Download DNA double strand break repair in mammalian - Mi Portal

GDA213.QXD
03/22/2000
02:27
Page 144
144
DNA double strand break repair in mammalian cells
Peter Karran
Human cells can process DNA double-strand breaks (DSBs)
by either homology directed or non-homologous repair
pathways. Defects in components of DSB repair pathways are
associated with a predisposition to cancer. The products of the
BRCA1 and BRCA2 genes, which normally confer protection
against breast cancer, are involved in homology-directed DSB
repair. Defects in another homology-directed pathway, singlestrand annealing, are associated with genome instability and
cancer predisposition in the Nijmegen breakage syndrome and
a radiation-sensitive ataxia-telangiectasia-like syndrome. Many
DSB repair proteins also participate in the signaling pathways
which underlie the cell’s response to DSBs.
Addresses
Imperial Cancer Research Fund, Clare Hall Laboratories, South
Mimms, UK EN6 3LD; e-mail: [email protected]
Current Opinion in Genetics & Development 2000, 10:144–150
0959-437X/00/$ — see front matter © 2000 Elsevier Science Ltd.
All rights reserved.
Abbreviations
A-T
ataxia telangiectasia
ATM
ataxia telangiectasia mutated
ATR
ataxia telangiectasis Rad3-related
DNA-PK
DNA-dependent protein kinase
DNA-PKcs DNA-PK catalytic subunit
DSB
double-strand break
HR
homology directed recombination
MMR
mismatch repair
NHEJ
non-homologous end joining
PCNA
proliferating cell nuclear antigen
RPA
replication protein A
SCID
severe combined immune deficiency
SSA
single-strand annealing
TCR
transcription-coupled repair
V(D)J
variable division joining
Xrcc4
X-ray cross-complementing 4
Introduction
DNA double-strand breaks (DSBs) are a common form of
DNA damage and DSB rejoining is a fundamental mechanism of genome protection. Breaks arise through direct
action of ionizing radiation or some chemicals, and indirectly as a product of blocked replication forks. The ability to
repair DSBs — and to ensure that repair is performed with
appropriate fidelity — is a fundamental part of genome protection. The three known DSB repair pathways are outlined
in Figure 1 and the participating proteins listed in Figure 2.
The pathways are conserved between Saccharomyces cerevisiae and mammalian cells although the relative importance in
each differs considerably. The prevailing view has been that
homologous recombination is the predominant pathway of
DSB repair in yeast whereas mammalian cells presented
with similar substrates use non-homologous or illegitimate
pathways. This still seems to be the consensus although
there is mounting evidence that DSB repair via homologous
recombination in mammalian cells is more significant than
was previously thought [1•,2•]. In addition, there are
indications that some proteins may participate in more than
one of the three repair pathways.
Non-homologous end rejoining
Non-homologous end rejoining (NHEJ) has been considered the major pathway of DSB repair in mammalian cells
(Figure 1). Repair is achieved without the need for extensive homology between the DNA ends to be joined.
NHEJ processes the site-specific DSBs introduced during
V(D)J (variable [division] joining) recombination and its
importance is emphasised by the severe combined
immune deficiency (SCID) and ionizing radiation sensitivity of mice with NHEJ defects. NHEJ involves a DNA
end-binding heterodimer of the Ku70 and Ku80 proteins
which activates the catalytic subunit (DNA-PKcs) of
DNA-dependent protein kinase (DNA-PK) by stabilising
its interaction with DNA ends. This facilitates rejoining by
a DNA ligase IV/Xrcc4 (X-ray cross-complementing 4) heterodimer. (For a recent review of DNA-PK, see [3].)
The presence of constitutively high steady-state levels of
DNA-PK is consistent with a role as a primary DNA damage recognition factor. The family of kinases to which
DNA-PKcs belongs contains the important ATM (ataxia
telangiectasia mutated) and ATR (ataxia telangiectasia
Rad3-related) signalling proteins. All three proteins are serine/threonine protein kinases which have some sequence
similarity to phosphatidylinositol kinases. DNA-PK can
phosphorylate p53 but the p53 response is apparently intact
in DNA-PK-defective mouse cells which express wild-type
p53 [4•]. This rules out DNA-PK as an essential requirement for activation of the p53 DNA damage response.
Other plausible targets for DNA-PK include the singlestranded binding protein RPA (replication protein A), the
DNA ligase IV cofactor Xrcc4, Ku, and DNA-PKcs itself. It
is noteworthy that, although the stimulation of DNA ligase IV activity by phosphorylated and unphosphorylated
forms of Xrcc4 is similar, phosphorylation of Xrcc4 may prevent its direct association with DNA [5•]. This could
provide a means to regulate rejoining by modulating the
interactions among Xrcc4, DNA and DNA ligase IV.
Both Ku subunits and DNA-PKcs are essential for repair
by NHEJ although defects in DNA-PKcs generally confer
a milder phenotype than defects in Ku. DNA-PK activity
is undetectable in Ku-defective cell lines, indicating that
DNA binding by the Ku70:80 heterodimer is essential for
its activation. The carboxy-terminal portion of Ku80 is also
important for DNA-PK function and distinct regions are
responsible for Ku70 interaction and DNA-PKcs activation. Deletion of the carboxy-terminal region of Chinese
hamster Ku80 imparts a phenotype similar to DNA-PK
deficiency — even when high levels of DNA-PKcs are
GDA213.QXD
03/22/2000
02:27
Page 145
DNA double strand break repair in mammalian cells Karran
145
Figure 1
Pathways of DSB repair. The termini of a DNA
DSB introduced by ionising radiation or other
means are bound either by the Ku
heterodimer/DNA-PKcs complex or by
hRad52. In the NHEJ rejoining pathway, repair
is completed by DNA ligase IV and XRCC4.
DNA strand invasion of the intact sister
chromatid, facilitated by hRad51, initiates
repair by homologous recombination.
Resection and annealing of short regions of
complementary sequence initiates repair by
the SSA pathway in which ligation is
preceded by the trimming of noncomplementary single-stranded DNA tails.
The scheme is based on [15].
present [6•]. The requirement for this carboxy-terminal
region in kinase activation is consistent with the absence of
an analogous Ku80 carboxy-terminal tail in S. cerevisiae
which also lack a homologous DNA-PKcs protein.
pass [7•]. These findings are consistent with DNA-PKcs
mediating the alignment of short stretches of singlestranded DNA prior to ligation. This would be in addition
to any role of DNA-PK in signalling.
The molecular architecture of DNA-PKcs suggests a structural role for the protein in the rejoining process. It has a
potential DNA-binding groove and an enclosed cavity with
three apertures through which single-stranded DNA could
Repair by NHEJ is completed by the DNA ligase IV/Xrcc4
complex. Mice with targeted inactivation of either component exhibit embryonic lethality [8,9,10•] Consistent with
their joint participation in the NHEJ pathway, the
Figure 2
Proteins involved in DSB repair. The circle on
the left contains a list of the known
participants in NHEJ. Gene products involved
in DSB repair by homology directed
recombination (HR) are indicated in the large
circle to the right. The SSA pathway which
requires some degree of homology between
joined termini is depicted as a subpathway of
HR. Possible overlaps with the human
excision (TCR) and MMR pathways are
indicated although it should be noted that
there is at present no direct indication of
involvement of hMSH2 or hMSH3 in the
human SSA pathway.
GDA213.QXD
03/22/2000
146
02:27
Page 146
Chromosomes and expression mechanisms
sensitivity of DNA ligase IV or Xrcc4 knockout mouse
cells to ionising radiation, and their defects in V(D)J
recombination mimic those of Ku null mice. As Ku and
DNA-PKcs deficient mice are viable, however, there
appears to be an additional requirement for DNA ligase IV
and Xrcc4 during embryonic development.
Reduced NHEJ activity may confer radiosensitivity in the
absence of overt defects in immune function. Radiation
sensitivity and a modest defect in DSB repair, but no significant impairment in V(D)J recombination, are
associated with a mutated DNA ligase IV in cells from a
radiation-sensitive leukaemia patient [11•]. The absence
of a detectable effect on V(D)J recombination indicates
that an alternative DNA-rejoining activity might partially
substitute for defective Xrcc4/DNA ligase IV in these
cells. The occurrence of a DNA ligase IV defect in a
leukaemia patient suggests that reduced NHEJ might be a
factor in the incidence of this disease.
Homologous recombination
S. cerevisiae has provided a useful model for homology
directed recombination (HR) processes. HR is performed
by the RAD52 epistasis group of proteins which includes
the products of RAD50–55, RAD57, and RAD59, MRE11
and XRS2 (for a recent review of HR, see [12]). Although
mammalian cells are considered to rely less on HR, they do
perform mitotic recombination and preferentially repair
DSBs by HR in late S and G2 phases of the cell cycle when
an undamaged sister chromatid is available. Indeed, the
DSB repair defect of murine scid (severe combined
immunodeficiency) cells is only apparent in G1/early
S phases [13•].
The mammalian Rad51 protein is a homolog of the
Escherichia coli RecA protein and is involved in both meiotic
and mitotic recombination. The S. cerevisiae and human
Rad51 proteins catalyse strand exchange in a reaction which
is stimulated by Rad52 and RPA [14,15]. Human Rad52 has
a DNA double-strand end binding activity like Ku [16•] and
it probably co-operates with hRad51 in DSB repair but this
is unlikely to be the only important function of Rad51.
Targeted inactivation of mRad51 results in early embryonic
lethality [17•] whereas Rad52–/– mice are viable and fertile
[18•]. Rad52–/– murine stem cells, although deficient in
recombination, are not detectably hypersensitive to the
presence of DSBs. Animals which are null for a third member of the RAD52 family, mRad54, are also viable and fertile
but, in this case, their cells are hypersensitive to DSB-inducing agents [19•]. Unravelling the biochemical functions of
these mammalian RAD52 family members — and defining
the essential functions of the Rad51 and Rad54 proteins —
is clearly a high priority. At present, it is clear that they are
not functionally identical to their yeast counterparts.
Human Rad51 forms discrete foci in the nuclei of cells
exposed to ionising radiation (but not UV) or chemicals. In
rodents, DNA damage-induced mRad51 focus formation
requires mRad54 [20•]. As both mRad51 and mRad54 null
cells are sensitive to ionising radiation, the direct participation of mRad54 and mRad51 in DSB repair is implied.
Human cells are known to express two other Rad51-related proteins — Xrcc2 and Xrcc3 — both of which interact
with Rad51 and influence DSB repair by HR [21•,22•].
Confusingly, although mRad54-null and Xrcc3-deficient
cells are both sensitive to ionising radiation, only the latter
are cross-sensitive to UV light.
An important recent development has been the realisation
that the products of the human breast cancer susceptibility genes BRCA1 and BRCA2 are involved in DNA repair.
In fact, the carboxy-terminal region (BRCT domain) of
these proteins defines a common motif among DNA-repair
proteins [23]. Loss of functional Brca1 results in sensitivity (albeit slight) to radiation and DNA-damaging
chemicals [24•–26•]. A fraction of hRad51 colocalises with
Brca1 and Brca2 in mitotic cells. Following DNA damage,
the three proteins are relocated to structures which also
contain PCNA (proliferating cell nuclear antigen) and may
represent sites of active repair [27].
Both Brca2–/– and Brca1–/– mouse fibroblasts develop
spontaneous chromosome aberrations consistent with the
participation of these proteins in the repair of spontaneous DSBs [28•]. Brca1 is phosphorylated by the ATM
protein [26•]. ATM, which is mutated in the radiationsensitive disorder ataxia-telangiectasia, is a candidate
primary sensor of certain types of DNA damage, particularly DSBs induced by ionising radiation. Brca1 in
therefore a target for a key protein which signals the
response to ionising radiation.
Brca2–/– cells are also sensitive to DNA-damaging agents,
although, curiously, display a much more pronounced sensitivity to UV than to γ-radiation. In addition, the massive
sensitisation to mitomycin C which accompanies loss of
Xrcc2 or Xrcc3 is not seen in the Brca2–/– cells [28•]. The
likely involvement of Brca1 in HR, which is suggested by
its association with mRad51, is supported by the observation that there is relatively more DSB repair by
non-homologous pathways in Brca1–/– cells [2•]. This is
unlikely to be the full story, however, because Brca1 also
displays a DNA-damage-dependent association with the
hRad50/Mre11/Nbs1 complex (see below) [25•]. This
important tumour suppressor therefore appears to participate in the two pathways of DSB repair.
Brca1 is also implicated in the removal of oxidised DNA
bases from DNA. Damage is selectively removed from the
transcribed DNA strand by a process known as transcription-coupled repair (TCR). Brca1-null cells are deficient in
the TCR of DNA thymine glycol produced by ionising
radiation or hydrogen peroxide but perform TCR of UVinduced DNA damage normally [24•,29•]. It is unclear
whether Brca1 participates directly in TCR or indirectly
via a signalling role. The involvement of Brca1 suggests
GDA213.QXD
03/22/2000
02:27
Page 147
DNA double strand break repair in mammalian cells Karran
that the TCR of oxidised bases may represent a form of
recombinational repair.
Single-strand annealing
The single-strand annealing (SSA) pathway of DSB rejoining (Figure 1) can be considered as a subpathway of HR.
Although it does not involve formation and resolution of
Holliday junctions, SSA relies on regions of homology with
which to align the strands of DNA to be rejoined. The
S. cerevisiae genes which define SSA belong to the Rad52
epistasis group of HR. Their human counterparts, hRad50,
hMre11 and Nbs1 (also known as p95 or Nibrin), define the
human SSA pathway (Figure 2).
Rad50 is an ATP-dependent DNA-binding protein. Mre11
has several activities, the most important of which may be
a 3′→5′ double-stranded DNA exonuclease which may
remove damaged or mismatched DNA termini and expose
short lengths of single-stranded DNA. This initiates a
search for short regions of homology between the fragments to be joined. If the sister chromatid is available, the
single-stranded tails may invade it to initiate rejoining by
the HR pathway. Alternatively, sites of limited homology
within the resected region may anneal to begin repair by
SSA. The Nbs1 protein is functionally homologous to the
S. cerevisiae Xrs2 protein. The NBS gene, which is located
on chromosome 8q21, is mutated in patients with the
Nijmegen breakage syndrome (NBS: for a recent review,
see [30]). Cells from NBS patients exhibit many features
associated with DSB-repair deficiency: sensitivity to ionising radiation, immune deficiency, chromosome instability,
and a predisposition to lymphoreticular tumours. The
pathway which is impaired in these cells clearly provides
significant protection against the effects of unrepaired
DSBs introduced either by external agents or through normal metabolic processes. Despite this, and in contrast to
NHEJ-defective cells, there is no overt defect in DSB
repair in NBS cells.
Two families with truncating or missense mutations in
hMRE11 have been reported [31•]. The similarity of their
clinical symptoms to the cancer-prone ataxia telangiectasia
(A-T) syndrome emphasises the importance of the SSA
pathway. The properties of cells from affected individuals
closely resemble those of NBS cells. As with NBS cells,
there is no detectable defect in DSB rejoining in
hMRE11-defective cells despite their ionising radiation
hypersensitivity and increased spontaneous and radiationinduced chromosome instability.
The effects of null mutations in mRad50 or mMre11 are
severe. Animals bearing homozygous disruptions of the
mRad50 gene exhibit early embryonic lethality [32•].
Embryonic mRad50–/– cells are extremely sensitive to ionising radiation and die during normal in vitro culture.
Similarly, Mre11-null mouse cells are inviable and chicken
cells in which Mre11 is conditionally disabled undergo proliferative arrest [33•]. These same cells sustain high levels
147
of chromosome aberrations and have a reduced capacity for
repair by homologous recombination. The differential
severity of Nbs1 and Rad50/Mre11 deficiency may reflect
the overlapping involvement of the mammalian
Rad50/Mre11 complex in different sub-pathways of DSB
repair or in cellular signalling.
The hRad50 and hMre11 proteins form an immunoprecipitable complex and colocalise in discrete nuclear foci
following irradiation of cells. hRad50/hMRE11 foci are
considered to be sited at DSBs. Focus formation requires
that both proteins are phosphorylated [34•]. Their phosphorylation is dependent on functional Nbs1 and
hMRE11 and cells defective in Nbs1 or expressing
mutated hMRE11 do not form foci [31•,35•]. In addition
to its targeting function, Nbs1 association with the
hRad50/ hMre11 complex has a significant effect on the
nuclease activity of the complex. It stimulates the
endonucleolytic cleavage of hairpin loops, suggesting that
there might be a greater overlap between the SSA, NHEJ
and homologous recombination pathways than was previously recognised [36•]. It may be significant in this regard
that translocations involving chromosomes 7 and 14 —
which harbour immunoglobulin and T cell receptor
genes, the substrates for NHEJ — are commonly
observed in NBS, A-T, and A-T-like lymphocytes
[30,31•]. Incorporation of Nbs1 into the hRad50/hMre11
complex also stimulates ATP-dependent endonucleoytic
cleavage at a single-strand/duplex DNA junction [36•].
This activity may be significant because it represents a
way of removing single-stranded DNA tails.
The single-stranded tails created by resection and
annealing of overlapping DNA ends are trimmed before
ligation. This is most probably performed by the structure-specific endonuclease activity of the XPF/Ercc1
complex which is the human counterpart of the S. cerevisiae Rad1/Rad10 complex. In addition to Rad1/Rad10,
clipping of the non-homologous 3′-single-stranded
regions also requires the yeast Msh2 and Msh3 proteins,
at least when the tails are relatively short [37,38•]. This
represents a second function for Msh2 and Msh3. An
Msh2/Msh3 heterodimer initiates the correction of some
mismatches by binding to DNA containing short singlestranded loops (for a recent review of human mismatch
repair (MMR), see [39]). This is another example of
overlap between DNA repair pathways and the apparent
dual roles of some repair proteins. The overlap does not
appear to extend further and downstream participants in
MMR, such as the Pms1 and Mlh1 proteins, are not
required for SSA. Indeed, the Msh2 functions which are
required for MMR are at least partially distinct and some
mutated Msh2 proteins which are inactive in MMR
retain full recombinational activity [40]. Human MMR
proteins certainly act as editors of recombination. The
extent to which they participate in the processing of
DSBs by the SSA pathway is clearly an important area for
further investigation.
GDA213.QXD
03/22/2000
148
02:27
Page 148
Chromosomes and expression mechanisms
Cellular signalling functions and DSB repair
Many of the participants in DSB repair — including
hMRE11, hRad50, DNA-PK, Ku and Xrcc4 — are phosphoproteins. Brca1 and Brca2 are both phosphorylated in a
cell-cycle-specific fashion and the former is a target for the
ATM protein kinase [26•]. Detailed description of signalling in the response to DNA damage is beyond the
scope of this review but ATM and the tyrosine kinase cAbl protein appear to be central to the control of DSB
repair. The c-Abl protein is itself activated by phosphorylation. This appears to be achieved by its interaction with
the ATM protein [41]. Human Rad51 is phosphorylated by
c-Abl, possibly via the formation of a tripartite complex
with the ATM protein [42•]. ATM/c-Abl-dependent phosphorylation promotes the interaction between hRad51 and
hRad52, suggesting that the HR recombination repair
pathway is subject, at least in part, to fine control by these
interactions. c-Abl is also implicated in activation of c-Jun
amino-terminal kinase. Significantly, this activation is
impaired in Nbs1 (and hMre11) as well as ATM-defective
human cells.
The ATM protein is implicated in the p53-related DNAdamage response. p53 plays a crucial role in determining
the fate of cells in which DSBs persist. In particular, the
embryonic lethality of both the Rad51 null [17•] and Brca1
null [29•] animals is attenuated in a p53–/– background and
some double knockout animals survive to full term. This
suggests that the massive failure of cellular proliferation,
which is a feature of Rad51–/– and Brca1–/– embryos, is to
some degree a direct consequence of an active p53. Put
another way, cells which fail to repair DSBs by the Rad51and Brca1-dependent HR pathways die in a p53-dependent fashion. Promoting the deletion of cells which might
otherwise perform mutation-prone DSB repair may be a
facet of p53’s role as ‘guardian of the genome’.
Conclusions and outstanding questions
The above speculation notwithstanding, there is a clear
need to define the details of the connection between
DSBs, their repair or persistence, and cellular responses
such as cell-cycle checkpoints and cell death/apoptosis.
DSBs may be introduced by several different routes.
These include direct introduction by agents such as ionising radiation or inhibitors of DNA topoisomerase II. DNA
replication itself carries an intrinsic probability of DSB formation and human cells are estimated to sustain about ten
DSBs per cell cycle [43]. This may be increased by the
reduced availability of deoxynucleotide triphosphates or
the presence of DNA lesions which either slow or block
the replication fork. It is possible that the type and timing
of DSBs influences the choice of repair pathway. There
may perhaps be subpathways which involve slightly different constellations of, say, HR participants which are more
likely to function on DSBs from a particular source.
heterodimer, Rad50/Mre11 or members of the hRad52
pathway in which the winner determines the course of
repair? Or is there some, more subtle means of signalling
which is the appropriate pathway? It would make sense if
repair by HR was activated in late S or G2 phases of the cell
cycle when a homologous copy is present. How might this
be achieved? Is this one of the functions of cell-cycle
checkpoints or is it a simple consequence of proximity to
replication forks of appropriate members of the hRad52
pathway? Detailed investigation of the signalling events
which accompany the formation of DSBs in mammalian
DNA is a clear priority.
Acknowledgements
I am grateful to Drs Bignami, West and Wood for their comments on the
manuscript.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
•
Sonoda E, Sasaki MS, Morrison C, Yamaguchi-Iwai Y, Takata M,
Takeda S: Sister chromatid exchanges are mediated by
homologous recombination in vertebrate cells. Mol Cell Biol 1999,
19:5166-5169.
Many DNA-damaging agents induce sister chromatid exchanges (SCEs).
Using the chicken DT40 cell line with defined defects in HR or NHEJ, it is
shown that Rad51 and Rad54, but not Ku70, affect the spontaneous or mitomycin C-induced SCE frequency.
2. Moynahan ME, Chiu JW, Koller BH, Jasin M: Brca1 controls
•
homology-directed repair. Mol Cell 1999, 4:511-518.
The authors demonstrate that Brca1–/– mouse cells are selectively deficient
in homologous recombination measured by targeted integration of transfected DNA as well as by DSB repair by homologous recombination.
3.
4.
•
Jimenez GS, Bryntesson F, Torres-Arzayus MI, Priestly A, Beeche M,
Saito S, Sakaguchi K, Appella E, Jeggo PA, Taccioli GE et al.: DNAdependent protein kinase is not required for the p53-dependent
response to DNA damage. Nature 1999, 400:81-83.
The defective p53 response in the DNA-PK deficient SCGR11 cell line —
which was considered to reflect the interdependence of p53 and DNA-PK —
is here shown to be a consequence of a p53 mutation. In cells expressing a
wild-type p53, p53 activation is demonstrated in the absence of a functional DNA-PK. This is in agreement with several other lines of evidence which
indicate that p53-related checkpoint functions are activated independently
of DNA-PK.
5.
•
Modesti M, Hesse JE, Gellert M: DNA binding of Xrcc4 protein is
associated with V(D)J recombination but not with stimulation of
DNA ligase IV. EMBO J 1999, 18:2008-2018.
XRCC4 is shown to exhibit cooperative binding to DNA. This stimulates the
adenylation of DNA ligase IV. The phosphorylated form of XRCC4 is shown
to lack DNA-binding activity.
6.
•
Singleton BK, Torres-Arzayus MI, Rottinghaus ST, Taccioli GE, Jeggo PA:
The C terminus of Ku80 activates the DNA-dependent protein
kinase catalytic subunit. Mol Cell Biol 1999, 19:3267-3277.
Analysis of mutations in the hamster Ku80 identifies a carboxy-terminal 178
amino acid region as being essential for stimulation of DNA-PK. This region
is distinct from the one which interacts with Ku70.
7.
•
Leuther KK, Hammarstan O, Kornberg RD, Chu G: Structure of DNA
dependent protein kinase: implications for its regulation by DNA.
EMBO J 1999, 18:1114-1123.
Analysis of DNA-PKcs crystallised on a lipid layer reveals an intriguing threedimensional structure. On the basis of its affinities for different DNA structures, a model for how DNA-PK might operate in DSB rejoining is proposed.
8.
A related issue concerns the selection of repair pathway. Is
there simply a direct competition between the Ku
Smith GSM, Jackson SP: The DNA-dependent protein kinase.
Genes Dev 1999, 13:916-934.
Frank KM, Sekiguchi JM, Seidl KJ, Swat W, Rathburn GA, Cheng HL,
Davidson L, Kangaloo L, Alt FW: Late embryonic lethality and
impaired V(D)J recombination in mice lacking DNA ligase IV.
Nature 1998, 396:173-177.
GDA213.QXD
03/22/2000
02:27
Page 149
DNA double strand break repair in mammalian cells Karran
9.
Barnes DE, Stamp G, Rosewell I, Denzel A, Lindahl T: Targeted
disruption of the gene encoding DNA ligase IV leads to lethality in
embryonic mice. Curr Biol 1998, 8:1395-1398.
10. Gao Y, Sun Y, Frank KM, Dikkes P, Fujiwara Y, Seidl KJ, Sekiguchi JM,
•
Rathburn GA, Swat W, Wang J et al.: A critical role for DNA endjoining proteins in both lymphomagenesis and neurogenesis. Cell
1998, 95:891-902.
References [6•,7•,8] together implicate mouse DNA ligase IV and XRCC4 in
an essential developmental function. Animals defective for either gene exhibit embryonic lethality with a characteristic impairment of central nervous system development as well as the anticipated defect in V(D)J recombination.
The inviability of these mice contrasts with the viability of Ku or DNA-PKcs
mutant mice which strongly suggests an additional role for DNA
ligase IV/XRCC4.
11. Riballo E, Crichlow SE, Teo S-H, Doherty AJ, Priestly A, Broughton B,
•
Kysela B, Beamish H, Plowman N, Arlett CF et al.: Identification of a
defect in DNA ligase IV in a radiosensitive leukaemia patient. Curr
Biol 1999, 9:699-702.
A human cell line from a radiation-sensitive leukaemia patient is shown to
have a mutated DNA ligase IV with a severely limited ability to undergo
adenylation. Yeast cells expressing the mutated ligase are extremely sensitive to ionizing radiation and deficient in NHEJ in vitro.
12. Baumann P, West SC: Role of the human RAD51 protein in
homologous recombination and double-stranded break repair.
Trends Biochem Sci 1998, 23:247-251.
13. Lee SE, Mitchell RA, Cheng A, Hendrickson EA: Evidence for DNA
•
PK-dependent and -independent DNA double-strand break repair
pathways in mammalian cells as a function of the cell cycle. Mol
Cell Biol 1997, 17:1425-1433.
DNA-PK activity is here shown to be maximal in the late G1/early S and
G2 phases of the cell cycle. The radiation-sensitivity and DSB rejoining defect
of mouse scid cells is apparent only in the G1/early S phase. The absence of
DNA-PK in the G2 phase does not affect radiation sensitivity or rejoining.
14. New JH, Sugiyama T, Zaitseva E, Kowalczylkowski SC: Rad52 protein
stimulates DNA strand exchange by Rad51 and replication protein A.
Nature 1998, 391:407-409.
15. Benson FE, Baumann P, West SC: Synergistic actions of Rad51
and Rad52 in recombination and DNA repair. Nature 1998,
391:401-404.
16. Van Dyck E, Stasiak AZ, Stasiak A, West SC: Binding of double
•
strand breaks in DNA by human Rad52 protein. Nature 1999,
398:728-731.
Human Rad52 protein is shown to be an alternative DNA end binding activity to Ku. By analogy to Ku/DNA-PK in NHEJ, it is proposed that end binding by hRad52 initiates the HR repair pathways.
149
chromosome stability and protect against DNA cross-links and
other damages. Mol Cell 1998, 1:783-793.
The cloning of these two important RAD51 family members by complementing the extreme mitomycin C sensitivity of deficient hamster cells is described.
The interaction between XRCC3/mRad51 is directly demonstrated.
22. Pierce AJ, Johnson RD, Thompson LH, Jasin M: XRCC3 promotes
•
homology-directed repair of DNA damage in mammalian cells.
Genes Dev 1999, 13:2633-2638.
The authors provide direct evidence for the participation of XRCC3 in
homologous recombinational repair of DSBs.
23. Callebaut I, Mornon J-P: From BRCA1 to RAP1: a widespread BRCT
module closely associated with DNA repair. FEBS Lett 1997,
400:25-30.
24. Gowen LC, Avrutskaya AA, Latour AM, Koller BH, Leadon SA: BRCA1
•
required for transcription-coupled repair of oxidative DNA
damage. Science 1998, 281:1009-1012.
Brca1–/– mouse ES cells shown to be more sensitive to H2O2 than –/+heterozygous or +/+wild-type cells and to be deficient in the selective removal of
oxidised thymine bases (thymine glycol) from the transcribed DNA strand.
25. Zhong Q, Chen C-F, Li S, Chen Y, Wang C-C, Xiao J, Chen P-L,
•
Sharp ZD, Lee W-H: Association of BRCA1 with the hRad50hMre11-p95 complex and the DNA damage response. Science
1999, 285:747-750.
Human Rad50 is shown to complex with Nibrin and hMre11. hRad50 focus
formation is reduced in a human breast carcinoma cell line with homozygous
mutation in Brca1. Focus formation is restored by a transfected wild-type
Brca1. Correction of the (slight) MMS sensitivity is also achieved by expressing a wild-type Brca1.
26. Cortez D, Wang Y, Qin J, Elledge SJ: Requirement of ATM
•
dependent phosphorylation of Brca-1 in the DNA damage
response to double-strand breaks. Science 1999, 286:1162-1166.
The authors provide a first indication that Brca1 might act downstream in the
signalling pathway initiated by ATM. The phosphorylation of Brca1 which
normally follows ionising radiation is not seen in A-T cells. Biochemical and
mutational analyses confirm that Brca1 is a substrate for phosphorylation by
ATM. There is some indication that this is related to the slight ionising radiation sensitivity of Brca1-defective cells. The γ-ray sensitivity of Brca1–/– cells
is, however, much less pronounced than A-T cells, indicating that Brca1 is
likely to be only one of the significant targets of ATM.
27.
Chen J, Silver DP, Walpita D, Cantor SB, Gazdar AF, Tomlinson G,
Couch FJ, Weber BL, Ashley T, Livingston DM, Scully R: Stable
interaction between the products of the BRCA1 and BRCA2
tumour suppressor genes in mitotic and meiotic cells. Mol Cell
1998, 2:317-328.
17.
•
28. Patel KJ, Yu VPCC, Lee H, Corcoran A, Thistlethwaite FC, Evans MJ,
•
Colledge WH, Friedman LS, Ponder BAJ, Venkitaraman AR:
Involvement of Brca2 in DNA repair. Mol Cell 1998, 1:347-357.
The authors report hypersensitivity to various DNA-damaging agents and
increased rates of spontaneous chromosome aberrations in mBrca2–/–
embryonic fibroblasts. Interestingly, this DNA-repair defect is associated
with apparently normal cell cycle checkpoint controls.
18. Rijkers T, van den Ouweland J, Morolli B, Rolink AG, Baarends WM,
•
van Sloun PP, Lohman PH, Pastink A: Targeted inactivation of
mouse RAD52 reduces homologous recombination but not
resistance to ionizing radiation. Mol Cell Biol 1998, 18:6423-6429.
mRad52–/– embryonic stem cells appear to be insensitive to DSB-inducing
agents. The same cells exhibit reduced homologous recombination but the
mice are viable, fertile and immunocompetent. This is in striking contrast to
the properties of mRad51–/– cells and mice.
29. Cressman VL, Backlund DC, Avrutskaya AV, Leadon SA, Godfrey V,
•
Koller BH: Growth retardation, DNA repair defects, and lack of
spermatogenesis in BRCA1-deficient mice. Mol Cell Biol 1999,
19:7061-7075.
An investigation of the relationship between mBrca1 and p53. The embryonic lethality of the mBrca1 defect is partially alleviated by a p53 defect.
Parallel observations are reported in cultured fibroblasts in which the poor
proliferation of mBrca1–/– cells is reversed by selection for an abrogated
p53 response. This is another example of p53 controlling the survival of cells
with unresolved DSBs.
Lim DS, Hasty P: A mutation in mouse rad51 results in an early
embryonic lethal that is suppressed by a mutation in p53. Mol
Cell Biol 1996, 16:7133-7143.
A report of embryonic lethality of mRad51–/– mice and radiation sensitivity in
embryonic cells. This also appears to be the first indication that the cytotoxic effects of impaired DSB rejoining may be mediated by p53.
19. Essers J, Hendricks RW, Swagemakers SM, Troelstra C, de Wit J,
•
Bootsma D, Hoeijmakers JH, Kanaar R: Disruption of mouse RAD54
reduces ionizing radiation resistance and homologous
recombination. Cell 1997, 89:194-204.
Inactivation of the third RAD51-related mouse gene produces a phenotype
intermediate between that of mRad51–/– and mRad52–/– mice. Like their
mRad51 counterparts, mRad54–/– cells exhibit reduced homologous recombination and are sensitive to DSB-inducing agents. In contrast, but as with
mRad52–/– mice, mRad54–/– animals are viable.
20. Tan TLR, Essers J, Citterio C, Swagemakers SMA, de Wit J, Benson FE,
•
Hoeijmakers JHJ, Kanar R: Mouse Rad54 affects DNA conformation
and DNA-damage-induced Rad51 foci formation. Curr Biol 1999,
9:325-328.
mRad54 is here shown to interact with mRad51 and to be required for formation of DNA damage-induced mRad51 foci.
21. Liu N, Lamerdin JE, Tebbs RS, Schild D, Tucker JD, Shen MR,
•
Brookman KW, Siciliano MJ, Walter CA, Fan W et al.: XRCC2 and
XRCC3, new human Rad51-family members, promote
30. Digweed M, Reis A, Sperling K: Nijmegen Breakage Syndrome:
consequences of defective DNA double strand break repair.
Bioessays 1999, 21:649-656.
31. Stewart GS, Maser RS, Stankovic T, Bressan DA, Kaplan MI,
•
Jaspers NGJ, Raams A, Byrd PJ, Petrini JHJ, Taylor AMR: The DNA
double-strand break repair gene hMRE11 is mutated in
individuals with an Ataxia-Telangiectasia-like disorder. Cell 1999,
99:577-587.
Two families diagnosed as A-T are shown to have truncating or missense
mutations in hMRE11. Skin fibroblasts from affected individuals exhibit ionising radiation sensitivity and levels of radiation-resistant DNA synthesis
comparable to A-T cells. As with A-T and NBS cells, no overt defect in DSB
rejoining is observed. hMRE11/Rad50/NBS focus formation is absent in
hMRE11 mutant cells which are also defective in JNK activation. The p53
response to ionising radiation serves to distinguish hMRE11 mutant cells
from A-T cells. Because of the similarity of clinical phenotypes, the authors
suggest that up to 6% of diagnosed A-T patients may actually harbour
hMRE11 mutations.
GDA213.QXD
03/22/2000
150
02:27
Page 150
Chromosomes and expression mechanisms
32. Luo G, Yao MS, Bender CF, Mills M, Bladl AR, Bradley A, Petrini JHJ:
•
Disruption of mRad50 causes embryonic stem cell lethality,
abnormal embryonic development, and sensitivity to ionising
radiation. Proc Natl Acad Sci USA 1999, 96:7376-7381.
Inactivation of mRad50 is lethal in both early embryos and embryonic stem
cells. Irradiation of blastocyst explants indicates a substantial sensitivity to
ionising radiation in the mRad50–/– cells.
33. Yamaguchi-Iwai Y, Sonoda E, Sasaki MS, Morrison C, Haraguchi T,
•
Hiraoka Y, Yamashita YM, Yagi T, Takata M, Price C et al.: Mre11 is
essential for the maintenance of chromosomal DNA in vertebrate
cells. EMBO J 1999, 18:6619-6629.
A cellular phenotype closely similar to that of mRad50–/– mouse cells is seen
in chicken cells in which MRE11 is conditionally inactivated. The absence of
Mre11 causes proliferative arrest and an increase in spontaneous and radiation-induced chromosome aberrations. The effects are more apparent when
combined with a Ku defect, indicating that the NHEJ and SSA HR pathways
process DSBs independently.
34. Dong Z, Zhong Q, Chen P-L: The Nijmegen Breakage Syndrome
•
protein is essential for Mre11 phosphorylation upon DNA
damage. J Biol Chem 1999, 274:19513-19516.
Mre11 phosphorylation in response to different DNA-damaging agents is
absent in NBS cells. Phosphorylation precedes DNA-damage-dependent
Mre11 focus formation, suggesting that a phosphorylated form of Mre11
may be required for participation in DSB repair.
35. Carney JP, Maser RS, Olivares H, Davis EM, le Beau M, Yates JR,
•
Hays L, Morgan WF, Petrini JH: The hMre11/hRad50 protein
complex and Nijmegen breakage syndrome: linkage of doublestrand break repair to the cellular DNA damage response. Cell
1998, 93:477-486.
The key identification of Nbs1 as p95 and confirmation that Nbs forms a
complex with hRad50 and Mre11 which localises to radiation-induced foci
in normal, but not NBS, cells. The report suggests a DNA-damage-sensing
role for the complex.
36. Paull TT, Gellert M: Nbs1 potentiates ATP-driven DNA unwinding
•
and endonuclease cleavage by the Mre11/Rad50 complex. Genes
Dev 1999, 13:1276-1288.
Analysis of the biochemical properties of a purified recombinant human
Rad50/Mre11/Nbs complex indicates that Nbs confers several additional
functions on the hRad50/Mre11 heterodimer. The authors also suggest that
the tripartite complex may serve a DNA-damage recognition function which
may control how DSBs are processed. This decision would be determined
partly by the ability of the complex to sense differences in the type of DNA
ends present.
37.
Paques F, Haber JE: Two pathways for removal of nonhomologous DNA ends during double-strand break repair in
Saccharomyces cerevisiae. Mol Cell Biol 1997, 17:6765-6771.
38. Sugawara N, Paques F, Colaiacovo M, Haber JE: Role of
•
Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in
double-strand break-induced recombination. Proc Natl Acad Sci
USA 1997, 94:9214-9219.
Evidence is presented that the MSH2 and MSH3 mismatch repair proteins
may participate in the removal of single-stranded DNA tails during DSB
repair by SSA.
39. O’Driscoll M, Humbert O, Karran P: DNA mismatch repair. Nucleic
Acids Mol Biol 1998, 12:173-197.
40. Studamire B, Price G, Sugawara N, Haber JE, Alani E: Separation-offunction mutations in Saccharomyces cervisiae MSH2 that confer
mismatch repair defects but do not affect nonhomologous-tail
removal during recombination. Mol Cell Biol 1999, 19:7558-7567.
41. Shafman T, Khannah KK, Kedar P, Spring K, Kozlov S, Yen T, Hobson
K, Gatei M, Zhang N, Watters D et al.: Interaction between ATM
protein and c-Abl in response to DNA damage. Nature 1997,
387:520-523.
42. Chen G, Yuan S-SF, Liu W, Xu Y, Trujillo K, Song B, Cong F, Goff SP,
•
Wu Y, Arlinghaus R et al.: Radiation-induced assembly of Rad51
and Rad52 recombination complex requires ATM and c-Abl. J Biol
Chem 1999, 274:12748-12752.
The authors demonstrate a radiation-induced ATM and c-Abl dependent
phosphorylation of Rad51 which enhances its interaction with Rad52.
43. Haber JE: DNA recombination the replication connection. Trends
Biochem Sci 1999, 224:271-275.