Compaction of replicated chromosomes into morphologically and spatially distinct sister chromatids is essential for faithful DNA segregation in all organisms. This is a complex, poorly understood process that is controlled by the physical and mechanical properties of DNA as well as the action of specific proteins and enzymes. In bacteria, only a small set of essential factors are required for chromosome compaction, offering the hope that it will be relatively straightforward to understand the principles by which they act. We have developed and adapted a new set of molecular and cytological assays that will allow us to address specific steps in DNA compaction and organization in the model organism Bacillus subtilis. Here, newly replicated DNA is compacted by the combined activities of topoisomerases that generate interwound (supercoiled) loops, small nucleoid-associated proteins that bend DNA, and SMC condensin complexes, which are thought to bridge DNA segments. We know that the organization of the compacted chromosome involves folding both at short and long length-scales, but what these two levels of organization look like and how they are coordinated are unclear. Using genome-wide chromosome conformation capture complemented by quantitative cytological assay, we will define compaction on short length-scales and chromosome organization on long length-scales. This new level of description will then allow us to establish the contributions of and interplay between the small set of essential factors that compact the chromosome and the transcription machinery that opens up the DNA. In particular, we aim to gain insight into the role of the highly conserved SMC condensin complex, for which we have the least information. Our preliminary data indicate that important factors that participate in chromosome compaction and segregation have been missed by traditional genetic screens, and we will take a novel cytological approach and high throughput synthetic lethal screens to identify them.
Our specific aims are to: 1) Determine how the SMC condensin complex contributes to chromosome conformation in vivo;define the role of ParB/parS and highly transcribed genes in SMC-mediated origin segregation;and establish how SMC is enriched at these loci. 2) Define how DNA is compacted on short length-scales throughout the chromosome using genome-wide interaction frequencies. 3) Identify and characterize new chromosome organization and segregation factors using high throughput cytological and synthetic lethal screens.

Public Health Relevance

In this proposal we investigate how bacterial chromosomes are compacted, organized, and segregated. Because chromosome segregation is essential, understanding the molecular mechanisms underlying this process could lead to the discovery of new targets appropriate for antimicrobial intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM086466-05A1
Application #
8756076
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Janes, Daniel E
Project Start
2009-08-01
Project End
2018-05-31
Budget Start
2014-09-18
Budget End
2015-05-31
Support Year
5
Fiscal Year
2014
Total Cost
$356,586
Indirect Cost
$146,211
Name
Harvard Medical School
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
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Rodrigues, Christopher D A; Henry, Xavier; Neumann, Emmanuelle et al. (2016) A ring-shaped conduit connects the mother cell and forespore during sporulation in Bacillus subtilis. Proc Natl Acad Sci U S A 113:11585-11590
Wang, Xindan; Le, Tung B K; Lajoie, Bryan R et al. (2015) Condensin promotes the juxtaposition of DNA flanking its loading site in Bacillus subtilis. Genes Dev 29:1661-75
Meeske, Alexander J; Sham, Lok-To; Kimsey, Harvey et al. (2015) MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis. Proc Natl Acad Sci U S A 112:6437-42
Wang, Xindan; Montero Llopis, Paula; Rudner, David Z (2014) Bacillus subtilis chromosome organization oscillates between two distinct patterns. Proc Natl Acad Sci U S A 111:12877-82
Wang, Xindan; Tang, Olive W; Riley, Eammon P et al. (2014) The SMC condensin complex is required for origin segregation in Bacillus subtilis. Curr Biol 24:287-92
Wang, Xindan; Rudner, David Z (2014) Spatial organization of bacterial chromosomes. Curr Opin Microbiol 22:66-72
Broedersz, Chase P; Wang, Xindan; Meir, Yigal et al. (2014) Condensation and localization of the partitioning protein ParB on the bacterial chromosome. Proc Natl Acad Sci U S A 111:8809-14
Graham, Thomas G W; Wang, Xindan; Song, Dan et al. (2014) ParB spreading requires DNA bridging. Genes Dev 28:1228-38
Meisner, Jeffrey; Montero Llopis, Paula; Sham, Lok-To et al. (2013) FtsEX is required for CwlO peptidoglycan hydrolase activity during cell wall elongation in Bacillus subtilis. Mol Microbiol 89:1069-83

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