Homologous chromosomes move and rapidly initiate contact at the sites of double-strand breaks in genes in G?-phase human cells.

2013·0.94 MB·English

by  Gandhi, Manoj

Extra ViEws Extra ViEw
Cell Cycle 12:4, 547–552; February 15, 2013; © 2013 Landes Bioscience
Homologous chromosomes move and rapidly initiate contact
at the sites of double-strand breaks in genes in G -phase human cells
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Manoj Gandhi,1,† Viktoria N. Evdokimova,1,† Karen T. Cuenco,2 Christopher J. Bakkenist3 and Yuri E. Nikiforov1,*
1Department of Pathology and Laboratory Medicine; University of Pittsburgh; Pittsburgh, PA USA; 2Department of Oral Biology; School of Dental Medicine;
Department of Human Genetics; Graduate School of Public Health; University of Pittsburgh; Pittsburgh, PA USA; 3Departments of Radiation Oncology and
Pharmacology and Chemical Biology; University of Pittsburgh; Pittsburgh, PA USA
†These authors contributed equally to this work.
We recently reported that homolo- the restriction endonuclease I-PpoI in
gous chromosomes make con- G /G-phase human cells.1 Using multi-
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tact at the sites of double-strand breaks color 3D-fluorescence in situ hybridiza-
(DSBs) induced by ionizing radiation tion (FISH) with chromosome arm paints
(IR) and the restriction endonuclease or gene-specific probes and high-resolu-
I-PpoI in G /G-phase somatic human tion confocal microscopy, we identified
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cells. The contact involves short seg- frequent contact between homologous
ments of homologous chromosomes and arms or homologous genes residing on
is centered on a DSB that occurs in a several different chromosomes follow-
gene; contact does not occur at a DSB ing the induction of DSBs. In contrast
in intergenic DNA. Contact between to homologous chromosome pairing that
homologous chromosomes is abro- occurs in meiosis, which involves multiple
gated by inhibition of transcription and interstitial interactions along the entire
requires the kinase activity of ATM, but length of homologs,2 the observed contact
not DNA-PK. Here, we report additional in somatic cells involves a very limited
insights into the mechanism underlying section of the chromosome surrounding
this novel phenomenon. We identify four the site of DSB.1 This phenomenon was
patterns of homologous chromosome identified in both primary epithelial thy-
contact, and show that contact between roid cells and fibroblasts suggesting that
homologous arms, but not centrosomes, it likely to be a common event in human
is induced by IR. Significantly, we dem- somatic cells.
onstrate that contact is induced by IR in We further showed that DSB-induced
non-proliferating, G -phase human cells contact between homologous chromo-
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derived from tissue explants. Finally, we somes was restricted to genes and required
show that contact between homologous active transcription and the kinase activity
chromosomes is detectable as early as 5 of ATM.1 Not all regions of homologous
min after IR. These results point to the chromosomes made contact following the
existence of a mechanism that rapidly induction of a DSB. While the induc-
Keywords: homologous chromosome localizes homologous chromosome arms tion of DSBs in genes using the restric-
contact, DNA repair, DNA breaks, at sites of DSBs in genes in G -phase tion endonuclease I-PpoI caused contact
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homologous recombination, nuclear human cells. between short segments of homologous
architecture, cell cycle chromosomes surrounding the restric-
Submitted: 12/29/12 Introduction tion site, the induction of DSBs in inter-
genic DNA regions did not cause contact
Accepted: 01/23/13
We recently reported that homolo- between homologous chromosomes.
http://dx.doi.org/10.4161/cc.23754 gous chromosomes make contact at the I-PpoI-induced contact between homolo-
sites of double-strand breaks (DSBs) gous chromosomes was abrogated when
*Correspondence to: Yuri E. Nikiforov;
Email: [email protected] induced by ionizing radiation (IR) or RNA polymerase was inhibited with
www.landesbioscience.com Cell Cycle 547
centromeres, is frequently induced by IR.
Second, we show that contact between
homologous chromosomes is induced by
IR in non-proliferating, G -phase human
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cells derived from tissue explants. Third,
we show that contact between homologous
chromosomes is detectable as early as 5
min after IR.
Results
Four patterns of homologous chromo-
some contact are observed in untreated
and irradiated cells. Multicolor 3D-FISH
and confocal microscopy identified four
patterns of homologous chromosome
contact in G /G-phase cells: (1) contact
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between two homologous p arms; (2) con-
tact between two homologous q arms; (3)
contact between homologous p arms and
q arms, i.e., p:p and q:q contact; and (4)
contact between homologous centromeres
(Fig. 1). Contact between centromeres
was observed far less frequently than con-
tacts between homologous chromosome
arms. In contacts involving a single arm
of each homologous chromosome (either
p-arm or q-arm contact), significant sepa-
ration was typically observed between the
centromeres, and the opposite arms fre-
quently extended away from each other.
Contact between both arms (p:p and q:q
contact) presented in three recurrent con-
formations: parallel arrangement of both
homologs, cross-shape conformation and
doughnut-shape conformation (Fig. 1).
The parallel arrangement resulted in the
most extended contact between homolo-
gous chromosome territories, allowing for
interactions between multiple reciprocal
regions scattered along both p and q arms.
Figure 1. Patterns of contact between homologous chromosomes in G/G human cells. 3D-FisH The cross-shape pattern involved interac-
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of cultured human thyroid cells (Ht) and human fibroblasts (HF) with p-arm paint (green), q-arm tion between the pericentromeric regions
paint (red) and centromeric probe (light blue). Left panel is a schematic representation of each
of both arms, while the telomeric parts
pattern of contact. ir, ionizing radiation; Chr, chromosome.
were repelled from each other. In con-
trast, the doughnut-shape pattern led to
either actinomycin D or α-amanitin at all kinase-dependent mechanism that induces the interaction of the telomeric regions of
five gene loci studied. Finally, we deter- contact between allelic regions of homol- the homologous p- and q-arms, while the
mined that contact required the kinase ogous chromosomes at sites of DSBs in remaining reciprocal regions of the homol-
activity of ATM but not DNA-PK, which human somatic cells. ogous chromosome were located on the
is consistent with an associated homolo- Here, we report additional insights largest possible separation from each other.
gous recombination repair rather than into the mechanism that leads to contact These patterns of contact were observed
non-homologous end-joining (NHEJ) between homologous chromosomes in with different frequencies in untreated and
mechanism of DSB repair. These find- G /G-phase human cells. First, we show irradiated cells (Table 1). In untreated
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ings provided the first documentation of that contact between either the q-arms cells, contact between single arms (p-arms
a common transcription-related and ATM or both the p- and q-arms, but not the or q-arms) was almost twice as frequent as
548 Cell Cycle Volume 12 issue 4
contact between both arms of homologous
chromosomes. Exposure to IR induced
contact between both chromosome arms
more frequently than contact between sin-
gle arms. Indeed, in thyroid cells, exposure
to 5 Gy IR increased contact between both
chromosome arms 2.9-fold and contact
between single arms 1.4-fold (p < 10−7). In
fibroblasts, IR increased contact between
both chromosome arms 2.2-fold and con-
tact between single arms 1.5-fold (p = 0.02).
As a result, in cells exposed to IR, contact
between both arms of homologous chro-
mosomes became the most common pat-
tern of contact (Table 1). Centromere-only
contact and centromere co-localization in
all other patterns of contact was rarely
seen in untreated and irradiated cells, sug-
gesting that contact between homologous
chromosomes does not require for interac-
tion of centromeres.
Contact between homologous chro-
mosomes is observed in non-proliferat-
ing, G -phase human cells. In our initial
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study, contact between homologous chro-
mosomes was observed in primary cul-
tured human cells that were largely in
G /G-phase.1 The occurrence of con-
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tact in G /G-phase cells was confirmed
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by a lack of immunoreactivity for PCNA
(a marker of S-phase cells) and cyclin A Figure 2. Homologous chromosome contact in G-phase cells. (A) Formation of γH2ax foci and
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(a marker of S- and G -phase cells).1 53BP1 nuclear foci in cells from human thyroid tissue explants 15 min after exposure to ionizing
2 radiation (ir) as detected by immunofluorescence. (B) 3D-FisH on ex-vivo cells showing contact
Here, we confirm and expand this
between chromosome 3 homologs in two of the three nuclei (arrows). the middle nucleus shows
finding by showing that contact between
a parallel-shape contact of both arms, while the lower nucleus shows a doughnut-shape contact
homologous chromosomes occurs in of both arms. (C) Frequency of contact between homologous chromosomes in cell from human
G -phase human cells from thyroid tis- thyroid tissue explants before and 15 min after ir. results of the analysis of two chromosomes
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sue explants. Adult human thyroid cells (chr. 3 and chr. 11) are combined. statistically significant difference between untreated and ir-
treated cells is denoted with an asterisk.
are known to have an exceedingly low
proliferation rate (≤ 0.1%) and are, there-
fore, among the most enriched G -phase Table 1. Frequency of various types of arm-specific contact between homologous chromosomes
population of human epithelial ce0lls that in untreated primary human cells and 15 min after exposure to ionizing radiation (ir)
can be obtained.3 The tissue explants Thyroid epithelial cells Fibroblasts
used in this study were obtained directly IR− IR+ IR− IR+
from surgically removed normal tissue
p-arm contact 5.39% 5.38% 3.42% 3.31%
fragments adjacent to a benign thyroid
q-arm contact 6.01% 11.36% 8.59% 11.73%
nodule and had a Ki-67 labeling index of
both arm contact 7.23% 21.48% 8.78% 15.07%
0.09 ± 0.03%, confirming that they were
G -phase cells. To confirm that the cells in centromere contact 0.3% 0.1% 0.2% 0.3%
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these tissue explants were viable and could
induce a DNA damage response after IR, identified in these ex-vivo cells, and the This finding conclusively demonstrates
we performed immunofluorescence for frequency of contact was increased after that homologous chromosome contact
γH2AX and 53BP1.4 Robust formation of IR (Fig. 2B and C). Exposure to 5 Gy IR occurs in G -phase cells in human tissues
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both γH2AX and 53BP1 foci was observed increased contact between both chromo- and is not a phenomenon associated with
15 min after IR (Fig. 2A). Using 3D-FISH some arms 2.4-fold and contact between cell propagation in culture.
and confocal microscopy, contact between single arms 1.8-fold, similar to the levels of Contact between homologous chro-
homologous chromosome arms was readily increase observed in primary cell cultures. mosomes is detectable as early as 5 min
www.landesbioscience.com Cell Cycle 549
Figure 3. Kinetics of homologous chromosome contact after ir. (A) FisH on primary cultures of human thyroid cells showing location of centromeres
for chromosomes 3, 10 and 17 labeled in red, green and light blue, respectively. Untreated cells (ir−) have random positioning of centromeres of
homologous chromosomes. after exposure to ionizing radiation (ir+), two cells (arrows) show at least two sets of centromeres arranged in a paired
fashion and separated by ≤ 3.25 μm. (B) Change in the proportion of nuclei with at least two of homologous chromosome centromeres paired and
separated by ≤ 3.25 μm. (C) P values for comparison of cumulative frequency distributions of distances between centromeres of homologous chro-
mosomes at different time points after ir as compared with untreated cells assessed by the two-sample Kolmogorov-smirnov test. P values that are
significant are shown in bold font. (D) Frequency of arm-specific contact between chromosome 3 homologs in untreated cells and 5 min after ir.
after IR. We investigated the kinetics of is the mean distance between homologous reduced for all three chromosomes at
homologous chromosome interaction by centromeres in those nuclei where contact 5 min, 30 min, 8 h and 24 h after IR
measuring the distances between the cen- between homologous chromosome arms (p values are shown in Fig. 3B).
tromeres of chromosomes 3, 10 and 17 was observed. The proportion of nuclei To confirm that the reduced distances
at various time intervals after 5 Gy IR. in which at least two pairs of the homolo- between centromeres observed 5 min after
The approach was based on the assump- gous centromeres were within ≤ 3.25 μm IR corresponded to the increased contact
tion that in order to interact at the limited increased 2.1-fold at 5 min after IR and between homologous chromosomes, we
sub-chromosomal regions, homologous fluctuated over the following 24 h in a directly analyzed the frequency of arm-
chromosomes must travel toward each bimodal manner before returning to the specific contact 5 min after cell irradiation.
other, which should result in the reduc- background level by 48–72 h (Fig. 3B). Using FISH with arm-specific paints for
tion of distances between homologous Similar findings were obtained by analyz- chromosome 3, we observed a significant
centromeres within the nucleus. This was ing the change in the entire distribution of increase in arm-specific contact 5 min after
indeed observed after IR as the increase distances between centromeres of each of IR (Fig. 3D).
in the proportion of nuclei, in which at the three homologous chromosomes mea-
least two of the three pairs of homolo- sured in 200 nuclei in two experimental Discussion
gous centromeres were compartmental- repeats. This analysis revealed that dis-
ized and separated by a distance of ≤ 3.25 tances between centromeres of homolo- Taken together, our recent studies,1
μm (Fig. 3A). The distance of 3.25 μm gous chromosomes were significantly and the observations presented here,
550 Cell Cycle Volume 12 issue 4
provide the first demonstration that con- so-called targeted recombination events performed using cells/tissues from two
tact between homologous chromosomal are more frequent when the chromosomal different donors.
regions is induced by DSBs in genes in target sustains a DSB.12,13 Although the Cell fixation, fluorescence in situ
human G - and G-phase cells. While our molecular mechanisms responsible for the hybridization (FISH), immunofluores-
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findings do not demonstrate that the con- homology search remain unknown, it is cence (IF) and immunohistochemistry
tact is involved in DSB repair, the fact that reasonable to propose that factors that gov- (IHC). For 3D analysis, primary cultures
contact is centered on a DSB and requires ern gene targeting also play a role in break- of cells were prepared as described before.1
ATM kinase activity raises this possibil- induced chromosome contact. Based on For 2D analysis, cells on coverslips were
ity. It is well established that DSBs are our data and findings reported by others, fixed by standard methanol-acetic acid
repaired via two principal repair pathways, it is likely that the mechanisms of homol- (MAA) treatment without hypotonic
non-homologous end-joining (NHEJ) ogy recognition are transcription-based. solution. For tissue explants, touch prepa-
and homologous recombination (HR).5,6 Indeed, recruitment of some DNA repair rations were made on positively charged
NHEJ seals the break by re-joining free factors such as 53BP1 to the sites of DNA slides and subjected to 3D-fixation.1
DNA ends after minimal processing and damage has been previously shown to Pre-labeled centromeric enumeration
can result in the deletion or insertion of one require direct interaction with RNA mol- probes were also purchased from Abbott
or more nucleotides at the site of joining. ecules,14 and homologous chromosomes laboratories. Chromosome arm paints
HR requires a homologous DNA sequence pairing in meiosis may involve RNA:RNA were obtained from Metasystems. Slide
that is copied for the error-free restoration base pairing.15 Therefore, there is at least pretreatment and hybridization was per-
of the broken DNA sequence. In verte- a theoretical possibility that sequence- formed as previously described.16 For
brates, NHEJ and HR are believed to be specific RNA molecules may be involved γH2AX and 53BP1 IF, cells were fixed
associated with specific phases of the cell in both the search for homology and the in 4% paraformaldehyde and permea-
cycle. NHEJ is believed to be the primary co-localization of homologous gene loci in bilized with 0.5% Triton X-100. Anti-
mechanism of DSB repair in G /G-phase somatic cells. Our results presented here γH2AX antibody (#05-636, Upstate
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cells, when no obvious homologous tem- suggest that although the co-localization Biotechnology) and anti-53BP1 antibody
plate is available, whereas HR is thought of homologous chromosomes at the sites (#ab21083, Abcam) were used. For Ki-67
to be the primary mechanism of DSB of breaks involves the movement of the proliferation index, frozen sections of tis-
repair during the late-S- and G -phases, entire chromosome territories, it does not sue explants were obtained, stained with
2
when a sister chromatid may be used as require physical contact between homolo- Ki-67 antibody and scored as described
a template for repair.6-8 Our observations gous centromeres and therefore may be elsewhere.3
allow for several additional possibilities. mediated by factors in the vicinity of the Image acquisition and analysis.
First, in G - and G-phase cells, a homolo- DSB. Confocal microscopy was performed
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gous chromosome may serve as a template While the molecular mechanisms using a Leica SP5 TCS 4D confocal laser
for DSB repair. Second, NHEJ and HR through which homologous chromosomes scanning fluorescence microscope and
may be differentially associated with the interact remain to be discovered, our data image stacks were reconstructed using the
repair of DSBs located in different DNA provide the first documentation of an Volocity software as previously described.1
regions, i.e., intergenic regions and genes. ATM kinase-dependent mechanism that The analysis of spatial contact between
In G /G-phase cells, HR mediated by rapidly induces contact between homolo- chromosome arms was performed using
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contact between homologous chromo- gous chromosomes at the sites of DSBs the intensity-based image segmentation
somes would allow error-free repair of the in genes that are being transcribed G / technique.17 3D measurement of distances
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relatively small number of DSBs that arise G-phase human cells. We suggest that between centromeric probe pairs was
1
in genes, which could be a highly signifi- such mechanisms may be significant for performed by selecting the image grav-
cant mechanism that prevents mutations genome stability and human health. ity centers corresponding to the signal by
in coding regions of DNA that are critical navigating through the image stack. For
for cell viability and function. Materials and Methods the 2D analysis of MAA-fixed cells, the
Our results clearly show that machinery maximum intensity projection image was
capable of identifying homologous chro- Primary cell cultures, tissue explants and used to obtain the distance measurements
mosomes regions and co-localizing them irradiation. Primary cultures of human between centromere signals.
within the G /G-phase nucleus exists in thyroid cells were established as previ- Analysis of kinetics using CFDs and
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human somatic cells. Mammalian cells are ously described.1 For tissue explants, the estimation of contact frequency in 2D.
known to have the ability to recombine a freshly excised normal human thyroid tis- Distances between centromeres of homol-
chromosomal locus with a homologous sues adjacent to a benign thyroid nodule ogous chromosomes were measured in 2D
DNA molecule that is introduced into were collected in DMEM, cut into 5 mm in two individuals (200 cells each) and
the nucleus,9-11 indicating that cells have fragments and assayed either untreated combined for the analysis. Cumulative fre-
a mechanism that uses sequence homol- or after exposure to 5 Gy of γ-irradiation quency distributions (CFDs) of distances
ogy to bring otherwise distant DNA from a cesium-137 source at a dose rate of were built by plotting the values on the X
molecules into contact. In fact, these 301.66 cGy/min. Each experiment was axis and the proportions of all data points
www.landesbioscience.com Cell Cycle 551
4. Schultz LB, Chehab NH, Malikzay A, Halazonetis 12. Silva G, Poirot L, Galetto R, Smith J, Montoya
that were smaller than this measurement
TD. p53 binding protein 1 (53BP1) is an early G, Duchateau P, et al. Meganucleases and other
on the Y axis. CFDs were compared using participant in the cellular response to DNA dou- tools for targeted genome engineering: perspectives
ble-strand breaks. J Cell Biol 2000; 151:1381-90; and challenges for gene therapy. Curr Gene Ther
the two-sample Kolmogorov-Smirnov
PMID:11134068; http://dx.doi.org/10.1083/ 2011; 11:11-27; PMID:21182466; http://dx.doi.
test.18 A mean 2D distance between cen- jcb.151.7.1381 org/10.2174/156652311794520111
tromeres of homologous chromosomes 3, 5. Lieber MR, Ma Y, Pannicke U, Schwarz K. 13. Smih F, Rouet P, Romanienko PJ, Jasin M. Double-
Mechanism and regulation of human non-homol- strand breaks at the target locus stimulate gene
10 and 17 was calculated based on direct
ogous DNA end-joining. Nat Rev Mol Cell Biol targeting in embryonic stem cells. Nucleic Acids Res
measurements in those nuclei that showed 2003; 4:712-20; PMID:14506474; http://dx.doi. 1995; 23:5012-9; PMID:8559659; http://dx.doi.
arm-specific contact on 3D analysis and org/10.1038/nrm1202 org/10.1093/nar/23.24.5012
6. Sung P, Klein H. Mechanism of homologous recom- 14. Pryde F, Khalili S, Robertson K, Selfridge J, Ritchie
was found to be 3.25 μm.
bination: mediators and helicases take on regula- AM, Melton DW, et al. 53BP1 exchanges slowly
tory functions. Nat Rev Mol Cell Biol 2006; 7:739- at the sites of DNA damage and appears to require
Disclosure of Potential Conflicts of Interest 50; PMID:16926856; http://dx.doi.org/10.1038/ RNA for its association with chromatin. J Cell Sci
nrm2008 2005; 118:2043-55; PMID:15840649; http://dx.doi.
No potential conflicts of interest were 7. Huertas P. DNA resection in eukaryotes: decid- org/10.1242/jcs.02336
disclosed. ing how to fix the break. Nat Struct Mol Biol 15. Xu M, Cook PR. The role of specialized transcription
2010; 17:11-6; PMID:20051983; http://dx.doi. factories in chromosome pairing. Biochim Biophys
org/10.1038/nsmb.1710 Acta 2008; 1783:2155-60; PMID:18706455; http://
Acknowledgments 8. Lieber MR. The mechanism of double-strand DNA dx.doi.org/10.1016/j.bbamcr.2008.07.013
This work was supported by NIH break repair by the nonhomologous DNA end- 16. Gandhi M, Nikiforov YE. Suitability of animal mod-
joining pathway. Annu Rev Biochem 2010; 79:181- els for studying radiation-induced thyroid cancer in
grants R01 CA88041 (Y.E.N.) and R01 211; PMID:20192759; http://dx.doi.org/10.1146/ humans: evidence from nuclear architecture. Thyroid
CA148644 (C.J.B.). annurev.biochem.052308.093131 2011; 21:1331-7; PMID:22136268; http://dx.doi.
9. Doetschman T, Gregg RG, Maeda N, Hooper ML, org/10.1089/thy.2011.0269
Melton DW, Thompson S, et al. Targetted correc- 17. Weierich C, Brero A, Stein S, von Hase J, Cremer
References
tion of a mutant HPRT gene in mouse embryonic C, Cremer T, et al. Three-dimensional arrange-
1. Gandhi M, Evdokimova VN, T Cuenco K, stem cells. Nature 1987; 330:576-8; PMID:3683574; ments of centromeres and telomeres in nuclei of
Nikiforova MN, Kelly LM, Stringer JR, et al. http://dx.doi.org/10.1038/330576a0 human and murine lymphocytes. Chromosome Res
Homologous chromosomes make contact at the 10. Shaw-White JR, Denko N, Albers L, Doetschman 2003; 11:485-502; PMID:12971724; http://dx.doi.
sites of double-strand breaks in genes in somatic TC, Stringer JR. Expression of the lacZ gene tar- org/10.1023/A:1025016828544
G/G-phase human cells. Proc Natl Acad Sci USA geted to the HPRT locus in embryonic stem cells 18. Conover WJ. Practical Nonparametric Statistics New
0 1
2012; 109:9454-9; PMID:22645362; http://dx.doi. and their derivatives. Transgenic Res 1993; 2:1- York: John Wiley & Sons, 1971
org/10.1073/pnas.1205759109 13; PMID:8513334; http://dx.doi.org/10.1007/
2. Gerton JL, Hawley RS. Homologous chromosome BF01977675
interactions in meiosis: diversity amidst conservation. 11. Vasquez KM, Marburger K, Intody Z, Wilson JH.
Nat Rev Genet 2005; 6:477-87; PMID:15931171; Manipulating the mammalian genome by homol-
http://dx.doi.org/10.1038/nrg1614 ogous recombination. Proc Natl Acad Sci USA
3. Saad AG, Kumar S, Ron E, Lubin JH, Stanek J, 2001; 98:8403-10; PMID:11459982; http://dx.doi.
Bove KE, et al. Proliferative activity of human org/10.1073/pnas.111009698
thyroid cells in various age groups and its correla-
tion with the risk of thyroid cancer after radiation
exposure. J Clin Endocrinol Metab 2006; 91:2672-
7; PMID:16670159; http://dx.doi.org/10.1210/
jc.2006-0417
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