA repair
pathways. NER-defective CHO mutants [38], which
provided a versatile model system for studying the
nature of the defects in the human cancer-prone
disorder xeroderma pigmentosum, displayed high sensitivity to cell killing and induction of SCE and
single-gene mutation for various bulky-adduct mutagens: UV-C, 7-bromomethylbenz[a]anthracene, and
the cooked-food mutagens Trp-P-2 and IQ [3941].
The finding of increased SCE was one of the
first to suggest that the inability of the cell to
efficiently repair replication-blocking lesions likely
results in fork collapse and the initiation of HR for
repair.
The first CHO mutant isolated as being hypersensitive
to killing by the potent mutagen ethyl methanesulfonate
(i.e. line EM9) was serendipitous. When Jay Minkler,
who did much of the chromosome analysis with Tony
and Larry, examined the metaphase cells of untreated
cultures for SCE, he observed an amazing abundance
of exchange in every cell (Fig. 1B) [15], a feature very
reminiscent of cells from Bloom syndrome (BS) patients
[42] (see Section 5). Tonys initial reaction upon seeing the results was to question Larry about whether the
cells had been accidentally exposed to fluorescent light
while growing in BrdUrd. Fortunately, that was not the
case. An obvious, more scientific question was whether
EM9 represented the CHO analog of BS, but that, too,
proved not to be the case [43] as detailed in Section
4.
3.2. Multiple DNA repair genes, including XRCC1,
on human chromosome 19
The isolated CHO repair mutants provided a powerful system in which one could identify the human
chromosome, and even chromosomal region, that contained the human ortholog that complemented the defect
in the hamster mutant line. In somatic cell hybrids
made between hamster and human cells (often fresh
peripheral blood lymphocytes), human chromosomes
are rapidly and extensively lost. Therefore, hybrid cells
grown under conditions of low-dose mutagen expo-

15

sure, which normally kills the repair-deficient hamster

mutant, can continue to proliferate by virtue of carrying a human chromosomal region that functionally
complements the repair deficiency. The first such study
performed by Tony and colleagues utilized the CHO
ercc1 UV20 mutant, which proved to be corrected for
MMC resistance by chromosome 19 [44]. During the
annual Directors review of 1983, Tony convinced Lou
Siminovitch to look into the microscope to see for himself chromosome 19 (which stood out cytologically as
brightly staining upon reverse banding) in a hybrid cell.
Lou then made a comment about the importance of
getting molecular biology methods on-line at LLNL,
so that the defective gene could be isolated. Although
Tony and Larry initially had hopes of cloning this gene
(ERCC1, excision repair cross-complementing 1), two
other groups soon published on its identification [45,46].
Tony was involved in a series of experiments that
lead to the eventual cloning of the XRCC1 gene, which
corrected the EM9 mutant [47,48]; he later oversaw
the comparative genomic analysis of the cloned human
and mouse XRCC1 homologs [49]. The discovery that
EM9 cells were killed efficiently upon incorporation of
chlorodeoxyuridine (CldUrd) [10,50], whereas parental
CHO cells were not, provided an effective selecting
agent to test for chromosomal complementation. When
CldUrd-resistant hybrid cell clones were analyzed, chromosome 19 was again in the limelight by carrying
the complementing repair gene [51]. During the initial analysis of the resistant clones, Tony told Larry
that something was wrong, that the cells were not corrected to normal levels of SCE. An explanation soon
emerged because the resistant clones had been grown
continuously in CldUrd before being frozen. The SCE
experiment was done with hybrid clones taken directly
from the freezer that had not been grown first in normal
medium to dilute out the pre-existing CldUrd. Presumably via a similar mechanism as BrdUrd (see Section
2.2), CldUrd is a potent inducer of SCE in normal cells
[52]. These untoward results authenticated the view that
a high burden of SCE is not inherently associated with
cytotoxicity when the underlying damage is efficiently
repaired.
Tonys fascination with human chromosome 19 was
sealed when the DNA repair gene ERCC2/XPD was
also discovered to reside on this chromosome [53,54].
Tony therefore choose chromosome 19, isolated from
one of the monochromosomal CHO-human hybrid cells
by flow sorting, as the target for cosmid-contig analysis and whole-chromosome sequencing in the Human
Genome Project funded by the Department of Energy
[55,56].

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D.M. Wilson III, L.H. Thompson / Mutation Research 616 (2007) 1123

4. The function of XRCC1

Since the isolation of EM9 and its complementing
gene (see Section 3 above), extensive cell biological and
biochemical analyses have revealed that XRCC1 is a
non-enzymatic factor that participates in the related pathways of base excision repair (BER) and single-strand
break repair (SSBR) (Fig. 3). BER and SSBR handle
most forms of spontaneous DNA damage, including
hydrolytic decay products (e.g. uracil bases and abasic
sites) and oxidative lesions (e.g. base modifications and
SSBs) [57]. Early alkaline elution studies revealed that
xrcc1 mutant cells (e.g. EM9 and other independently
isolated CHO mutant lines in the same complementation
group [58]) exhibit a clear decrease in SSBR efficiency
[15,48]. A defect in SSBR in cells lacking XRCC1, both
CHO and human, has since been corroborated using the
more recently developed Comet assay [59,60].
The major biochemical defect of xrcc1 mutant cells
appears to be in nick ligation, the final step of BER/SSBR
(Fig. 3B) [61]. This observation is consistent with the
known role of XRCC1 as a stabilizing factor for DNA

ligase 3 (LIG3) [6264]. Specifically, cells deficient

in XRCC1 exhibit a four- to six-fold decrease in the
LIG3 protein level, which is directly correlated with the
XRCC1 cellular concentration [65]. Other interactions
with DNA polymerase (POL) [6669], polynucleotide kinase/phosphatase (PNKP) [70,71], and the
strand-break binding protein PARP-1 [66,7274] also
appear to help promote efficient BER/SSBR (Fig. 3B).
While XRCC1 may co-exist in a complex with additional BER proteins, namely certain DNA glycosylases
[75,76] and/or the abasic endonuclease APE1 [77], a cellular role for XRCC1 in the early steps of BER has not
been demonstrated [61].
Current data clearly indicate that XRCC1 plays a significant role as a stabilizing factor for DNA LIG3, and
as a non-enzymatic scaffold protein for assembly and
coordination of the BER/SSBR processes. In addition to
its numerous protein interactions (Fig. 3A), XRCC1 has
also been shown to exhibit DNA binding activity [78,79].
Despite the fact that the protein displays some preference
for nicks and gaps in DNA, XRCC1 exhibits a general
or non-specific DNA binding affinity as well ([79] and

Fig. 3. (A) Linear diagram of XRCC1. XRCC1 N = N-terminal DNA binding and POL interaction domain; BRCT1 and BRCT2 = the central and
C-terminal BRCT (BRCA1 C-terminus) domains. Direct physical interactors of XRCC1 are denoted, as are the regions within XRCC1 that they
interact. Amino acid locations are indicated (from 1 to 633). NLS = location of several putative nuclear localization signals. PCNA = proliferating
cellular nuclear antigen; OGG1 = 8-oxoguanine DNA glycosylase. See text for additional details. (B) Role of XRCC1 in BER/SSBR. Major steps of
BER/SSBR are denoted 15 to the left. In brief, a DNA glycosylase (e.g. OGG1) excises a damaged base from DNA (gray square), leaving behind
an apurinic/apyrimidinic (AP) site. The AP site is then typically incised by an AP endonuclease, primarily APE1 in mammals. The termini of the
resulting strand break are processed (i.e. the 5 -deoxyribose phosphate is removed, gray circle), and DNA POL (primarily) fills the nucleotide
gap (gray line). Finally, the remaining nick is sealed by a DNA ligase, such as LIG3. SSBs are also products of certain multifunctional DNA
glycosylases or the action of reactive oxygen species, both of which can generate 3 -obstructive ends such as phosphate groups (black circle).
Through direct physical interactions: PARP-1 recruits XRCC1 to a SSB site; XRCC1 facilitates POL organization at the SSB; XRCC1 promotes
PNKP 3 -phosphatase activity; XRCC1 both stabilizes LIG3 protein levels and facilitates nick ligation.

D.M. Wilson III, L.H. Thompson / Mutation Research 616 (2007) 1123

unpublished observations). In fact, the BRCT2 domain

of XRCC1 has been shown to associate with DNA DSB
ends [80]. Research to more thoroughly characterize the
DNA binding properties of XRCC1 is warranted. Of
course, documentation that the DNA binding function
of XRCC1 contributes to the cell biology of this protein
is imperative as well.
In addition to operating in SSBR, recent evidence also
suggests that XRCC1 acts as part of a complex to foster DSBR, and that defects in XRCC1 specifically lead
to impaired NHEJ [81]. However, the contribution of
XRCC1 to DSBR is largely in a back-up capacity, and
thus, is not the primary function of this protein. Instead,
the deficiency of xrcc1 cells in repairing SSBs is thought
to be responsible for the increased SCEs, which were
originally shown by Tony to arise when DNA replication
occurred on a BrdUrd-substituted template strand and to
be closely linked with chromatid breaks (e.g. incomplete
SCEs in which one chromatid is broken at the site of the
exchange) [82]. As discussed earlier, unrepaired SSBs
encountered by the progressing replication machinery
will be converted to DSBs that are potential substrates for
HR, which determines exchange frequencies. Interestingly, LIG3 knockout mouse cells also exhibit elevated
SCEs, albeit not as pronounced [83], suggesting that the
stabilizing role of XRCC1 for DNA LIG3 (and its nick
ligation activity) is an important element in the increased
SCE events. Presumably the other physical associations
(Fig. 3) contribute to the remainder of the impaired SSBR
and the highly increased SCE of xrcc1 cells. We direct
readers to other review articles for a more exhaustive
discussion of the biochemical and biological functions
of XRCC1 [8486], as well as its recent association
with proteins defective in human spinocerebellar ataxias,
namely Aprataxin (APTX) and tyrosyl-DNA phosphodiesterase (TDP1) [87,88].
5. Effectors of SCE
5.1. Proteins that impact SSBR
As outlined above, evidence strongly implicates SSBs
as a primary genetic intermediate for natural SCE.
Specifically, defects in SSBR lead to increased SCE
frequencies, likely through a mechanism that involves
an unrepaired SSB, DSB formation upon replication
fork collapse at the SSB site, and HR (with the sister
chromatid) to facilitate accurate repair (Fig. 1A). Thus,
defects in proteins such as XRCC1, PARP-1, and DNA
LIG3, which play central roles in mediating SSBR, are
expectedly associated with elevated SCEs (Table 1). Surprisingly, mouse cells lacking DNA POL, the major

17

gap-filling DNA polymerase, exhibit normal levels of

spontaneous SCEs, although they display higher than
normal SCE levels following alkylating agent exposure
[89].
5.2. BS helicase and other regulators of
recombination
In addition to SSBR factors, which play indirect roles
in suppressing SCE events, defects in proteins that regulate HR have also been associated with altered SCE
frequencies (Table 1). Most notably, cells derived from
patients with the autosomal recessive disorder BS are
genetically unstable and exhibit a hallmark high occurrence of SCE [42], presumably due to dysregulation
of HR [90]. In fact, markedly increased SCE is used
clinically as a diagnostic marker for this disease. The
defective protein, the BLM helicase, is a member of
the highly conserved RecQ family of helicases, and
possesses both DNA unwinding and DNA strand annealing activities [90,91]. The key biochemical features of
BLM thought to modulate SCE are its ability to: (i) in
cooperation with topoisomerase III and BLAP75, dissolve double Holliday junctions to yield non-crossover
recombinants [9294], (ii) dissociate (or unwind) D-loop
recombination intermediates, thus operating as an antirecombinase [95], and (iii) promote the regression of
a stalled replication fork [96]. Defects in one or more
of these processes are presumably responsible for the
genetic instability, increased SCE, and high incidence of
cancer early in life that are associated with BS. As noted
in Section 2.2 and Table 1, a role for HR in regulating
SCE is also consistent with the fact that cells defective in
homologous exchange (e.g. rad51 cells) typically exhibit
lower SCE frequencies.
5.3. Translesion synthesis (TLS) and Fanconi
anemia pathways
In the chicken DT40 system, knockout mutations that
abolish the E3 ligase Rad18 [97] or the translesion polymerases Rev1 and Pol- (Rev3 and Rev7 subunits) result
in increased SCE levels [98100] (Table 1). Curiously,
rev1 rev3 rev7 triple mutants show only slightly elevated
SCE. However, it is difficult to know what causes this loss
of phenotype in rev triple mutants because reduced SCEs
can reflect fewer lesions being processed by HR or fewer
crossover recombination events (or both). Mouse rad18
embryonic stem (ES) cells also show a two-fold increase
in spontaneous SCE [101], which is similar to the two- to
three-fold increase seen in rad18 DT40 cells [97]. However, mouse rev1 ES cells show no change in spontaneous

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D.M. Wilson III, L.H. Thompson / Mutation Research 616 (2007) 1123

Table 1
Proteins that regulate spontaneous SCEa
Protein function
Effectors of SSBR
XRCC1
Stabilizes LIG3 protein, and acts as a non-enzymatic
assembly factor in BER/SSBR
PARP-1
Binds DNA break ends and coordinates a strand break
repair response
LIG3
Ligates DNA nicks
Effectors of HR
BLM
Rad51
Rad54
XRCC2
XRCC3
Rad51B
Rad51C
Rad51D

Dissociates D-loop recombination intermediates and promotes dissolution of double Holliday junctions
Mediates strand transfer in HR
Facilitates HR
Promotes Rad51 focus formation and HR
Promotes Rad51 focus formation and HR
Promotes Rad51 focus formation and HR
Promotes Rad51 focus formation and HR
Promotes Rad51 focus formation and HR

Effectors of TLS
Rad18
Cofactor of Rad6 (ubiquitin conjugating enzyme) in TLS
Revl
Deoxycytidyl transferase in TLS
Rev3
Polymerase catalytic subunit
Rev7
Polymerase regulatory subunit
rev1 rev3 rev 7 triple  mutant

SCE level in mutant

References for role in SCE

[5,15,117]

[16,17]

[83]

[42,118,119]

[29]
[29,32,119]
[121123]
[120,121]
[121]
[121,124,125]
[30,31,121]

[97,101]
[98,100]
[99,100]
[100]
[100]

Not listed are FANC genes, which seem to influence SCE in DT40 cells but not mammalian cells. The main categories for proteins modulating
SCE are indicated in bold. See text for more details. The arrows depict the increase (), decrease (), or no change () in spontaneous SCE level in
mutant cells. The size of each arrow represents the degree of change: small arrow, <two-fold; medium arrow, two- to five-fold; large arrow 10-fold.
Each arrow represents a separate study.

or UV-C induced SCE [102]. Whenever increased SCE

is seen, it can likely be explained as follows: a defect
in TLS may result in an increase in the number of postreplication gapped regions in one chromatid, such that
the gaps are repaired by HR, producing SCE. Alternatively, decreased TLS capacity may cause an increase
in blocked forks that collapse and are restarted by HR,
thereby producing more SCE (Fig. 1A).
Fanconi anemia (FA) is an autosomal recessive and
X-linked disorder associated with cancer susceptibility
[103,104]. Cells from FA patients are characterized by
chromosomal instability (mainly chromatid breaks and
exchanges), and evidence indicates that the FA proteins
function to promote TLS as well as restart of broken
replication forks by HR and end joining [105,106]. In
human and rodent cells, including isogenic fancg CHO
cells [107], mutations in this pathway do not result in
increased spontaneous SCEs. However, in the chicken
DT40 system, SCEs are consistently increased in fancc
and fancd2 mutants [108110]. This disparity in phenotype implies that the molecular details of HR and
SCE formation differ between the lower vertebrate and
mammalian cells.

6. Future directions
It is evident from the above discussion that an
enhancement of SCE in repair-proficient cells can be
expected to occur as part of the response of cycling cells
to virtually any genotoxic agent [19,34,111]. Replication forks collapse when they encounter DNA blocking
lesions introduced by mutagens or upon drug inhibition of replication, especially at doses that produce cell
killing. Broken forks are subsequently subject to repair
mainly by HR, which results in increased SCE. Thus, by
extension, altered SCE levels will be a feature of most,
but clearly not all, cells defective in the repair of DNA
damage. We suggest that future studies more exhaustively evaluate this endpoint in available DNA repair
mutant cells, with and without exposure to exogenous
sources of DNA lesions. Mutants that do not show a
change in SCE frequency may reflect: (i) that the substrates of the missing protein do not influence exchange,
or (ii) there exists an altered bias in resolving Holliday
junctions, such as presumably in rad51d rodent cells
[30,31]. This latter issue deserves study, and may prove
insightful about the functions of the Rad51 paralogs.

D.M. Wilson III, L.H. Thompson / Mutation Research 616 (2007) 1123

Another fact that has become apparent is that mutant

non-mammalian vertebrate cells exhibit a markedly different SCE profile than the corresponding human and
rodent cells. Thus, it will be particularly interesting to
examine SCE frequencies in genetically engineered or
siRNA-targeted repair-deficient human cell lines. Our
initial data using transient siRNAs for XRCC1 indicate
that a deficiency in this protein in both HeLa and normal human fibroblasts (NHF1) leads to increased SCEs,
although not as pronounced as seen in the presumed null
CHO mutant EM9 [112] (our unpublished results).
Other questions that are relevant to address in future
studies are:
(a) Why do rodent Rad51 paralog mutants show modest,
or no, reduction in SCE while exhibiting severely
elevated chromosomal aberrations?
(b) Why do mutants in the FA pathway in DT40 cells
consistently show increased levels of spontaneous
SCE (the opposite of what one might expect, assuming the FA pathway promotes HR)?
(c) Will TLS mutants of human cells have increased
SCE, like in DT40 cells, or not (as for Rev1 in mouse
ES cells)?
These questions underscore the importance of creating null mutants for numerous DNA repair genes
in human cells having diploid [113] and near diploid
[114] karyotypes. Conditions for improving genetargeting efficiency, for example by knocking down
BLM or PARP1 during transfection for gene knockout,
hold considerable promise and warrant extensive study
[115,116].

Acknowledgements
We thank Drs. John Hinz (LLNL), Sudha Sharma
(NIA), and Jinshui Fan (NIA) for their insightful comments. We extend a special thanks to Dr. Paul Wilson
(LLNL) for providing the image in Fig. 1C, and J. Fan
for sharing his unpublished results. This research was
supported (in part) by the Intramural Research Program
of the NIH, National Institute on Aging. A portion of
this work (LHT) was performed under the auspices of
the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory under
Contract No. W-7405-Eng-48 and funded by the DOE
Low-Dose Radiation Research Program and NCI/NIH
grant CA11256.

19

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