Clara S. Chan

Mec1 Is One of Multiple Kinases that Prime the Mcm2-7 Helicase for Phosphorylation by Cdc7

Activation of the eukaryotic replicative DNA helicase, the Mcm2-7 complex, requires phosphorylation by Cdc7/Dbf4 (Dbf4-dependent kinase or DDK), which, in turn, depends on prior phosphorylation of Mcm2-7 by an unknown kinase (or kinases). We identified DDK phosphorylation sites on Mcm4 and Mcm6 and found that phosphorylation of either subunit suffices for cell proliferation. Importantly, prior phosphorylation of either S/T-P or S/T-Q motifs on these subunits is required for DDK phosphorylation of Mcm2-7 and for normal S phase passage. Phosphomimetic mutations of DDK target sites bypass both DDK function and mutation of the priming phosphorylation sites. Mrc1 facilitates Mec1 phosphorylation of the S/T-Q motifs of chromatin-bound Mcm2-7 during S phase to activate replication. Genetic interactions between priming site mutations and MRC1 or TOF1 deletion support a role for these modifications in replication fork stability. These findings identify regulatory mechanisms that modulate origin firing and replication fork assembly during cell cycle progression.

Evidence of abundant stop codon readthrough in Drosophila and other metazoa

While translational stop codon readthrough is often used by viral genomes, it has been observed for only a handful of eukaryotic genes. We previously used comparative genomics evidence to recognize protein-coding regions in 12 species of Drosophila and showed that for 149 genes, the open reading frame following the stop codon has a protein-coding conservation signature, hinting that stop codon readthrough might be common in Drosophila. We return to this observation armed with deep RNA sequence data from the modENCODE project, an improved higher-resolution comparative genomics metric for detecting protein-coding regions, comparative sequence information from additional species, and directed experimental evidence. We report an expanded set of 283 readthrough candidates, including 16 double-readthrough candidates; these were manually curated to rule out alternatives such as A-to-I editing, alternative splicing, dicistronic translation, and selenocysteine incorporation. We report experimental evidence of translation using GFP tagging and mass spectrometry for several readthrough regions. We find that the set of readthrough candidates differs from other genes in length, composition, conservation, stop codon context, and in some cases, conserved stem-loops, providing clues about readthrough regulation and potential mechanisms. Lastly, we expand our studies beyond Drosophila and find evidence of abundant readthrough in several other insect species and one crustacean, and several readthrough candidates in nematode and human, suggesting that functionally important translational stop codon readthrough is significantly more prevalent in Metazoa than previously recognized.

On the volume of the polytope of doubly stochastic matrices

We study the calculation of the volume of the polytope Bn of n × n doubly stochastic matrices (real nonnegative matrices with row and column sums equal to one). We describe two methods. The first involves a decompos ition of the polytope into simplices. The second involves the enumeration of “magic squares”, that is, n × n nonnegative integer matrices whose rows and columns all sum to the same integer. We have used the first method to confirm the previously known values through n = 7. This method can also be used to compute the volumes of faces of Bn For example, we have observed that the volume of a particular face of Bn appears to be a product of Catalan numbers. We have used the second method to find the volume for n = 8, which we believe was not previously known.

On the expected value of the minimum assignment

The minimum k-assignment of an m × n matrix X is the minimum sum of k entries of X, no two of which belong to the same row or column. Coppersmith and Sorkin conjectured that if X is generated by choosing each entry independently from the exponential distribution with mean 1, then the expected value of its minimum k-assignment is given by an explicit formula, which has been proven only in a few cases. In this paper we describe our efforts to prove the Coppersmith-Sorkin conjecture by considering the more general situation where the entries xij of X are chosen independently from different distributions. In particular, we require that xij be chosen from the exponential distribution with mean 1/ricj. We conjecture an explicit formula for the expected value of the minimum k-assignment of such X and give evidence for this formula.

Separation of DNA replication from the assembly of break-competent meiotic chromosomes.

The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not strictly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms.

Plane trees and H -vectors of shellable cubical complexes

Stanley first defined the generalized toric h-vector, a fundamental combinatorial invariant of polyhedral complexes (and more general objects). In the case where the complex is simplicial, this invariant can be computed by shelling, or taking apart the complex in a certain order. This paper shows how any shellable complex with cubical facets can be dealt with analogously. Based on a result of Shapiro the h-vector of any shellable cubical complex is formulated in terms of certain classes of plane trees.

Evolutionary Dynamics of Abundant Stop Codon Readthrough

Translational stop codon readthrough emerged as a major regulatory mechanism affecting hundreds of genes in animal genomes, based on recent comparative genomics and ribosomal profiling evidence, but its evolutionary properties remain unknown. Here, we leverage comparative genomic evidence across 21 Anopheles mosquitoes to systematically annotate readthrough genes in the malaria vector Anopheles gambiae, and to provide the first study of abundant readthrough evolution, by comparison with 20 Drosophila species. Using improved comparative genomics methods for detecting readthrough, we identify evolutionary signatures of conserved, functional readthrough of 353 stop codons in the malaria vector, Anopheles gambiae, and of 51 additional Drosophila melanogaster stop codons, including several cases of double and triple readthrough and of readthrough of two adjacent stop codons. We find that most differences between the readthrough repertoires of the two species arose from readthrough gain or loss in existing genes, rather than birth of new genes or gene death; that readthrough-associated RNA structures are sometimes gained or lost while readthrough persists; that readthrough is more likely to be lost at TAA and TAG stop codons; and that readthrough is under continued purifying evolutionary selection in mosquito, based on population genetic evidence. We also determine readthrough-associated gene properties that predate readthrough, and identify differences in the characteristic properties of readthrough genes between clades. We estimate more than 600 functional readthrough stop codons in mosquito and 900 in fruit fly, provide evidence of readthrough control of peroxisomal targeting, and refine the phylogenetic extent of abundant readthrough as following divergence from centipede.

Heterologous Stop Codon Readthrough of Metazoan Readthrough Candidates in Yeast

Recent analysis of genomic signatures in mammals, flies, and worms indicates that functional translational stop codon readthrough is considerably more abundant in metazoa than previously recognized, but this analysis provides only limited clues about the function or mechanism of readthrough. If an mRNA known to be read through in one species is also read through in another, perhaps these questions can be studied in a simpler setting. With this end in mind, we have investigated whether some of the readthrough genes in human, fly, and worm also exhibit readthrough when expressed in S. cerevisiae. We found that readthrough was highest in a gene with a post-stop hexamer known to trigger readthrough, while other metazoan readthrough genes exhibit borderline readthrough in S. cerevisiae.

Buchsbaum and Eulerian complexes

Let Δ be a finite simplicial complex, and K [Δ] its Stanley-Reisner ring. We show that if Δ is Eulerian, then every depth of K[Δ] is possible, and we find all possible depths of K [Δ] for Buchsbaum Eulerian Δ, dealing with the orientable and nonorientable cases separately. In addition, we find all possible betti sequences of Buchsbaum-Eulerian, Eulerian, and semi-Eulerian complexes.

Evolutionary dynamics of abundant stop codon readthrough in Anopheles and Drosophila

Translational stop codon readthrough was virtually unknown in eukaryotic genomes until recent developments in comparative genomics and new experimental techniques revealed evidence of readthrough in hundreds of fly genes and several human, worm, and yeast genes. Here, we use the genomes of 21 species of Anopheles mosquitoes and improved comparative techniques to identify evolutionary signatures of conserved, functional readthrough of 353 stop codons in the malaria vector, Anopheles gambiae, and 51 additional Drosophila melanogaster stop codons, with several cases of double and triple readthrough including readthrough of two adjacent stop codons, supporting our earlier prediction of abundant readthrough in pancrustacea genomes. Comparisons between Anopheles and Drosophila allow us to transcend the static picture provided by single-clade analysis to explore the evolutionary dynamics of abundant readthrough. We find that most differences between the readthrough repertoires of the two species are due to readthrough gain or loss in existing genes, rather than to birth of new genes or to gene death; that RNA structures are sometimes gained or lost while readthrough persists; and that readthrough is more likely to be lost at TAA and TAG stop codons. We also determine which characteristic properties of readthrough predate readthrough and which are clade-specific. We estimate that there are more than 600 functional readthrough stop codons in A. gambiae and 900 in D. melanogaster. We find evidence that readthrough is used to regulate peroxisomal targeting in two genes. Finally, we use the sequenced centipede genome to refine the phylogenetic extent of abundant readthrough.