After chromosomal replication, faithful segregation of the resulting sister chromatids requires the correct bipolar attachment of sister centromeres to microtubules emanating from opposite poles of the mitotic spindle [ 7 , 8 ]. This bi-orientation of sister chromatids is established in mitosis at kinetochore complexes, which are assembled onto centromeres and serve as attachment sites for microtubules.
For proper bi-orientation each sister chromatid must be attached to microtubules from only one pole. Rings of cohesin complexes are thought to embrace both sister chromatids with greatest density around their centromeres, preventing their premature separation and allowing the detection of tension across sisters when they become bi-oriented [ 9 ].
The absence of this tension is sensed by the spindle assembly checkpoint, which prevents anaphase until all sister chromatid pairs become bi-oriented [ 10 ]. When anaphase proceeds, the cohesin rings are cleaved, releasing each chromatid to be pulled to the spindle pole to which they are attached. Importantly, the faithful segregation of sister chromatids depends on proper assembly of kinetochores, correct establishment of centromeric cohesion, and the presence of only one centromere per sister chromatid.
In principle, each of these factors can be disrupted by re-replication through a centromere, raising the possibility that the fidelity of reinitiation control is important for the fidelity of chromosome segregation.
To test the dependence of segregation fidelity on reinitiation control, we asked whether transient and localized re-replication of a centromere could disrupt the segregation of a chromosome in the budding yeast Saccharomyces cerevisiae. We find that centromeric re-replication is a potent way of inducing missegregation of both sister chromatids to one daughter cell.
Surprisingly, we also discover that centromeric re-replication can induce aneuploidy by formation of an extra sister chromatid. This formation is dependent on homologous recombination, suggesting that centromeric re-replication can lead to chromosomal breaks that then undergo homologous recombination to reconstitute intact chromatids.
Finally, microscopic examination of re-replicated centromeres suggests that they have the ability to reassemble functional kinetochores and be placed under tension. In summary, the deregulation of DNA replication initiation can have a significant impact on the mitotic mechanisms that ensure faithful chromosome segregation and can provide a potential new source of chromosomal instability and aneuploidy.
These findings have potential relevance to cancer where both compromised reinitiation control and defective segregation fidelity can be found. We previously demonstrated that the cell-cycle controls preventing reinitiation of replication are critical for genome stability by showing that compromising these controls leads to intrachromosomal amplifications [ 6 ].
In those studies we developed a system in which conditional deregulation of replication initiation proteins can induce transient and localized re-replication of any chromosome segment of interest.
This system provided an opportunity to explore how the regulation of DNA replication influences the execution of mitosis by allowing us to induce the re-replication of a centromere.
To examine if re-replication could affect chromosomal stability, we designed an assay to quantify the segregation fidelity of a budding yeast chromosome following the transient, localized re-replication of its centromere. We had previously shown that conditional deregulation of a specific subset of DNA replication controls makes origins susceptible to reinitiation, with the most prominent and detectable reinitiation occurring at the origin ARS [ 4 , 6 ].
To induce overt re-replication of a centromere, we inserted a cassette containing ARS 8 kb from the Chromosome V centromere CEN5 on one of the two homologs in a diploid re-replicating strain. To monitor the copy number of the re-replicating chromosome, the cassette also carried the copy-number reporter ade3—2p , which makes cells containing zero, one, or more copies white, pink, or red, respectively [ 11 ].
In our assay Fig. At the arrest, re-replication was transiently induced until half of the ARS in the population had reinitiated, as measured by array comparative genomic hybridization aCGH Fig. Cells were released from the arrest either before or after the induction of re-replication by plating for individual colonies. These colonies were screened for colored sectors that suggested the ade3—2p marked Chromosome V homolog missegregated in the mitosis following the induced re-replication.
Normal segregation of one sister chromatid to each daughter cell segregation produces a uniformly pink colony. However, missegregation of both sister chromatids to one daughter cell segregation would leave that cell with both copies of the ade3—2p reporter and the other daughter with none, generating colonies divided into large red and white sectors Fig. A Experimental flowchart starting with diploid re-replicating cells containing one Chromosome V homolog marked with the ade3—2p copy number reporter.
B Re-replication profile of Chromosome V for diploid cells arrested in metaphase with baseline copy number of 4C and induced to re-replicate see S1 Table. ARS and ade3—2p mark integration sites of the preferentially reinitiating origin and the copy number reporter, respectively.
Inset shows schematic of re-replication bubbles inferred from profiles. Circles on X-axis and in schematic represent centromere CEN5. Diploid re-replicating strains characterized in Fig. C Estimated frequency of segregation events after 3 hr of re-replication.
The average sectoring frequency for each strain shown in A was multiplied by the fraction of aCGH-analyzed isolates that showed segregation of the ade2—3p marked Chromosome V homolog see S2 and S8 Tables. Assuming that the unmarked non-re-replicating homolog segregates normally , we would expect the red sector to contain a total of three copies of Chromosome V and the white sector to contain one copy.
Centromeric re-replication induced by ARS caused a missegregation frequency of 6. We suspect that the latter frequency is itself elevated both because of the prolonged nocodazole arrest [ 12 , 13 ] and because of cryptic re-replication occurring throughout the genome independent of ARS and possibly involving CEN5 [ 4 , 14 ].
Hence, we were most interested in comparing the re-replication-induced frequency of Chromosome V missegregation events to the spontaneous frequency of these events. Although the latter has not been directly measured for Chromosome V in wild-type diploid cells, upper limits can be estimated by the spontaneous rate of Chromosome V loss 2—8 x 10 —6 per cell division [ 15 , 16 ] and gain 3 x 10 —7 per cell division [ 17 ] , respectively.
Thus the frequency of missegregation events induced by transient centromeric re-replication in a single cell cycle is approximately 10 3 —10 4 higher than the expected spontaneous frequency of these events. We conclude that centromeric re-replication can be a potent inducer of chromosomal instability and aneuploidy. Surprisingly, we also discovered a second source of aneuploidy induced by centromeric re-replication from ARS Colonies divided into large red and pink sectors were observed following centromeric re-replication at a frequency of 2.
The color of the sectors suggested the presence of one copy of the ade3—2p marked Chromosome V homolog in the pink sector and at least two copies in the red sector, consistent with there being three copies of the re-replicating homolog segregating in a manner. This induction of segregation suggests that centromeric re-replication can induce chromosome gain by forming a whole additional copy of the chromosome.
Upper limits on the spontaneous frequency of these events can be estimated by the spontaneous rate of chromosome gain, 3 x 10 —7 per cell division [ 17 ]. Thus the frequency of missegregation events induced by transient centromeric re-replication in a single cell cycle is approximately 10 4 to 10 5 higher than the expected spontaneous frequency of these events.
B Estimated frequency of segregation events after 3 hr of re-replication. The average sectoring frequency for each strain shown in A was multiplied by the fraction of aCGH-analyzed isolates that showed segregation of the ade2—3p marked Chromosome V homolog see S3 and S8 Tables.
D Dependence of segregation events induced by centromeric re-replication on homologous recombination. Segregation events were estimated as described in B using the frequencies reported in C see S3 and S8 Tables. A trivial explanation for chromosome gain is that, despite the predominantly localized nature of the re-replication induced by ARS on Chromosome V Fig.
If that were the case, however, we would expect the red-pink colony frequency to be independent of the chromosomal location of ARS An alternative route for generating these extra chromosomes is suggested by our previous observation that re-replication forks are highly susceptible to breakage and subsequent recombination [ 18 ]. Eukaryotes rely primarily on two pathways to repair double-strand breaks [ 19 — 21 ].
One is homologous recombination, which in budding yeast is dependent of Rad52, a protein that can facilitate complementary strand annealing and single-strand exchange [ 22 , 23 ]. Thus a significant proportion of segregation events induced by centromeric re-replication depend on homologous recombination and are unlikely to be due to complete re-replication of Chromosome V.
Importantly, none of the changes in frequencies observed with any of the deletions could be attributed to changes in re-replication efficiency because the re-replication profiles of the deletion mutants were comparable to that of the wild-type strain S2 Fig.
In addition, the effect of the deletions was specific to the segregation events because the red-white colony frequencies associated with missegregation events were not affected by the deletions S3 Fig. Together these results suggest that DNA damage induced by centromeric re-replication can be efficiently repaired by homologous recombination in a manner that generates an extra whole sister chromatid.
This route to aneuploidy appears to be partially inhibited by nonhomologous end joining, possibly by competition for the damage substrate [ 19 , 20 ]. By temporally isolating the centromeric re-replication of a chromosome from its normal replication and segregation, the experimental strategy used above provided the cleanest demonstration that re-replication induces and segregation events.
Hence, we asked if centromeric re-replication induced in unarrested, asynchronously-dividing cells was sufficient to induce and events. We activated re-replication for three hours in these cells, which induced equivalent amounts of centromeric re-replication as that induced by three hours of re-replication in nocodazole-arrested cells S4 Fig.
The state of the cells plated after the re-replication was also comparable in that they had undergone re-replication for approximately one cell cycle before the DNA damage caused by re-replication [ 25 — 28 ] triggered their transient arrest in mitosis S5 Fig. Analysis of chromosomal copy number using aCGH confirmed that this colony induction reflected increases in the frequencies of and segregation events Fig. Thus centromeric re-replication induced in cycling cells is sufficient to generate these events.
Re-replication was induced for 3 hr in unarrested cycling cells see S4 Fig , which were then analyzed as described in Fig. B Estimates of segregation frequencies after re-replication were calculated as described for Fig.
D Estimates of segregation frequencies after re-replication were calculated as described for Fig. As a first step toward exploring the molecular events that lead from centromeric re-replication to chromosome missegregation or breakage we examined the mitotic behavior of re-replicated centromeres by fluorescence microscopy. The re-replicating CEN5 was fluorescently marked with tet operator arrays placed 2 kb to the left of the centromere in haploid cells expressing tdTomato-tagged Tet repressors.
At this distance, bipolar spindle tension placed on normal bi-oriented sister centromeres in metaphase can be detected by the separation of sister arrays into two resolvable fluorescent spots [ 29 — 31 ]. To monitor the position of the marked centromeres relative to the mitotic spindle, the microtubule subunit Tub1 was tagged with GFP.
To examine the interaction of the spindle with re-replicated centromeres in metaphase, we took advantage of the fact that cycling cells induced to re-replicate trigger a DNA damage response that causes them to arrest in metaphase S5 Fig , [ 26 ]. At this point, we can observe the opposing action of bipolar spindle tension and pericentromeric cohesion on centromeres. When ARS was not present to reinitiate replication near CEN5 , all metaphase-arrested cells displayed two resolvable fluorescent spots during periodic imaging over a 20 min period Fig.
This implies that a large proportion of the re-replicated centromeres of a sister chromatid can be pulled apart from each other as well as from the centromere or possibly re-replicated centromeres of the other sister chromatid.
During the periodic imaging, both the non-re-replicated and re-replicated centromeres remained mostly separated while the spindle and centromeres were pulled in various directions around the nucleus S6 Fig.
The dynamic nature of these movements can be seen with higher resolution time-lapse imaging of re-replicated cells containing three spots Fig. These results are consistent with many of the re-replicated centromeres undergoing separation due to bipolar spindle tension.
During this time, the DNA damage response triggered by re-replication caused both strains to arrest in metaphase with intact mitotic spindles. Cells were shifted to dextrose containing media to limit further induction of re-replication before being imaged live, initially in the absence of nocodazole then 2 hr after addition of nocodazole. The number of cells scored pre-nocodazole is charted based on initial spot number, with each bar divided into the number of cells retaining one, two, three, or four spots after nocodazole treatment see S6 Table.
C Video microscopy in a single Z-plane of a live cell that has undergone centromeric re-replication. To determine if the separation of re-replicated centromeres was indeed dependent on spindle tension, the cells we scored for spot numbers were continuously imaged after treatment with nocodazole, which inhibits microtubule polymerization.
This collapse is due to pericentromeric cohesion, which resists the spindle tension placed on bi-oriented sister centromeres. This collapse of three spots to one indicates that many of these re-replicated centromeres were separated because of spindle tension. It further implies that these re-replicated centromeres reassembled functional kinetochores, maintained pericentromeric cohesion, and underwent bipolar spindle attachments. We note that, although many cells with four resolvable spots showed a reduction in the number and motion of spots upon nocodazole addition, most did not collapse down to a single spot.
There were also a few cells with three spots that retained all three after nocodazole treatment. These observations suggests that in some cases pericentromeric cohesion of re-replicated centromeres may be compromised, particularly if more than one centromere is re-replicated. We have shown that centromeric re-replication provides a highly potent way to induce chromosomal instability and aneuploidy in cells where the mitotic segregation machinery is intrinsically intact. This suggests that the mitotic mechanisms that preserve segregation fidelity are not designed to handle the problems that arise when centromeres are re-replicated.
As a consequence chromosome segregation fidelity is dependent on the fidelity of re-replication control, establishing an important connection between these very distinct processes.
Moreover, as discussed later, it raises the possibility that the decreased fidelity of both processes that is seen in cancer cells may be related.
The aneuploidy arising from centromeric re-replication is generated in part through missegregation of both sister chromatids to one daughter cell. Exactly how this missegregation occurs remains to be determined, but centromeric re-replication has the potential to perturb the segregation machinery in at least three ways: 1 disruption of kinetochores; 2 disruption of centromeric cohesion; and 3 attachment of a single sister chromatid to microtubules from both spindle poles i.
In budding yeast, kinetochores inherited from the previous cell cycle are disrupted by passage of the replication fork but then rapidly reassemble onto the newly replicated centromeres [ 34 ].
In principle, a similar disruption followed by reassembly could occur with re-replication forks since budding yeast kinetochores can assemble and become functional throughout the cell cycle [ 35 ].
Moreover, our observation that more than two centromeres in a re-replicating strain can be microscopically resolved in a microtubule dependent manner is consistent with the presence of functional kinetochores on many re-replicated centromeres.
Nonetheless, we cannot rule out some of the missegregation we detected in our sectored colony assay arising from a failure to reassemble kinetochores on a minority of re-replicated centromeres.
The microtubule dependence of the separation of re-replicated centromeres also suggests that pericentromeric cohesion is often preserved following centromeric re-replication.
Such cohesion is presumably responsible for the collapse of re-replicated and separated CEN5 spots to a single spot following the disruption of microtubules.
We note, however, that in the less frequent cases when four spots were present in a cell, they often collapsed to two spots rather than one, suggesting that in some instances, particularly when more than one centromere re-replicates, pericentromeric cohesion may be compromised.
For the majority of centromeric re-replication bubbles that retain functional kinetochores and pericentromeric cohesion, attachment of re-replicated centromeres to microtubules from opposite poles will result in bipolar attachment of the re-replicated sister chromatid to the mitotic spindle Fig. Our observation of three or four resolvable centromeres moving around the nucleus in a microtubule-dependent manner is consistent with such bipolar attachments.
Normal sister chromatids are bilaterally symmetric and held together via cohesin to ensure their bi-orientation with respect to the spindle poles. Centromere re-replication disrupts this bilateral symmetry and can lead to abnormal bipolar attachment of a single chromatid to both spindle poles.
During anaphase, this bipolar attachment could lead to a segregation pattern. Alternatively the affected sister chromatid could break and repair in a RAD dependent manner to produce a segregation pattern.
Also conceivable but not shown are disruption of kinetochore function or pericentromeric cohesion by re-replication. Importantly, this potential source of missegregation can, in principle, be established without triggering the two surveillance mechanisms that normally ensure faithful segregation.
One of these mechanisms prevents merotelic kinetochore attachments, i. Normally, because each sister chromatid has only a single kinetochore, this mechanism plays a critical role in preventing bipolar attachment of the chromatid to the mitotic spindle.
However, if centromeres re-replicate, the presence of two kinetochores on a sister chromatid permits its bipolar attachment to the spindle without requiring merotelic attachment to either kinetochore. The second mechanism, the spindle assembly checkpoint, which detects the absence of tension on kinetochores, would also not be triggered because the tension generated by bipolar attachment to re-replicated kinetochores would be transmitted to their sister kinetochore s via sister chromatid cohesion.
The inability of either surveillance mechanisms to sense and correct bipolar attachment to centromerically re-replicated sister chromatids could help explain why centromeric re-replication is such a potent inducer of missegregation and aneuploidy.
It should be noted that the re-replication bubbles that enable these bipolar attachments are transient chromosomal structure.
Exactly how these bubbles eventually disappear is not clear, although they can conceivably be resolved during the normal course of a subsequent S phase. Nonetheless, because the bubbles are transient, centromeric re-replication can induce simple aneuploidy in a hit-and-run fashion.
This stands in contrast to dicentric chromosomes, whose bipolar attachments inevitably lead to chromosome breakage and rearrangement [ 36 — 38 ]. In addition to inducing aneuploidy by missegregating sister chromatids, centromeric re-replication can also induce aneuploidy by the formation of extra sister chromatids, as manifested by the appearance of segregation events.
The dependence of at least half of these events on RAD52 , a gene essential for most homologous recombination in budding yeast [ 39 ], implies that many of these extra chromatids are generated by chromosome breakage and recombinational repair. The breakage is not surprising. It may arise from bipolar spindle tension being placed on centromeric re-replication bubbles by, or it might arise simply because of the susceptibility of re-replication forks to breakage [ 18 ].
What is striking, is the apparent efficiency with which the repair of these breaks can be channeled into the formation of extra sister chromatids.
Again, this outcome contrasts sharply with the chromosomal rearrangements that result from dicentric chromosome breaks [ 37 ], and it reinforces the notion that chromosomes with re-replicated centromeres are not simply dicentric chromosomes in a different guise.
Whether similar generation of aneuploidy by extra sister chromatid formation can occur in mammalian cells with their much larger chromosomes remains to be seen. Nonetheless, our studies have uncovered a novel way in which aneuploidy can be generated.
It is possible that centromeric re-replication can also lead to other chromosomal consequences that we did not observe either because they are lethal or because we did not score them. For example, the large rise in RAD52 -dependent segregation events in a dnl4 mutant background suggests that there may be other competing fates for chromosome breakage events that involve nonhomologous end-joining.
In our primary colony screen, we focused on colonies that were significantly induced by centromeric re-replication and that we anticipated would be most straightforward to interpret, namely red-white and red-pink sectored colonies. Analysis of other colonies with different or more complex shapes and color patterns may uncover other types of chromosomal loss, gain, or rearrangements induced by centromeric re-replication.
The connection we have established between decreased fidelity of re-replication control and decreased fidelity of chromosome segregation may be relevant to cancer as compromised fidelity for both processes have been observed in cancer cells. The decreased fidelity of chromosome segregation is well established in cancer [ 40 ]. Moreover, there are increasing hints that aneuploidy may contribute to tumorigenesis by promoting genomic instability [ 43 , 44 ].
How chromosomal instability arises in cancers is still an open question. Increasing attention has been placed on non-mitotic perturbations that can disrupt chromosome segregation because mitotic genes directly involved in kinetochore function, spindle function, cohesion, or the spindle assembly checkpoint are rarely mutated in sequenced cancer genomes [ 40 , 45 ].
One such perturbation is the accumulation of excess centrosomes, the microtubule organizing centers at the poles of spindles, as this can lead to incorrect merotelic attachment of sister chromatids to more than one spindle pole [ 46 ]. However, despite being present in many cancers, excess centrosomes are not observed in all cancers displaying chromosomal instability [ 47 ], raising the question of what other non-mitotic perturbations contribute to this instability. Our observation that centromeric re-replication is a potent inducer of aneuploidy offers one such perturbation.
This possibility is encouraged by accumulating evidence that re-replication may occur in cancer and contribute to oncogenesis [ 3 , 48 , 49 ]. Specifically, moderately elevated levels of the replication initiation proteins Cdc6 and Cdt1, which in high amounts induces detectable re-replication in cell culture and model organisms [ 27 , 50 — 55 ], has been observed in multiple types of primary tumors [ 56 — 61 ]. In addition, moderate overexpression of Cdt1 has been shown to potentiate carcinogenesis in mouse models [ 58 , 62 , 63 ].
Re-replication has yet to be directly confirmed in any of these settings [ 50 , 58 , 62 , 63 ], but this is likely due to the fact that levels of re-replication currently detectable by conventional replication assays cause extensive DNA damage and cell lethality [ 5 , 25 — 28 , 50 , 51 , 64 — 66 ].
Hence, only lower levels of re-replication can be compatible with cancer cell viability, and detecting such cryptic re-replication will require the development of more sensitive replication assays [ 67 ].
We reasoned that if centromeres can influence origin activation time, then the origins that are closest to the moved centromeres would have the greatest chance of being affected. Centromere XIV resides in its endogenous position located 6. Haploid cells grown in the presence of dense 13 C and 15 N isotopes were arrested prior to S-phase. The cells were synchronously released into medium containing isotopically light carbon and nitrogen, and samples were collected at various times during the ensuing S-phase.
DNA was extracted from each cell sample, digested with restriction enzyme EcoRI, and subjected to ultracentrifugation in cesium chloride gradients. The gradients were then drip fractionated, and the kinetics with which the EcoRI restriction fragments containing each of the three loci of interest shifted from heavy to hybrid density were compared via slot blot analysis Figure 2A ; also see Materials and Methods. A Cartoon depiction of experimental setup.
Cells were grown in medium containing heavy carbon 13 C and nitrogen 15 N isotopes. Upon genome saturation with the heavy isotopes, cells were arrested by the addition of alpha factor and released synchronously in medium containing light carbon 12 C and nitrogen 14 N isotopes.
The cells were then collected over the next minutes and their DNA was extracted, digested with EcoRI, and separated via ultra centrifugation in cesium chloride gradients such that unreplicated DNA resides lower in the gradient than newly replicated DNA.
DNA samples were then collected and analyzed through drip fractionation. B S phase progression of WT left and rearranged right cells as measured by flow cytometry. The kinetic curves for ARS and R11 are shown as dashed and dotted lines, respectively. T rep is the time of half-maximal replication for each locus see Materials and Methods.
ARS black dashed arrow and R11 black dotted arrow were used as early and late timing standards, respectively. Direction of the black arrows indicates the direction of the shift in replication index for each locus between WT and rearranged strains. WT cells entered S-phase by 40 minutes after release from alpha factor arrest, and most of the cells reached 2C DNA content between and minutes Figure 2B.
The time of replication T rep for each locus was calculated as the time it reached half maximal replication see Materials and Methods. ARS , one of the earliest known origins, and R11, a late replicating fragment on chromosome V, were used as timing standards for comparison.
To facilitate comparison between cultures, these T rep values were converted to replication indices [22] by assigning ARS a replication index RI of 0 and R11 an RI of 1. Most other genomic loci have RIs between 0 and 1. Similar to WT cells, the cells with the relocated centromere entered S-phase by 40 minutes following release from alpha factor arrest and reached 2C DNA content between and minutes Figure 2B. Consistent with ARS being the origin from which met2 replicates, ARS maintained a slight timing advantage over met2.
Similar results were obtained using an independent segregant see Figure S1. Highly efficient origins display a more intense bubble-arc signal relative to the Y-arc [29]. Based on the similarity of bubble- to Y-arc ratios we conclude that the centromere has no obvious effect on the efficiency of ARS Figure 3A.
A similar result was obtained for ARS Figure 3A supporting the idea that the observed timing changes are not due to changes in efficiency of existing origins. No bubble-arc was observed for the MET2 locus in the WT strain or the met2 locus in the rearranged strain Figure 3B , indicating that an origin was not created by insertion of the centromere into the MET2 locus.
Together these data suggest that the presence of a nearby centromere or some feature of its pericentric DNA induces early activation of origins. DNA fragments containing a functional origin are detected as a bubble arc depicted by the bubble fragment while fragments that are passively replicated are detected as a Y-arc depicted as a Y shaped fragment.
The centromere in the rearranged construct was detected as a pause site black arrowhead visualized as a dot of relatively increased intensity on the descending Y-arc. DNA sequences that determine origin activation time have remained largely elusive. However, previous work has shown that sequences flanking a subset of origins on chromosome XIV delay the activation of those origins [22].
Therefore, it is possible that by integrating the centromere into the MET2 locus in the rearranged strain, a delay element responsible for making ARS late activating was disrupted or pushed out of its effective range, thereby causing the origin to fire early. Although this scenario would explain the change in the replication times of met2 and ARS , the observation that ARS became later replicating when the centromere was removed from its endogenous position argues in favor of the timing changes being a consequence of centromere proximity.
Alternatively, it is conceivable that there is an uncharacterized sequence element, distinct from centromeric sequence, residing in pericentric DNA that is promoting early activation of nearby origins. We tested these possibilities as described below. At the sequence level, S. CDEIII has been found to be the element most important for centromere function as it is the binding site for essential inner kinetochore proteins, notably members of the CBF3 complex [1] , [19] , [30].
To ask if a functional centromere is required for early activation of nearby origins, we engineered a strain to have a non-functional centromere with a mutated CDEIII motif integrated at the MET2 locus while the functional centromere remained in its endogenous position Figure 4A.
This strain was also subjected to flow cytometry Figure 4B and replication timing analysis as described above Figure 4C. Unlike the dramatic replication timing change observed when we introduced a functional centromere, introducing the mutated centromere caused no replication timing change at met2 and ARS RI of 0. Therefore, we conclude that centromere function is needed to effect a timing change on nearby origins.
Furthermore, the late replication of this region was not due to inactivation of ARS through insertion of the mutated centromere as indicated by 2D gel analysis Figure 4E. Together, these data demonstrate that functional centromeres actively advance the activation times of origins over a distance of This chromosome is maintained through its wild type centromere at the endogenous location black circle. B Flow cytometry of cells with a non-functional centromere in the MET2 locus.
C Replication kinetic curves for metcen7 and ARS D Replication indices for met2:cen7 green diamond and ARS blue diamond. E 2D gel analysis of ARS Presence of a bubble arc black arrow for ARS in the strain in which the non-functional centromere was integrated at MET2 compare with Figure 1B indicates that ARS is a functional origin in this strain. Upon finding that centromeres advance the activation time of origins to a distance of at least Raghuraman et al.
Interestingly, at least one early replicating origin can be found within A recent study using an S-phase cyclin mutant [7] showed that early replicating domains that include a centromere can be well over kb, implying that the range over which centromeres can regulate origin activation time might be quite broad.
To determine the range over which a centromere can influence replication time we performed a genome wide analysis of replication in the WT and rearranged strains. Cells were grown in dense medium see Materials and Methods and timed samples were collected following release into S-phase in light medium. To obtain a genome-wide view, the HH and HL DNAs from each timed sample were labeled with different fluorophores, cohybridized to microarray slides, and replication profiles were generated Figure 5A , Figures S2 and S3.
Peak locations in the profile correspond to the locations of origins while the timed sample in which the peaks first appear gives an indication of the time at which the corresponding origins become active during S-phase [6]. Percent replication was monitored across chromosome XIV at 40 magenta , 45 orange , 55 green , and 65 blue minutes following release from alpha factor arrest. When the native centromere yellow circle is present near ARS, a prominent peak is seen in the 40 and 45 minute time samples.
In this strain, the peak at ARS is shallow in the 40 and 45 minute samples. When the centromere is repositioned orange circle near ARS in the rearranged strain, both the time of appearance and the prominence of the peaks at ARS and ARS are inverted with respect to the WT strain.
Replication kinetic profiles from the 40 minute sample were normalized by converting percent replication values to Z-score values see Materials and Methods. Replication profiles generated for the WT strain Figure S2 were consistent with previous studies [8].
Conversely, replication profiles in the rearranged strain displayed a shallow and late appearing peak at ARS while the peak at ARS appeared strong and early Figure 5A and Figure S3 confirming the observations made by slot blot analysis of individual restriction fragments.
To directly compare replication profiles from the two strains, the percent replication values from the 40, 45, and 65 minute samples were normalized by conversion to Z-scores see Materials and Methods and superimposed on the same axes Figure 5B ; Figures S4 , S5 , and S6. Comparison of the Z-scores on chromosome XIV Figure 5B indicates that centromeres have a drastic influence over the activation times of their closest origins, suggesting that centromeres, mechanistically, operate locally.
Z-score profiles for WT and rearranged chromosomes display a prominent early appearing peak centered about 19 kb to the left of the endogenous centromere. However, 2D gel analysis demonstrated that ARS is the origin likely responsible for this peak as ARS is not active in either strain data not shown. That this mild effect on ARS is not reflected in the Z-score overlays is likely due to the higher resolution of slot blot analysis compared to microarray analysis.
We then asked if the moved centromere influenced the activation times of origins located on other chromosomes. The remaining profiles were examined by Z-score comparisons looking for possible trans effects of centromere position. The profiles were strikingly similar Figures S4 , S5 , and S6. A prominent peak of high percent replication corresponding to ARS was present in the WT strain while no peak was observed in the rearranged strain.
However, further examination using 2D gel electrophoresis revealed that although ARS is not an active origin in the isolate of the rearranged strain used for microarray analysis it is active in the WT strain as well as in the rearranged independent segregant used in the slot blot analysis of timing Figure 6B ; see Figure S1 for kinetic data for the independent segregant.
These data suggest that the apparent timing change at this chromosome location is likely to be a consequence of a polymorphism affecting the ability of ARS to function as an origin rather than from any long-range effect of the centromere.
A change of the T to a C at this position has been shown to entirely abolish origin activity in ARS [32]. See Figure S4 for all chromosomes. B 2D gel analysis of ARS in the WT left , the rearranged strain used in microarray analysis middle , and the rearranged strain used for prior slot blot analysis right. The presence of a bubble arc in the WT black arrow indicates that ARS is a functional origin in this strain. The presence of a bubble arc in one of the two rearranged strains confirms that the absence of origin activity in rearranged A used in microarray analysis is not due to relocation of the centromere on chromosome XIV.
In this study we investigated the long-standing question of why centromeres replicate early in S-phase. We considered two possibilities: 1 that some component required for centromere function is also involved in early origin activation, or 2 that evolution has favored the migration of centromeres to early replicating regions.
It has been hypothesized that early origin initiation in S. However, the observation that early centromere replication is conserved [9] — [11] in conjunction with the identification of a DNA element that is not associated with centromeres but capable of advancing origin activation time [25] , [26] indicates that establishment of early origin activation time is more complex than previously thought.
Consistent with this idea, a recent study shows that the centromeres of C. Because the neocentromere in C. These results raised the question of whether the relocation of a centromere would have a similar effect in organisms with point centromeres. In this study, we took advantage of the well-characterized centromeres and origins in S. We show that centromeres in S. We find that centromere-mediated early origin activation requires an intact CDEIII region, suggesting that early origin activation is dependent on at least some portion of the DNA-protein complex normally formed at the centromere.
Thus, centromeres and at least a subset of the kinetochore proteins they assemble participate as cis -acting regulatory elements of origin firing time. Furthermore, our 2D gel results indicate that centromeres do not affect origin efficiency, suggesting that the mechanisms responsible for centromere-mediated early origin activation are distinct from those that determine efficiency.
In light of the finding that centromeres regulate the activation times of their closest origins, we were interested in determining over what distance centromeres exert their effect. This result implies that there are at least two distinct mechanisms by which origins can fire early.
In contrast to what has been observed in C. The genome is not randomly organized within the nucleus but particular genomic regions co-localize or cluster into functional foci during processes such as DNA replication [33] , [34]. In particular, centromeres in S. Therefore, it is plausible that centromeres, their neighboring origins, as well as other portions of the genome that interact with them, are clustered in G1 phase when timing decisions are made.
This clustering could provide a way for centromeres to mediate replication time through a trans -acting mechanism. To determine if the mechanism responsible for centromere mediated early origin activation is capable of acting in trans , we examined the genome wide replication timing data for other timing changes occurring in the genome as a result of the repositioned centromere on chromosome XIV. We looked for differences in the Z-score profiles between matched S-phase samples, demanding that they persist over the course of S-phase See Figures S4 , S5 , S6 to be considered significant.
Other than these three locations, the replication profiles are remarkably similar, suggesting that the mechanisms by which centromeres influence origin activation time are restricted to relatively limited adjacent regions.
Because the timing change at this location was only present in two of the three Z-scored samples, we did not consider it to be a significant timing difference.
Here we show direct evidence of a molecular link between the establishment of the kinetochore and replication initiation machinery. Although the mechanism of centromere-mediated early origin activation is unknown, we show that such a mechanism is dependent on at least some of the protein components associated with the kinetochore that require an intact CDEIII region.
We propose four possible models, which are not mutually exclusive Figure 7 : 1 The nuclear environment near the microtubule organizing center MTOC is particularly enriched in replication initiation factors; 2 The tension exerted by the microtubule is translated along the nearby DNA, altering its chromatin structure, thereby influencing the accessibility of the imbedded origins to initiation factors; 3 Proteins within the kinetochore directly or indirectly interact with initiation factors, recruiting them to nearby origins; and 4 The C-loop architecture of the pericentric chromatin see below ensures the origins within the C-loop will be at the periphery of the chromatin mass and are therefore more exposed to initiation factors.
Studies examining the concentration of replication initiation proteins near MTOCs have not been conducted; however, nuclear pore complexes are enriched near MTOCs in S. While it is tempting to invoke localization to the nuclear periphery in the vicinity of MTOCs as a potential link between replication timing and centromeres, there is as yet no clear causal connection between nuclear localization and replication timing.
Of these two processes, mitosis is more common. In fact, whereas only sexually reproducing eukaryotes can engage in meiosis, all eukaryotes — regardless of size or number of cells — can engage in mitosis.
But how does this process proceed, and what sorts of cells does it produce? During mitosis, a eukaryotic cell undergoes a carefully coordinated nuclear division that results in the formation of two genetically identical daughter cells.
Mitosis itself consists of five active steps, or phases: prophase, prometaphase, metaphase, anaphase, and telophase. Before a cell can enter the active phases of mitosis, however, it must go through a period known as interphase , during which it grows and produces the various proteins necessary for division. Then, at a critical point during interphase called the S phase , the cell duplicates its chromosomes and ensures its systems are ready for cell division.
If all conditions are ideal, the cell is now ready to move into the first phase of mitosis. This page appears in the following eBook. Aa Aa Aa. Walther Flemming's drawing of chromosomes. What happens during mitosis? Figure 1: During prophase, the chromosomes in a cell's nucleus condense to the point that they can be viewed using a light microscope. Prophase is the first phase of mitosis. During this phase, the chromosomes inside the cell's nucleus condense and form tight structures. In fact, the chromosomes become so dense that they appear as curvy, dark lines when viewed under a microscope Figure 1.
Because each chromosome was duplicated during S phase, it now consists of two identical copies called sister chromatids that are attached at a common center point called the centromere. Figure 2: The mitotic spindle white begins to form outside the cell's nucleus. Important changes also take place outside of the nucleus during prophase. In particular, two structures called centrosomes move to opposite sides of the cell during this phase and begin building the mitotic spindle.
The mitotic spindle plays a critical role during the later phases of mitosis as it orchestrates the movement of sister chromatids to opposite poles of the cell Figure 2. After prophase is complete, the cell enters prometaphase. During prometaphase, the nuclear membrane disintegrates and the mitotic spindle gains access to the chromosomes. During this phase, a protein structure called the kinetochore is associated with the centromere on each sister chromatid.
Stringlike structures called microtubules grow out from the spindle and connect to the sister chromatids at their kinetochores; one microtubule from one side of the spindle attaches to one sister chromatid in each chromosome, and one microtubule from the other side of the spindle attaches to the other sister chromatid Figure 3a.
Figure 3: a Metaphase and b Anaphase. In metaphase a , the microtubules of the spindle white have attached and the chromosomes have lined up on the metaphase plate.
0コメント