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Trends in Genetics,16:143-146, 2000

Radiation-Produced Chromosome Aberrations: Colourful Clues

Rainer K. Sachs [1], Lynn R. Hlatky [2], and Barbara J. Trask [3]

[1] Departments of Mathematics and of Physics
Evans Hall, MC 3840, University of California, Berkeley, CA 94720
Phones: 510-642-4384, 510-658-5790; Fax 510-642-8204
Email: sachs@math.berkeley.edu
URL: / sachs/

[2] Dana-Farber Cancer Institute
Department of Radiation Oncology, Harvard Medical School
330 Brookline Avenue, Boston, MA 02215
Email: hlatky@speedy.dfci.harvard.edu

[3] Department of Molecular Biotechnology
Box 357730, HSC K336B; 1705 NE Pacific Ave NE
University of Washington, Seattle WA 98195
Email: btrask@u.washington.edu

Number of words (excluding figure captions): 1650. Number of words in figure captions: 500.
Number of figures: 2. Tables: none. Boxes: none

Key Words: Ionizing Radiation; DNA Double Strand Break Repair/Misrepair; Chromosome Aberrations; FISH; Interphase Nuclear Organization; Large-Scale Chromatin Geometry.

Software used for text: AMS-Latex. Software used for figures: postscript.

Ionizing radiation produces chromosome aberrations prolifically. A brilliant variety of aberration types can now be seen with chromosome painting. Apart from being important in medicine and public health, radiation-produced aberrations act as colourful molecular clues to damage processing mechanisms and, because juxtaposition of different parts of the genome is involved, to interphase nuclear organization. Recent analyses using chromosome painting help identify DNA double strand break repair/misrepair pathways, determine the extent of chromosome territories and motions, and characterize different aberration patterns left behind by different kinds of radiation.

Irradiating cells during the G0/G1 phase of the cell cycle produces chromosome-type aberrations $^{\ref{r:cor98}}$. DNA double strand breaks (DSBs) are formed and then, during the next hour or so, misrejoining induces a large-scale reshuffling of chromatin pieces (Fig. 1), usually scored at the metaphase following irradiation. Recently, the scope of aberration studies has increased dramatically, due to fluorescent in situ hybridization (FISH) chromosome painting $^{\ref{r:pin88}}$. A rich diversity of different aberration types is now seen (e.g. $^{\ref{r:sim99},\ref{r:kne99}}$).
The primary impetus for aberration studies has been the association of chromosomal structural changes with cancer (reviewed in $^{\ref{r:pop97}})$ and the importance of aberrations to the three main applications of radiobiology: radiotherapy; biodosimetry (in which aberrations of peripheral blood lymphocytes are used to estimate how much radiation dose an individual received); and carcinogenesis risk estimation $^{\ref{r:cor98},\ref{r:edw97}}$. However, as will now be outlined, aberrations also give quantitative insights into DNA damage processing and into the arrangement of chromosomes within the cell nucleus.

Randomness.
A long-standing question is whether radiation induction of DSBs, and subsequent DSB misrejoining, are approximately random when averages over long DNA stretches are taken $^{\ref{r:sav82}}$. Recent experimental evidence on human cells probably somewhat favors randomness, apart from the territory/proximity effects discussed below $^{\ref{r:kov94} - \ref{r:sac99}}$. For example larger chromosomes are involved in aberrations more frequently than are smaller ones, the frequency ratio being approximately that which is expected from the respective chromosome sizes (with moderate discrepancies $^{\ref{r:kne96}}$). There is no convincing evidence for pronounced hot spots as large as a chromosome arm or for systematic associations, prior to clonal selection, of particular chromosome pairs.

Molecular mechanisms of misrejoining.
Aberration formation is usually quantified using the venerable breakage-and-reunion model. According to current versions of this model $^{\ref{r:sac99},\ref{r:sav98}}$, an aberration is formed when free ends from two or more different radiation-induced DSBs interact (Fig. 1); the interaction is via non-homologous end-joining (reviewed in $^{\ref{r:kan98}}$); the two free ends of any one DSB are able to misrejoin at different sites in the genome (e.g. Figs. 1(g), 1(j), or 1(k)). It is the final exchange configurations (panels (b), (c), (e), (g), (j), or (k)) which are observed; the breakage-and-reunion scenario described in the figure caption remains conjectural.
Molecular evidence indicates that a different, homologous recombination, pathway also sometimes makes aberrations. The specific scenario proposed $^{\ref{r:sav98},\ref{r:kan98},\ref{r:tha99}}$ is that a single radiation-induced DSB interacts with an otherwise undamaged site on the genome having some (possibly quite limited) DNA sequence homology; both free ends of the radiation-induced DSB can misrejoin at this site, resulting in a simple chromosome aberration. Examples of the final exchange configurations that can be generated under this scenario are shown in Figs. 1(b), 1(c), and 1(e). These final exchange configurations are thus consistent both with the breakage-and-reunion scenario described in the caption to Fig. 1 and with the homologous recombination scenario.
However, in recent radiation experiments with human cells, complex aberration types where the two free ends of a single DSB appear to misrejoin independently at different sites (e.g one of the final exchange configurations shown in Figs. 1(g), 1(j), or 1(k)) are frequently seen. The high frequency is consistent with the breakage-and-reunion model and shows that recombinational repair with the two free ends of any one DSB constrained to misrejoin at the same site is not the dominant aberration formation pathway for complex aberrations $^{\ref{r:sac99}}$.

Chromosome geometry.

Producing an aberration involves bringing two or more different genomic locations together; consequently, aberration frequencies are influenced by, and indicative of, large-scale chromatin geometry and motion.

Chromosome territories and proximity effects. It is now known that, even during interphase, chromosomes are localized to territories $^{\ref{r:cre96}}$. That is, at any one instant a particular chromosome is largely confined within a fraction of the nucleus, although its chromatin fibre follows a tortuous, multiply folded, partially random path $^{\ref{r:van92},\ref{r:yok95},\ref{r:mar97}}$. There are certain systematic deviations from randomness in aberration formation due to ``proximity'' effects, related to chromosome territories. Proximity effects result from limited chromatin motion and the fact that only free ends from DSBs formed near to each other have time to find each other and interact before being restituted (that is, repaired with at worst some local changes in the DNA sequence).

Biasing of aberration production. Territory/proximity effects are reflected, for example, in a statistical bias against aberrations involving two different chromosomes compared to aberrations involving a single chromosome $^{\ref{r:sav73},\ref{r:sac97}}$. Fig. 2(a) shows the basic phenomenon in simplified form. Suppose chromosomes are localized to territories as indicated in the figure. Suppose, in addition, that DSB free ends can interact only over a limited range, i.e. there are proximity effects. The proximity effects are indicated schematically in the figure by the dotted line, implying that a DSB free end can interact only with the other free ends in its own half. For example the free end $u$ of DSB $uu'$ can restitute with $u'$ or misrejoin with $v, v', w,$ or $w'$, but not misrejoin with $x, x', z,$ or $z'$.
For this situation there will be a bias, relative to expectations based on randomness with all five DSBs capable of interacting, against forming a a translocation (Fig. 1(b)) or a dicentric (Fig. 1(c)), compared to forming a ring (Fig. 1(e)), i.e. a bias against 2-chromosome aberrations compared to 1-chromosome aberrations. Some reactions that can form dicentrics or translocations are allowed (e.g. the reaction $xz,x'z'$, forming a translocation or dicentric, with the other three DSBs restituting as $uu',vv',ww'$; or the reaction $vw, v'w', uu',xx',zz'$; etc.). But territory/proximity effects prohibit some dicentrics or translocations that could otherwise occur (e.g. $ux, u'x',vv',ww',zz'$; etc.). In this illustrative cartoon, territory/proximity effects do not affect any of the reactions that can form a ring (e.g. $uw,u'w',vv',xx',zz'$). Overall, the ratio of dicentrics and translocations to rings is smaller than it would be if territory/proximity effects were absent.
This schematic, qualitative argument can be made more realistic and quantified. Remarkably, Savage and Papworth $^{\ref{r:sav73}}$ were thereby able to infer chromosome localization to territories long before direct FISH observations were available. The phenomenon is now well established. For example, in human cells the ratio of rings containing a centromere to dicentrics, although less than 1, is not nearly as small as would be expected from randomness ignoring territory/proximity effects. Modeling and numerical measurements of this ratio indicate that at any one instant most of the DNA in any one chromosome is confined to less than about 10% of the nuclear volume $^{\ref{r:sac97},\ref{r:che97}}$.
Other aberration types also show a similar bias. For example, in Fig. 2, some 3-DSB reactions that involve two chromosomes are possible (e.g. $uv,wv',u'w',xx',zz'$, compare Fig. 1(j)), but analogous 3-DSB reactions involving three different chromosomes (Fig. 1(g)) are prevented by territory/proximity effects; a corresponding bias is observed experimentally $^{\ref{r:che97}}$. The general rule during G0/G1 appears to be that for two different reaction types, involving the same number of DSBs but different numbers of chromosomes, territory/proximity effects bias against the reaction type involving more chromosomes.

Implications for nuclear structure. More direct observations of chromosome geometry and chromosome motion (e.g. $^{\ref{r:cre96},\ref{r:yok95},\ref{r:mar97}}$) can be compared to estimates based on territory/proximity effects for aberrations. By and large the results match, e.g. for the approximate size of chromosome territories and for the limited extent of chromosome motion. There are some discrepancies; for example the aberration results favor substantial spaghetti-like intertwining of different chromosomes since interactions among multiple chromosomes are observed, while direct measurements suggest more distinct territories instead. Together, the aberration measurements and direct measurements lead to quantitative models for chromatin at the largest scales, up to the full length of a chromosome. The main models use polymer configurations, related to random walks, in which chromosome configurations are stochastic, being complicated and different for different nuclei, with comparatively very simple laws holding for the average geometric properties $^{\ref{r:van92},\ref{r:yok95},\ref{r:ost98},\ref{r:mun99}}$. There is evidence that chromatin may undergo constrained Brownian (i.e. diffusional) motion, where the average properties are also simpler and easier to characterize than the motion of any one chromosome at any one instant $^{\ref{r:mar97}}$.

Densely ionizing radiations.
Fig. 2(a) is appropriate for sparsely ionizing radiations, such as gamma rays, which typically make at most one DSB per radiation track and therefore make DSBs more or less uniformly, although stochastically, throughout the genome. There are also densely ionizing radiations, alpha particles and neutrons being the ones most important in practice, which behave differently; they can give additional information on damage processing and chromosome territories.
For example, a single alpha particle typically inflicts a number of DSBs concentrated near the alpha particle track, as shown schematically in Fig. 2(b). This DSB concentration influences the kind of chromosome aberrations made by alpha particles. With all 5 DSBs in the same half of nucleus (Fig. 2(b)) instead of being partitioned more or less randomly between halves (Fig. 2(a)) it is immediately clear that there is a greater tendency to form complex aberrations (such as those shown in Figs. 1(g), 1(h) or 1(k)), compared to simple aberrations (e.g. those shown in Figs. 1(b), 1(c) and 1(e)). Extra complex aberrations are in fact observed for densely ionizing radiations $^{\ref{r:kne99},\ref{r:sab87},\ref{r:gri95},\ref{r:dur98}}$, and it has recently been suggested that this excess could perhaps act as a ``fingerprint'' of past exposure to such radiations $^{\ref{r:and99}}$.
The argument is strengthened by the fact that for densely ionizing radiations the ratio of complex to simple aberrations is observed to be approximately dose-independent $^{\ref{r:and99}}$, just as one would expect if both kinds of aberrations are formed mainly in one-track action, i.e. if all the DSB free ends involved in any one interaction are typically from a single radiation track, as described above. For sparsely ionizing radiations, where complex aberrations typically require interaction among DSB free ends from three or more independent radiation tracks, one expects instead, and duly finds (e.g. $^{\ref{r:sim96}}$), that the ratio of complex to simple aberrations decreases as dose decreases, because the likelihood of three or more independent tracks making nearby DSBs is small at low doses.

Summary.
Radiation produces an abundant and informative variety of chromosome aberrations. The information is now available in vivid detail, due to FISH. Recent results favor randomness of DSB induction and misrepair; they point, in mammalian cells, to an aberration formation pathway (e.g. non-homologous end-joining) which permits the two free ends of a single DSB to sometimes misrejoin at different sites in the genome; they confirm chromosome localization to territories; they indicate limited chromatin motion; and they characterize densely ionizing radiations as comparatively prolific producers of complex aberrations.

Acknowledgements. Research supported by the NIH (RKS and BJT), DOE (BJT), and NSF (LRH).

References

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Legends

Figure 1. Chromosome aberration types.
The figure schematically shows the following: chromosomes, painted various colours; radiation-induced DSBs (DNA double strand breaks) such as $uu'$; centromeres (relevant only in the first three panels) indicated by constrictions; and a few examples of common chromosome aberrations.
Panel (a) shows a simple situation, with just two DSBs on two chromosomes. According to the most usually accepted aberration formation model, the breakage-and-reunion model, end-joining reactions can occur among the four DSB free ends $u$, $u'$ $v$ and $v'$. An overall interaction removing both DSBs consists of two individual reactions (e.g. $u'v$ and $v'u$ shown in panel (b)); each of these individual reactions (e.g. $u'v$) involves two free DSB ends (e.g. $u'$ and $v$), and each DSB free end consists of two single-strand ends; polarity is preserved in the reactions. In an aberration assay, one does not notice whether or not some local alterations of the base-pair sequences occur near the ends during the reactions.
Under these rules, three different outcomes are possible for the initial situation shown in panel (a). Panel (b) shows one outcome, called a translocation. A cell containing a translocation usually remains clonogenically viable. However, genes are relocated, which may result in transformation. On the other hand, a dicentric (panel (c)) is usually clonogenically lethal. The third possible outcome of the situation in panel (a) is the double restitution $uu',vv'$ (not shown).
Panel (d) again shows two DSBs, but now on one chromosome instead of two. One possible outcome is a ring, shown in panel (e). Panel (f) shows three DSBs, on three chromosomes, and panel (g) shows one possible outcome (out of 15). Panel (h) also shows three DSBs, but now on only two chromosomes. Two of the possible outcomes are shown in panels (j) and (k).
The final exchange configurations on the top line (panels (b), (c), and (e)) involve only two breaks, and are consequently called ``simple''; final exchange configurations involving three or more breaks, called ``complex'', are illustrated on the bottom line. If there are more chromosomes, more misrejoining DSBs, and more paints, a cornucopia of complex, multi-coloured exchange configurations is generated.

Figure 2. Spatial effects bias aberration frequencies.
The figure schematically shows 6 chromosomes in a cell nucleus, with 5 DSBs.
(a). As explained in the text, proximity effects (here indicated, very schematically, by the dotted line) bias for aberrations involving fewer chromosomes relative to aberrations involving more chromosomes. Conversely, measuring aberration frequencies helps quantify the size of chromosome territories and helps estimate the DSB interaction range, dependent on chromosome motion.
(b). For alpha particles instead of gamma rays, the 5 DSBs would typically be near the track of a single alpha particle, as indicated by the crosses, instead of being randomly located in the nucleus as in panel (a). This difference in spatial DSB patterns leads to a different mix of aberration types.

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