Duncan J. Smith

Intrinsic coupling of lagging-strand synthesis to chromatin assembly.

Fifty per cent of the genome is discontinuously replicated on the lagging strand as Okazaki fragments. Eukaryotic Okazaki fragments remain poorly characterized and, because nucleosomes are rapidly deposited on nascent DNA, Okazaki fragment processing and nucleosome assembly potentially affect one another. Here we show that ligation-competent Okazaki fragments in Saccharomyces cerevisiae are sized according to the nucleosome repeat. Using deep sequencing, we demonstrate that ligation junctions preferentially occur near nucleosome midpoints rather than in internucleosomal linker regions. Disrupting chromatin assembly or lagging-strand polymerase processivity affects both the size and the distribution of Okazaki fragments, suggesting a role for nascent chromatin, assembled immediately after the passage of the replication fork, in the termination of Okazaki fragment synthesis. Our studies represent the first high-resolution analysis—to our knowledge—of eukaryotic Okazaki fragments in vivo, and reveal the interconnection between lagging-strand synthesis and chromatin assembly.

“Nought May Endure but Mutability”: Spliceosome Dynamics and the Regulation of Splicing

The spliceosome is both compositionally and conformationally dynamic. Each transition along the splicing pathway presents an opportunity for progression, pausing, or discard, allowing splice site choice to be regulated throughout both the assembly and catalytic phases of the reaction.

Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation.

Ribonucleotides are frequently incorporated into DNA during replication in eukaryotes. Here we map genome-wide distribution of these ribonucleotides as markers of replication enzymology in budding yeast, using a new 5′ DNA end–mapping method, hydrolytic end sequencing (HydEn-seq). HydEn-seq of DNA from ribonucleotide excision repair–deficient strains reveals replicase- and strand-specific patterns of ribonucleotides in the nuclear genome. These patterns support the roles of DNA polymerases α and δ in lagging-strand replication and of DNA polymerase ɛ in leading-strand replication. They identify replication origins, termination zones and variations in ribonucleotide incorporation frequency across the genome that exceed three orders of magnitude. HydEn-seq also reveals strand-specific 5′ DNA ends at mitochondrial replication origins, thus suggesting unidirectional replication of a circular genome. Given the conservation of enzymes that incorporate and process ribonucleotides in DNA, HydEn-seq can be used to track replication enzymology in other organisms.

Quantitative, Genome-Wide Analysis of Eukaryotic Replication Initiation and Termination

Many fundamental aspects of DNA replication, such as the exact locations where DNA synthesis is initiated and terminated, how frequently origins are used, and how fork progression is influenced by transcription, are poorly understood. Via the deep sequencing of Okazaki fragments, we comprehensively document replication fork directionality throughout the S. cerevisiae genome, which permits the systematic analysis of initiation, origin efficiency, fork progression, and termination. We show that leading-strand initiation preferentially occurs within a nucleosome-free region at replication origins. Using a strain in which late origins can be induced to fire early, we show that replication termination is a largely passive phenomenon that does not rely on cis-acting sequences or replication fork pausing. The replication profile is predominantly determined by the kinetics of origin firing, allowing us to reconstruct chromosome-wide timing profiles from an asynchronous culture.

An Eco1-independent sister chromatid cohesion establishment pathway in S. cerevisiae

Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, is a prerequisite for their alignment on the spindle apparatus and segregation in mitosis. Budding yeast cohesin first associates with chromosomes in G1. Then, during DNA replication in S-phase, the replication fork-associated acetyltransferase Eco1 acetylates the cohesin subunit Smc3 to make cohesin’s DNA binding resistant to destabilization by the Wapl protein. Whether stabilization of cohesin molecules that happen to link sister chromatids is sufficient to build sister chromatid cohesion, or whether additional reactions are required to establish these links, is not known. In addition to Eco1, several other factors contribute to cohesion establishment, including Ctf4, Ctf18, Tof1, Csm3, Chl1 and Mrc1, but little is known about their roles. Here, we show that each of these factors facilitates cohesin acetylation. Moreover, the absence of Ctf4 and Chl1, but not of the other factors, causes a synthetic growth defect in cells lacking Eco1. Distinct from acetylation defects, sister chromatid cohesion in ctf4Δ and chl1Δ cells is not improved by removing Wapl. Unlike previously thought, we do not find evidence for a role of Ctf4 and Chl1 in Okazaki fragment processing, or of Okazaki fragment processing in sister chromatid cohesion. Thus, Ctf4 and Chl1 delineate an additional acetylation-independent pathway that might hold important clues as to the mechanism of sister chromatid cohesion establishment.

Insights into Branch Nucleophile Positioning and Activation from an Orthogonal Pre-mRNA Splicing System in Yeast

The duplex formed between the branch site (BS) of a spliceosomal intron and its cognate sequence in U2 snRNA is important for spliceosome assembly and the first catalytic step of splicing. We describe the development of an orthogonal BS-U2 system in S. cerevisiae in which spliceosomes containing a grossly substituted second-copy U2 snRNA mediate the in vivo splicing of a single reporter transcript carrying a cognate substitution. Systematic use of this approach to investigate requirements for branching catalysis reveals considerable flexibility in the sequence of the BS-U2 duplex and its positioning relative to the catalytic center. Branching efficiency depends on the identity of the branch nucleotide, its position within the BS-U2 duplex, and its distance from U2/U6 helix Ia. These results provide insights into substrate selection during spliceosomal branching catalysis; additionally, this system provides a foundation and tool for future mechanistic splicing research.

Chromatin dynamics at the replication fork: there's more to life than histones

Before each division, eukaryotic cells face the daunting task of completely and accurately replicating a heterogeneous, chromatinized genome and repackaging both resulting daughters. Because replication requires strand separation, interactions between the DNA and its many associated proteins--including histones--must be transiently broken to allow the passage of the replication fork. Here, we will discuss the disruption and re-establishment of chromatin structure during replication, and the consequences of these processes for epigenetic inheritance.

A critical assessment of the utility of protein-free splicing systems

U2 and U6 snRNAs form part of the catalytic spliceosome and represent strong candidates for components of its active site. Over the past decade it has become clear that these snRNAs are capable of catalyzing several different chemical reactions, leading to the widespread conclusion that the spliceosome is a ribozyme. Here, we discuss the advances in both protein-free and fully spliceosomal systems that would be required to conclude that the reactions observed to be catalyzed by protein-free snRNAs are related to splicing and question the reliability of snRNA-only systems as tools for mechanistic splicing research.

Mechanistic insights from reversible splicing catalysis

Recent work demonstrating the ability of spliceosomes purified after the second catalytic step of splicing to efficiently reverse both steps of the reaction provides answers to several unresolved questions regarding the splicing reaction, and raises many more.

Pif1-family helicases cooperatively suppress widespread replication-fork arrest at tRNA genes

Saccharomyces cerevisiae expresses two Pif1-family helicases—Pif1 and Rrm3—which have been reported to play distinct roles in numerous nuclear processes. Here, we systematically characterized the roles of Pif1 helicases in replisome progression and lagging-strand synthesis in S. cerevisiae. We demonstrate that either Pif1 or Rrm3 redundantly stimulates strand displacement by DNA polymerase δ during lagging-strand synthesis. By analyzing replisome mobility in pif1 and rrm3 mutants, we show that Rrm3, with a partially redundant contribution from Pif1, suppresses widespread terminal arrest of the replisome at tRNA genes. Although both head-on and codirectional collisions induce replication-fork arrest at tRNA genes, head-on collisions arrest a higher proportion of replisomes. In agreement with this observation, we found that head-on collisions between tRNA transcription and replication are under-represented in the S. cerevisiae genome. We demonstrate that tRNA-mediated arrest is R-loop independent and propose that replisome arrest and DNA damage are mechanistically separable.