SCIENTIFIC PUBLICATIONS
Publications
1. Nucleic Acids Res. 2019 Feb 28;47(4):1814-1822. doi: 10.1093/nar/gky1242.
Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replication in vivo.
Kahli M(1), Osmundson JS(1), Yeung R(1), Smith DJ(1).
Prior to ligation, each Okazaki fragment synthesized on the lagging strand in eukaryotes must be nucleolytically processed. Nuclease cleavage takes place in the context of 5' flap structures generated via strand-displacement synthesis by DNA polymerase delta. At least three DNA nucleases: Rad27 (Fen1), Dna2 and Exo1, have been implicated in processing Okazaki fragment flaps. However, neither the contributions of individual nucleases to lagging-strand synthesis nor the structure of the DNA intermediates formed in their absence have been fully defined in vivo. By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki fragments synthesized in vivo in Saccharomyces cerevisiae, we conduct a systematic evaluation of the impact of Rad27, Dna2 and Exo1 on lagging-strand synthesis. We find that Rad27 processes the majority of lagging-strand flaps, with a significant additional contribution from Exo1 but not from Dna2. When nuclease cleavage is impaired, we observe a reduction in strand-displacement synthesis as opposed to the widespread generation of long Okazaki fragment 5' flaps, as predicted by some models. Further, using cell cycle-restricted constructs, we demonstrate that both the nucleolytic processing and the ligation of Okazaki fragments can be uncoupled from DNA replication and delayed until after synthesis of the majority of the genome is complete.
PMCID: PMC6393292
2. Nat Struct Mol Biol. 2017 Feb;24(2):162-170. doi: 10.1038/nsmb.3342. Epub 2016 Dec 19.
Pif1-family helicases cooperatively suppress widespread replication-fork arrest at tRNA genes.
Osmundson JS(1), Kumar J(1), Yeung R(1), Smith DJ(1).
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.
PMID: 27991904 [Indexed for MEDLINE]
3. Biochem Mol Biol Educ. 2015 May-Jun;43(3):145-53. doi: 10.1002/bmb.20844. Epub 2015 Mar 4.
Doing that thing that scientists do: A discovery-driven module on protein purification and characterization for the undergraduate biochemistry laboratory classroom.
Garrett TA(1), Osmundson J(2), Isaacson M(2), Herrera J(1).
In traditional introductory biochemistry laboratory classes students learn techniques for protein purification and analysis by following provided, established, step-by-step procedures. Students are exposed to a variety of biochemical techniques but are often not developing procedures or collecting new, original data. In this laboratory module, students develop research skills through work on an original research project and gain confidence in their ability to design and execute an experiment while faculty can enhance their scholarly pursuits through the acquisition of original data in the classroom laboratory. Students are prepared for a 6-8 week discovery-driven project on the purification of the Escherichia coli cytidylate kinase (CMP kinase) through in class problems and other laboratory exercises on bioinformatics and protein structure analysis. After a minimal amount of guidance on how to perform the CMP kinase in vitro enzyme assay, SDS-PAGE, and the basics of protein purification, students, working in groups of three to four, develop a protein purification protocol based on the scientific literature and investigate some aspect of CMP kinase that interests them. Through this process, students learn how to implement a new but perhaps previously worked out procedure to answer their research question. In addition, they learn the importance of keeping a clear and thorough laboratory notebook and how to interpret their data and use that data to inform the next set of experiments. Following this module, students had increased confidence in their ability to do basic biochemistry techniques and reported that the "self-directed" nature of this lab increased their engagement in the project.
PMID: 25735767 [Indexed for MEDLINE]
4. PLoS One. 2013 Oct 7;8(10):e76572. doi: 10.1371/journal.pone.0076572. eCollection 2013.
RNA-Seq reveals differential gene expression in Staphylococcus aureus with single-nucleotide resolution.
Osmundson J(1), Dewell S, Darst SA.
Staphylococcus aureus is a gram-positive cocci and an important human commensal bacteria and pathogen. S. aureus infections are increasingly difficult to treat because of the emergence of highly resistant MRSA (methicillin-resistant S. aureus) strains. Here we present a method to study differential gene expression in S. aureus using high-throughput RNA-sequencing (RNA-seq). We used RNA-seq to examine gene expression in S. aureus RN4220 cells containing an exogenously expressed transcription factor and between two S. aureus strains (RN4220 and NCTC8325-4). We investigated the sequence and gene expression differences between RN4220 and NCTC8325-4 and used the RNA-seq data to identify S. aureus promoters suitable for in vitro analysis. We used RNA-seq to describe, on a genome wide scale, genes positively and negatively regulated by the phage encoded transcription factor gp67. RNA-seq offers the ability to study differential gene expression with single-nucleotide resolution, and is a considerable improvement over the predominant genome-wide transcriptome technologies used in S. aureus.
PMID: 24116120 [Indexed for MEDLINE]
5. Proc Natl Acad Sci U S A. 2013 Jul 30;110(31):12619-24. doi: 10.1073/pnas.1308270110. Epub 2013 Jul 15.
Structure and function of CarD, an essential mycobacterial transcription factor.
Srivastava DB(1), Leon K, Osmundson J, Garner AL, Weiss LA, Westblade LF, Glickman MS, Landick R, Darst SA, Stallings CL, Campbell EA.
CarD, an essential transcription regulator in Mycobacterium tuberculosis, directly interacts with the RNA polymerase (RNAP). We used a combination of in vivo and in vitro approaches to establish that CarD is a global regulator that stimulates the formation of RNAP-holoenzyme open promoter (RPo) complexes. We determined the X-ray crystal structure of Thermus thermophilus CarD, allowing us to generate a structural model of the CarD/RPo complex. On the basis of our structural and functional analyses, we propose that CarD functions by forming protein/protein and protein/DNA interactions that bridge the RNAP to the promoter DNA. CarD appears poised to interact with a DNA structure uniquely presented by the RPo: the splayed minor groove at the double-stranded/single-stranded DNA junction at the upstream edge of the transcription bubble. Thus, CarD uses an unusual mechanism for regulating transcription, sensing the DNA conformation where transcription bubble formation initiates.
PMID: 23858468 [Indexed for MEDLINE]
6. Bacteriophage. 2013 Jan 1;3(1):e24767. doi: 10.4161/bact.24767.
Biochemical insights into the function of phage G1 gp67 in Staphylococcus aureus.
Osmundson J(1), Darst SA.
Author information: (1)The Rockefeller University; New York, NY USA.
Bacteriophage (phage) are among the most diverse and abundant life forms on Earth. Studies have recently used phage diversity to identify novel antimicrobial peptides and proteins. We showed that one such phage protein, Staphylococcus aureus (Sau) phage G1 gp67, inhibits cell growth in Sau by an unusual mechanism. Gp67 binds to the host RNA polymerase (RNAP) through an interaction with the promoter specificity σ subunit, but unlike many other σ-binding phage proteins, gp67 does not disrupt transcription at most promoters. Rather, gp67 prevents binding of another RNAP domain, the α-C-terminal domain, to upstream A/T-rich elements required for robust transcription at rRNA promoters. Here, we discuss additional biochemical insights on gp67, how phage promoters escape the inhibitory function of gp67, and methodological advancements that were foundational to our work.
PMID: 23819108
7. J Bacteriol. 2013 Aug;195(16):3621-8. doi: 10.1128/JB.00499-13. Epub 2013 Jun 7.
Phage-encoded inhibitor of Staphylococcus aureus transcription exerts context-dependent effects on promoter function in a modified Escherichia coli-based transcription system.
Montero-Diez C(1), Deighan P, Osmundson J, Darst SA, Hochschild A.
Promoter recognition in bacteria is mediated primarily by the σ subunit of RNA polymerase (RNAP), which makes sequence-specific contacts with the promoter -10 and -35 elements in the context of the RNAP holoenzyme. However, the RNAP α subunit can also contribute to promoter recognition by making sequence-specific contacts with upstream (UP) elements that are associated with a subset of promoters, including the rRNA promoters. In Escherichia coli, these interactions between the RNAP α subunit (its C-terminal domain [CTD], in particular) and UP element DNA result in significant stimulation of rRNA transcription. Among the many cellular and bacteriophage-encoded regulators of transcription initiation that have been functionally dissected, most exert their effects via a direct interaction with either the σ or the α subunit. An unusual example is provided by a phage-encoded inhibitor of RNA synthesis in Staphylococcus aureus. This protein, phage G1 gp67, which binds tightly to σ in the context of the S. aureus RNAP holoenzyme, has recently been shown to exert selective effects on transcription by inhibiting the function of the α subunit CTD (αCTD). Here we report the development of a gp67-responsive E. coli-based transcription system. We examine transcription in vitro from promoters that do or do not carry the UP element associated with a well-characterized E. coli rRNA promoter. Our findings indicate that the αCTD can increase promoter activity significantly even in the absence of an UP element. We also find that gp67 can exert αCTD-dependent or αCTD-independent effects on transcription depending on the particular promoter, indicating that the mechanism of gp67 action is context dependent.
PMID: 23749973 [Indexed for MEDLINE]
8. Cell. 2012 Nov 21;151(5):1005-16. doi: 10.1016/j.cell.2012.10.034.
Promoter-specific transcription inhibition in Staphylococcus aureus by a phage protein.
Osmundson J(1), Montero-Diez C, Westblade LF, Hochschild A, Darst SA.
Phage G1 gp67 is a 23 kDa protein that binds to the Staphylococcus aureus (Sau) RNA polymerase (RNAP) σ(A) subunit and blocks cell growth by inhibiting transcription. We show that gp67 has little to no effect on transcription from most promoters but is a potent inhibitor of ribosomal RNA transcription. A 2.0-Å-resolution crystal structure of the complex between gp67 and Sau σ(A) domain 4 (σ(A)(4)) explains how gp67 joins the RNAP promoter complex through σ(A)(4) without significantly affecting σ(A)(4) function. Our results indicate that gp67 forms a complex with RNAP at most, if not all, σ(A)-dependent promoters, but selectively inhibits promoters that depend on an interaction between upstream DNA and the RNAP α-subunit C-terminal domain (αCTD). Thus, we reveal a promoter-specific transcription inhibition mechanism by which gp67 interacts with the RNAP promoter complex through one subunit (σ(A)), and selectively affects the function of another subunit (αCTD) depending on promoter usage.
PMID: 23178120 [Indexed for MEDLINE]
9. Cell Microbiol. 2007 Dec;9(12):2870-9. doi: 10.1111/j.1462-5822.2007.01002.x.
Activation of classical pathway of complement cascade by soluble oligomers of prion.
Dumestre-Pérard C(1), Osmundson J, Lemaire-Vieille C, Thielens N, Grives A, Favier B, Csopaki F, Jamin M, Gagnon J, Cesbron JY.
Mice defective for C1q complement factor show enhanced resistance to peripheral prion inoculation, and previous work demonstrated a direct interaction between C1q and conformationally modified PrP. However, the nature and physiological consequences of this interaction remain uncharacterized. PrP amino acids 141-159 has been identified as a potential C1q binding site; we show, by both surface plasmon resonance (SPR) spectroscopy and ELISA, that C1q and its globular region bind to PrP mutagenized in the region of interest with comparable efficiency to that of wild-type protein. To test PrP's ability to activate complement, soluble oligomers of the PrP constructs were made. Only PrP and mutagenized PrP oligomers activate the classical complement cascade while PrP monomer and the C-terminal domain, both in oligomeric and in monomeric form, failed to induce activation. This suggests that a conformational change in PrP, which occurs both when PrP is bound to an SPR sensor chip and when it undergoes oligomerization, is requisite for PrP/C1q interaction and activation of the complement cascade. We propose that C1q may act as a natural sensor for prions, leading to activation of the classical complement cascade, which could result in local inflammation and subsequent recruitment of the immune cells that prions initially infect.
PMID: 17991046 [Indexed for MEDLINE]