David H. Collingwood

Replication in Hydroxyurea: It's a Matter of Time

ABSTRACT Hydroxyurea (HU) is a DNA replication inhibitor that negatively affects both the elongation and initiation phases of replication and triggers the “intra-S phase checkpoint.” Previous work with budding yeast has shown that, during a short exposure to HU, MEC1/RAD53 prevent initiation at some late S phase origins. In this study, we have performed microarray experiments to follow the fate of all origins over an extended exposure to HU. We show that the genome-wide progression of DNA synthesis, including origin activation, follows the same pattern in the presence of HU as in its absence, although the time frames are very different. We find no evidence for a specific effect that excludes initiation from late origins. Rather, HU causes S phase to proceed in slow motion; all temporal classes of origins are affected, but the order in which they become active is maintained. We propose a revised model for the checkpoint response to HU that accounts for the continued but slowed pace of the temporal program of origin activation.

The Temporal Program of Chromosome Replication : Genomewide Replication in clb5Δ Saccharomyces cerevisiae

Temporal regulation of origin activation is widely thought to explain the pattern of early- and late-replicating domains in the Saccharomyces cerevisiae genome. Recently, single-molecule analysis of replication suggested that stochastic processes acting on origins with different probabilities of activation could generate the observed kinetics of replication without requiring an underlying temporal order. To distinguish between these possibilities, we examined a clb5Δ strain, where origin firing is largely limited to the first half of S phase, to ask whether all origins nonspecifically show decreased firing (as expected for disordered firing) or if only some origins (“late” origins) are affected. Approximately half the origins in the mutant genome show delayed replication while the remainder replicate largely on time. The delayed regions can encompass hundreds of kilobases and generally correspond to regions that replicate late in wild-type cells. Kinetic analysis of replication in wild-type cells reveals broad windows of origin firing for both early and late origins. Our results are consistent with a temporal model in which origins can show some heterogeneity in both time and probability of origin firing, but clustering of temporally like origins nevertheless yields a genome that is organized into blocks showing different replication times.

The effect of Ku on telomere replication time is mediated by telomere length but is independent of histone tail acetylation

Ku controls telomere replication timing. We test the mechanism and find that Ku does not bind telomere-proximal origins directly or alter their histone acetylation state. Instead, Kus effect on replication timing is mediated through telomere length and requires the TG1-3 repeat-counting component Rif1.

Fragile Genomic Sites Are Associated with Origins of Replication

Genome rearrangements are mediators of evolution and disease. Such rearrangements are frequently bounded by transfer RNAs (tRNAs), transposable elements, and other repeated elements, suggesting a functional role for these elements in creating or repairing breakpoints. Though not well explored, there is evidence that origins of replication also colocalize with breakpoints. To investigate a potential correlation between breakpoints and origins, we analyzed evolutionary breakpoints defined between Saccharomyces cerevisiae and Kluyveromyces waltii and S. cerevisiae and a hypothetical ancestor of both yeasts, as well as breakpoints reported in the experimental literature. We find that origins correlate strongly with both evolutionary breakpoints and those described in the literature. Specifically, we find that origins firing earlier in S phase are more strongly correlated with breakpoints than are later-firing origins. Despite origins being located in genomic regions also bearing tRNAs and Ty elements, the correlation we observe between origins and breakpoints appears to be independent of these genomic features. This study lays the groundwork for understanding the mechanisms by which origins of replication may impact genome architecture and disease.

Centromere Replication Timing Determines Different Forms of Genomic Instability in Saccharomyces cerevisiae Checkpoint Mutants During Replication Stress

Yeast replication checkpoint mutants lose viability following transient exposure to hydroxyurea, a replication-impeding drug. In an effort to understand the basis for this lethality, we discovered that different events are responsible for inviability in checkpoint-deficient cells harboring mutations in the mec1 and rad53 genes. By monitoring genomewide replication dynamics of cells exposed to hydroxyurea, we show that cells with a checkpoint deficient allele of RAD53, rad53K227A, fail to duplicate centromeres. Following removal of the drug, however, rad53K227A cells recover substantial DNA replication, including replication through centromeres. Despite this recovery, the rad53K227A mutant fails to achieve biorientation of sister centromeres during recovery from hydroxyurea, leading to secondary activation of the spindle assembly checkpoint (SAC), aneuploidy, and lethal chromosome segregation errors. We demonstrate that cell lethality from this segregation defect could be partially remedied by reinforcing bipolar attachment. In contrast, cells with the mec1-1 sml1-1 mutations suffer from severely impaired replication resumption upon removal of hydroxyurea. mec1-1 sml1-1 cells can, however, duplicate at least some of their centromeres and achieve bipolar attachment, leading to abortive segregation and fragmentation of incompletely replicated chromosomes. Our results highlight the importance of replicating yeast centromeres early and reveal different mechanisms of cell death due to differences in replication fork progression.

Orbits and characters associated to highest weight representations

We relate two different orbit decompositions of the flag variety. This allows us to pass from the closed formulas of Boe, Enright, and Shelton for the formal character of an irreducible highest weight representation to closed formulas for the distributional character written as a sum of characters of generalized principal series representations. Otherwise put, we give a dictionary between certain Lusztig-Vogan polynomials arising in Harish-Chandra module theory and the Kazhdan-Lusztig polynomials associated to a relative category a of Hermitian symmetric type.

Jacquet modules for semisimple Lie groups having Verma module filtrations

We begin with a connected semisimple real matrix group G. Given a finite dimensional representation F of G, we may associate to it two categories of representations: a category %‘%F?~ of Harish-Chandra modules for G and a highest weight module category 0;7. The exact covariant Jacquetfunctor .I assigns to each irreducible Harish-Chandra module 7c in &+5ZF a Jacquet module J(rc), which lies in the category 0;. This paper solves the problem of determining when J(Z) is one of the basic modules arising in the Kazhdan-Lusztig theory of category 0;. More carefully, recall that the category Sl, admits four basic families of indecomposable modules, each of which forms a basis for the Grothendieck group of virtual characters: the irreducible highest weight modules; the Verma modules; the indecomposable projective modules; and the indecomposable self-dual Verma flag modules. In this paper, we answer a question of Jim Humphreys, by characterizing those rc for which the Jacquet module J(Z) is indecomposable with a Verma flag. This leads to a characterization of the projective Jacquet modules. Such Jacquet modules arise from irreducible principal series representations of split groups. If we assume ,G has no simple factor locally isomorphic to Sp(n, R), then the following are equivalent: (i) J(rc) is projective in the category 0;; (ii) J(n) is indebomposable with a Verma flag in 0;; (iii) E is an irreducible principal series representation in XvF of a split group G; and (iv) J(rc) is the unique self-dual projective indecomposable module in 0;. When G is locally isomorphic to Sp(n, R), then the classification of projective Jacquet modules is as above, ;but an additional Verma flag Jacquet module can occur in 0;; this Jacquet module will arise from two different irreducible representations 7~ in &%F?~. The Loewy filtrations of any such Verma flag Jacquet module are known, leading to a complete determination of their