Browsing by Subject "DNA replication"

DNA replication is an intricate process within eukaryotic cells that must be precisely executed to preserve genetic information. This process begins at multiple start sites, or origins of replication, along each chromosome which are selected, licensed, and activated through cell-cycle regulated steps. Powerful reconstitution studies have identified the proteins involved in these processes, but they do not fully recapitulate the nuclear environment. Within the nucleus, the genome is organized in a chromatin structure consisting of DNA and all associated factors. At origins of replication, local chromatin contributes to origin identity and activation, but the precise chromatin dynamics that occur at these sites during helicase activation and initial DNA unwinding have not been fully explored. Additionally, how these steps are regulated to ensure genomic stability remain unstudied within the context of chromatin.

To address these questions, I have developed a conditional system that removes polymerase α function to capture helicase activation at replication origins in the budding yeast. Under restrictive conditions, these cells (cdc17-ts-FRB) do not initiate replication. When allowed to recover, replication appears to initiate outside origins, necessitating a delay in G2/M phase to repair unreplicated gaps at origins. To investigate origin chromatin and helicase movement prior to replication, I used MNase chromatin profiling alongside ChIP-seq for various replication factors. Chromatin in a 1 kb region around early, efficient replication origins is disrupted under restrictive conditions. The active helicase unwinds DNA out to 1 kb from these origins and is likely the source of the chromatin disruption. I next used the cdc17-ts-FRB conditional system to investigate the regulation of helicase progression in the absence of replication. I first tested whether the intra-S-phase checkpoint had a role in stalling the helicase 1 kb from the origin. Though removing checkpoint activation distributed helicase movement and chromatin disruption to late, inefficient origins, it did not alter the distance the helicase progressed from the origin. Instead, the helicase stalls as it leaves the AT-rich origin region and encounters sequences with higher GC content. These results provide in vivo support for the recently proposed “dead man’s switch” model for decreased helicase processivity when uncoupled from replication.

Helicase activation and origin unwinding are essential steps during DNA replication that expose ssDNA and thus have the potential to cause genomic instability. My studies have captured origin chromatin dynamics caused by an active helicase unwinding DNA, and have contributed evidence that the helicase may be intrinsically less processive in the absence of leading strand synthesis. These results may have implications for the mechanisms underlying human diseases involving polymerase α, and contribute to our growing understanding of how the eukaryotic cell preserves the integrity of the genome.

The complete and faithful duplication of the genome is essential to ensure normal cell division and organismal development. Eukaryotic DNA replication is initiated at multiple sites termed origins of replication that are activated at different time through S phase. The replication timing program is regulated by the S-phase checkpoint, which signals and repairs replicative stress. Eukaryotic DNA is packaged with histones into chromatin, thus DNA-templated processes including replication are modulated by the local chromatin environment such as post-translational modifications (PTMs) of histones.

One such epigenetic mark, methylation of lysine 20 on histone H4 (H4K20), has been linked to chromatin compaction, transcription, DNA repair and DNA replication. H4K20 can be mono-, di- and tri-methylated. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7 and subsequent di-/tri- methylation is catalyzed by Suv4-20. Prior studies have shown that PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which may be partially attributed to defects in origin selection and activation. Meanwhile, overexpression of mammalian PR-Set7 recruits components of pre-Replication Complex (pre-RC) onto chromatin and licenses replication origins for re-replication. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 impacts the replication program on a genomic scale. Finally, the methylation substrates of PR-Set7 include both histone (H4K20) and non-histone targets, therefore it is necessary to directly test the role of H4K20 methylation in PR-Set7 regulated phenotypes.

I employed genetic, cytological, and genomic approaches to better understand the role of H4K20 methylation in regulating DNA replication and genome stability in Drosophila melanogaster cells. Depletion of Drosophila PR-Set7 by RNAi in cultured Kc167 cells led to an ATR-dependent cell cycle arrest with near 4N DNA content and the accumulation of DNA damage, indicating a defect in completing S phase. The cells were arrested at the second S phase following PR-Set7 downregulation, suggesting that it was an epigenetic effect that coupled to the dilution of histone modification over multiple cell cycles. To directly test the role of H4K20 methylation in regulating genome integrity, I collaborated with the Duronio Lab and observed spontaneous DNA damage on the imaginal wing discs of third instar mutant larvae that had an alanine substitution on H4K20 (H4K20A) thus unable to be methylated, confirming that H4K20 is a bona fide target of PR-Set7 in maintaining genome integrity.

One possible source of DNA damage due to loss of PR-Set7 is reduced origin activity. I used BrdU-seq to profile the genome-wide origin activation pattern. However, I found that deregulation of H4K20 methylation states by manipulating the H4K20 methyltransferases PR-Set7 and Suv4-20 had no impact on origin activation throughout the genome. I then mapped the genomic distribution of DNA damage upon PR-Set7 depletion. Surprisingly, ChIP-seq of the DNA damage marker γ-H2A.v located the DNA damage to late replicating euchromatic regions of the Drosophila genome, and the strength of γ-H2A.v signal was uniformly distributed and spanned the entire late replication domain, implying stochastic replication fork collapse within late replicating regions. Together these data suggest that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains, presumably via stabilization of late replicating forks.

In addition to investigating the function of H4K20me, I also used immunofluorescence to characterize the cell cycle regulated chromatin loading of Mcm2-7 complex, the DNA helicase that licenses replication origins, using H4K20me1 level as a proxy for cell cycle stages. In parallel with chromatin spindown data by Powell et al. (Powell et al. 2015), we showed a continuous loading of Mcm2-7 during G1 and a progressive removal from chromatin through S phase.

All organisms must accurately replicate their genomic DNA in order to transmit genetic information from generation to generation. The cognate Watson-Crick base pairs (dA•dT, dG•dC) adopt near identical ‘Watson-Crick geometry’ as defined by the hydrogen bonding pattern (height and depth) and the distance between the nucleoside sugar C1’ atoms (width). The shape complementarity of Watson-Crick pairs is a significant determinant in the selection of a correct nucleotide for a given template base during replication. Since the discovery of the DNA double helix, more than 60 years ago, the formation of Watson-Crick-like mismatch base pairs, stabilized by rare, energetically less favorable tautomeric or anionic bases, has been hypothesized as a cause of spontaneous mutation. However, these proposed lowly-populated and short-lived Watson-Crick-like conformational ‘excited states’ are characterized by subtle movements of protons and π-bonds that have proven difficult to visualize experimentally, even with modern biophysical techniques.

We have utilized nuclear magnetic resonance relaxation dispersion experiments in conjunction with kinetic modeling and in vitro assays to characterize the formation of tautomeric and anionic Watson-Crick-like dG•dT excited states in DNA duplexes and investigate their involvement in DNA replication errors. Insertion of the sequence- dependent tautomerization or ionization step into minimal kinetic mechanisms for correct incorporation during replication after the initial binding of the nucleotide, leads to accurate predictions of the probability of dG•dT misincorporation across different polymerases and pH conditions and for a chemically modified nucleotide, and providing mechanisms for sequence-dependent misincorporation. Our results indicate that the system is under thermodynamic control and that the energetic penalty for tautomerization and/or ionization accounts for an approximately 10-2 to 10-3-fold discrimination against misincorporation, which proceeds primarily via tautomeric dGenol•dT and dG•dTenol, with contributions from anionic dG•dT- dominant at pH 8.4 and above, or for some mutagenic nucleotides. Kinetic modeling reveals additional plausible pathways for dG•dT misincorporation in which the tautomerization event takes place prior to binding or in which the polymerase alters the kinetics of tautomerization within the active site.

The conformational landscape of the dA•dG mismatch has been characterized with the use of NMR relaxation dispersion in DNA duplexes. The mismatch has been shown to adopt three predominant forms: Aanti•Ganti, Asyn•Ganti, and A+anti•Gsyn. We have characterized sequence-specific conformational exchange between all three of these base pair forms in multiple sequence contexts. In addition, we find that nearest-neighbor base changes can alter the ground state conformation of the dA•dG base pair between Aanti•Ganti and Asyn•Ganti. Such sequence-specific alterations to the conformational landscape have been proposed to alter reaction rates of an adenine glycosylase repair protein, MutY. Notably, this work shows for the first time that the Asyn•Ganti base pair is able to form in solution both as a ground state and excited state base pair; and may influence the activity of MutY. In addition, two tautomeric forms of the dA•dG base pair have been proposed to form WC-like base pairs but R1ρ experiments targeting the NH2 functional groups of dA and dG have been thus far unable to observe these proposed states.

Abstract

DNA replication is an integral part of the cell cycle. Every time a cell divides, the entire genome has to be copied once and only once in a timely manner. In order to accomplish this, DNA replication begins at many points throughout the genome. These start sites are called origins of replication, and they are initiated in a temporal manner throughout S phase. How these origins are selected and regulated is poorly understood. Saccharomyces cerevisiae and Schizosaccharomyces pombe have autonomously replicating sequences (ARS) that can replicate plasmids extrachromosomally and function as origins in the genome. Metazoans, however, have shown no evidence of ARS activity.

DNA replication is a multistep process with several opportunities for regulation. Potential origins are marked with the origin recognition complex (ORC), a six subunit complex. In S. cerevisiae, ORC binds to the ARS consensus sequence (ACS), but no sequence specificity is seen in S. pombe or in metazoans. Therefore, factors other than sequence play a role in origin selection.

In G1, the pre-replicative (pre-RC) complex assembles at potential origins. This involves the recruitment of Cdc6 and Cdt1 to ORC, which then recruits MCM2-7 to the origin. In S phase, a subset of these pre-RC marked origins are initiated for replication. These origins are not fired simultaneously; instead, origins are fired in a temporal manner, with some firing early, some firing late, and some not firing at all.

The temporal firing of origins leads to wide regions of the genome being copied at different times during S phase. , which makes up the replication timing profile of the genome. These regions are not random, and several correlations between replication timing and both transcriptional activity and chromosomal landscape. Regions of the genome with high transcriptional activity tend to replicate earlier in S phase, and it is well know that the gene rich euchromatin replicates earlier than the gene poor heterochromatin. Additionally, areas of the genome with activating chromatin marks also replicate earlier than regions with repressive marks. Though many correlations have been observed, no single mark or transcriptional player has been shown to directly influence replication timing.

We mapped the replication timing profiles of three cell lines derived from Drosophila melanogaster by pulsing cells with the nucleotide analog bromodeoxyuridine (BrdU), enriching for actively replicating DNA labeled with BrdU, sequencing with high throughput sequencing and mapping the sequences back to the genome. We found that the X chromosome of the male cell lines replicated earlier than the X chromosome in the female cell line or the autosomes. We were then able to compare the replication timing profiles to data sets for chromatin marks acquired through the modENCODE (model organism Encyclopedia Of DNA Elements). We found that the early replicating regions of the male X chromosomes correlates with acetylation of lysine 16 on histone 4 (H4K16).

Hyperacetylation of H4K16 on the X chromosome in males is a consequence of dosage compensation in D. melanogaster. Like many organisms, D. melanogaster females have two X chromosomes while males have one. To compensate for this difference, males upregulate the genes on the X chromosome two-fold. This upregulation is regulated by the dosage compensation complex (DCC), which is restricted to the X chromosome. This complex includes a histone acetyl transferase, MOF, which acetylates H4K16. This hyperacetylation allows for increased transcription of the X chromosome.

We hypothesized that the activities of the DCC and the hyperacetylation of H4K16 also influences DNA replication timing. To test this, I knocked down components of the DCC (MSL2 and MOF) using RNAi. Cells were arrested in early S phase with hydroxyurea, released, and pulsed with the nucleotide analog EdU. The cells were arrested in metaphase and labeled for H4K16 acetylation and EdU. We found that male cells were preferentially labeled with EdU on the X chromosome, which corresponded with H4k16 acetylation. When the DCC was knocked down, H4K16 acetylation was lost along with preferential EdU labeling on the X chromosome. These results suggest that the DCC and H4K16 acetylation are necessary for early replication of the X chromosome. Additionally, early origin mapping of different cell lines showed that while ORC density does not differ between male and female cell lines, early origin usage is increased on the X chromosome of males, suggesting that this phenomenon is regulated at the level of activation, not pre-RC formation. Other experiments in female cell lines have been unclear about whether the DCC and subsequent H4K16Ac is sufficient for early X replication. However, these results are exciting because this is, to our knowledge, the first mark that has been found to directly influence replication timing.

In addition to these timing studies, I attempted to design a new way to map origins. A consequence of unidirectional replication with bidirectional replication fork movement is Okazaki fragments. These are short nascent strands on the lagging strand of replicating DNA. Because these fragments are small, we can isolate them by size and map them back to the genome. Okazaki density could tell us about origin usage and any directional preferences of origins. The process proved to be tedious, and although they mapped back with a higher density around ORC binding sites than randomly sheared DNA, little information about origin usage was garnered from the data. Additionally, the process proved difficult to repeat.

In these studies, we examined the replication timing program in D. melanogaster. We found that the male X chromosome replicates earlier in S phase, and this early replication is regulated by the DCC. However, it is unclear if the change in chromatin landscape directly influences replication or if the replication program is responding to other dosage compensation cues on the X chromosome. Regardless, we have found one the first conditions in which a mark directly influences the DNA replication timing program. 

All DNA-templated events, including replication and gene transcription, occur in the context of the local chromatin environment. The passage of the replication machinery results in disassembly of chromatin, which must be re-assembled behind the replication fork to re-establish the epigenetic state of the cell. Many of the factors and mechanisms regulating DNA replication and chromatin assembly have been identified from elegant in vitro biochemical experiments, work in model systems like Saccharomyces cerevisiae, or novel proteomic approaches. In spite of current advances in the field, it is still not clear how the chromatin landscape is organized and re-assembled during this process.

Current methods, while informative, lack the genome-wide base-pair resolution required to assess the dynamics of chromatin assembly and maturation in a spatial-temporal manner. To overcome the limitations of these studies, I have taken advantage of an epigenome mapping technique based on micrococcal nuclease (MNase) digestion followed by paired-end sequencing. This approach facilitates the analysis of chromatin structure by capturing not only nucleosomes, but also smaller DNA binding protein footprints in a factor-agnostic manner. I have developed a technique based on this approach that generates Nascent Chromatin Occupancy Profiles (NCOPs) to study the dynamics of chromatin assembly following passage of the DNA replication fork at a genome-wide level and at single base-pair resolution in S. cerevisiae. It employs a nucleoside analog to specifically enrich for nascent chromatin, which can be captured following a chase over different periods of time. Thus, NCOPs resolve the structure of nascent and mature chromatin, facilitating the analysis of chromatin maturation across the entire genome.

Using NCOPs, I provide a comprehensive description of the maturation process across different genomic regions and the dynamics of small DNA binding factor association with nascent and mature chromatin states. Our results support previous work characterizing the structure of nascent chromatin as being more disorganized and having poorly positioned nucleosomes. Importantly, using positioning and occupancy scores, I provide new details on the structure of nascent and mature chromatin at intergenic regions, including replication origins, and at highly transcribed and poorly transcribed genes. I uncovered that local epigenetic footprints have the potential to shape the dynamics of chromatin assembly, generating a chromatin maturation landscape that is dependent on the parental chromatin. Finally, I resolved patterns of transcription factor occupancy with nascent and mature chromatin, and observed transient factor association in the nascent state.

In all, this work provides insight into the dynamics of chromatin assembly, and allows for genome-wide and base-pair resolution investigation of chromatin maturation. The genomic and bioinformatic approaches developed here open the door for further investigation of the dynamics of epigenetic inheritance and the role of known and unknown players in re-establishing the eukaryotic epigenome following passage of the DNA replication fork.

Eukaryotic genomes have extensive flexibility and plasticity to modify transcription and replication programs, yielding a myriad of differentiated cell types and survival mechanisms to adverse environmental conditions. As these genomic processes require precise localization of DNA-binding factors, their dynamic temporal and spatial distributions provide dramatically different interpretations of a static genome sequence. DNA-binding factors must compete with nucleosomes, the basic subunit of chromatin, for access to the underlying DNA sequence. Even though the spatial preferences of these proteins are partially explained by DNA sequence alone, the complete genome occupancy profile has remained elusive, and we currently have a limited understanding of how DNA-binding protein configurations directly impact transcription and replication function.

Profiling the entire chromatin environment has typically required multiple experiments to capture both DNA-binding factors and nucleosomes. Here, we have extended the traditional micrococcal nuclease (MNase) digestion assay to simultaneously resolve both nucleosomes and smaller DNA-binding footprints in Saccharomyces cerevisiae. Visualization of protected DNA fragments revealed a nucleotide-resolution view of the chromatin architecture at individual genomic loci. We show that different MNase digestion times can capture nucleosomes partially unwrapped or complexed with chromatin remodelers. Stereotypical DNA-binding footprints are evident across all promoters, even at low-transcribed and silent genes. By aggregating the chromatin profiles across transcription-factor--binding sites, we precisely resolve protein footprints, yielding in vivo insights into protein-DNA interactions. Together, our MNase method, in one experiment, provides an unprecedented assessment of the entire chromatin structure genome-wide.

We utilized this approach to interrogate how the replication program is regulated by the chromatin environment surrounding DNA replication initiation sites. Pre-replicative complex (pre-RC) formation commences with recruitment of the origin recognition complex (ORC) to specific locations in the genome, termed replication origins. Although successful pre-RC assembly primes each site for S-phase initiation by loading the Mcm2-7 helicase, replication origins have substantially different activation times and efficiencies. We posited that replication origin function is substantially impacted by the local chromatin environment. Here, we resolved a high-resolution ORC-dependent footprint at 269 replication origins genome-wide. Even though ORC in S. cerevisiae remains bound at replication origins throughout the cell cycle, we detected a subset of inefficient origins that did not yield a footprint until G1, suggesting a more transient ORC interaction prior to pre-RC assembly. Nucleosome movement accommodated the pre-RC-induced expansion of the ORC-dependent footprint in G1, leading to increased activation efficiency. Mcm2-7 loading is preferentially directed to one side of each replication origin, in close proximity to the origin-flanking nucleosome. Our data demonstrates that pre-RC components are assembled into multiple configurations in vivo.

We anticipate that extending chromatin occupancy profiling to many different cell types will reveal further insights into genome regulation.

In eukaryotic cells, The local chromatin structures play a critical role in regulating all DNA-mediated events. However, the process of DNA replication is highly disruptive to the chromatin structure. Specifically, the parental chromatin needs to be disassembled before the passing of the replication fork and re-assembled afterward. Additionally, a maturation process is required for newly assembled chromatin to completely recover to the pre-replicative state. Rapid and faithful assembly and maturation of chromatin are essential for preserving the epigenome and maintaining genome integrity. Elegant in vitro biochemistry studies have allowed the characterization of numerous factors in the process of replication-coupled chromatin assembly. Recent advancements in genomic technologies further expanded our understanding of the process, providing genome-wide views of the spatial-temporal dynamics of chromatin maturation. However, there is still a lack of mechanistic details on many aspects of chromatin assembly, and the broader phenotypic significance of this process also remains unstudied.

One such example is Chromatin Assembly Factor 1 (CAF-1). It is one of the first histone chaperones identified that deposit histones onto the newly replicated DNA, yet there remain missing puzzles of CAF-1 ranging from the mechanisms by which CAF-1 interacts with DNA and the replisomes to its impact on chromatin maturation and how that relates to genome stability. Using nascent chromatin occupancy profiling (NCOP), I tracked the chromatin maturation kinetics in WT and CAF-1 mutant cells with high spatiotemporal resolution. I found that loss of CAF-1 results in a heterogeneous rate of nucleosome assembly, with individual nucleosomes exhibiting either rapid or slow maturation kinetics, ultimately leading to a global delay in chromatin maturation. The slow-to-mature nucleosomes are enriched with poly(dA:dT) sequences, suggesting CAF-1 facilitates (H3-H4)2 tetramer deposition and nucleosome maturation on sequences resistant to nucleosome formation. The assembly defect caused by loss of CAF-1 leads to the accumulation of nucleosome intermediates whose position is also influenced by underlying DNA sequences. The finding offers a new perspective on the pathway for nucleosome assembly in vivo. In addition, there exists a long-standing paradox that CAF-1 mutant cells exhibit loss of gene silencing and increased cryptic transcription, yet their steady-state chromatin landscape is nearly indistinguishable from that of WT cells. Our work illustrates that the dysregulation of transcription is temporary and occurs specifically in S phase, presumably as a result of transient defects in chromatin maturation. These results demonstrate how the DNA replication program can directly shape the chromatin landscape and regulate gene expression through the process of chromatin maturation.

During replication, the leading and lagging strands are replicated via distinct mechanisms. Numerous studies showed that different histone chaperones are employed for nucleosome assembly on the two strands; however, it remains unclear if or how the kinetics of chromatin maturation differ between the two strands. The current NCOP method does not distinguish actions on the leading and lagging strands. To address this, I developed strand-specific NCOP that captures the kinetics of chromatin maturation on the two daughter strands separately. Strand-specific NCOP experiments on CAF-1 and Mcm2 mutant cells provide evidence for the coordinated nucleosome assembly by Mcm2 and CAF-1 to ensure symmetric histone segregation and assembly on the daughter strands. Together, this work provides mechanistic insights into the role of multiple histone chaperones in chromatin assembly and maturation and opens up new avenues for understanding how disruption to these processes might contribute to defects in gene expression programs commonly found in disease states.

RecQ4, a member of the conserved RecQ family of helicases, is involved in replication and associated with several clinical syndromes. Although biologically important, the biochemistry of RecQ4 has remained elusive. We have expressed and purified Drosophila melanogaster RecQ4 from a baculovirus expression system. Biochemical characterization of the helicase, ATP hydrolysis, annealing, and binding activities of the enzyme has been performed, using native and non-native gel electrophoresis and thin layer chromatography, among other techniques. These reveal that RecQ4 is a 3' to 5' helicase that is stimulated by the presence of single-stranded DNA 3' of the duplex DNA region to be unwound. The enzyme is also capable of annealing complementary DNA strands, though this is inhibited by AMPPNP, a non-hydrolyzable analog of ATP. RecQ4 also forms a stable complex with single-stranded DNA in the presence of AMPPNP. We argue that the helicase activity of RecQ4 is important to the process of DNA replication. This leads to the conclusion that two helicases, RecQ4 and the Mcm2-7 complex, are involved in replication. The manner of their simultaneous involvement is not intuitive, and so models by which the two enzymes may cooperate are discussed.