Chromosomes in all organisms are highly organized and divided into multiple chromosomal interaction domains, or topological domains. Regions of active, high transcription help establish and maintain ...domain boundaries, but precisely how this occurs remains unclear. Here, using fluorescence microscopy and chromosome conformation capture in conjunction with deep sequencing (Hi‐C), we show that in Caulobacter crescentus, both transcription rate and transcript length, independent of concurrent translation, drive the formation of domain boundaries. We find that long, highly expressed genes do not form topological boundaries simply through the inhibition of supercoil diffusion. Instead, our results support a model in which long, active regions of transcription drive local decompaction of the chromosome, with these more open regions of the chromosome forming spatial gaps in vivo that diminish contacts between DNA in neighboring domains. These insights into the molecular forces responsible for domain formation in Caulobacter likely generalize to other bacteria and possibly eukaryotes.
Synopsis
Chromosomes universally consist of multiple topological interaction domains. Chromosome conformation capture in conjunction with deep sequencing (Hi‐C) and fluorescence microscopy identify mechanisms for topological domain in Caulobacter crescentus chromosomes.
The boundaries between chromosomal interaction domains seen in Hi‐C maps of Caulobacter are frequently associated with long, highly expressed genes.
Domain boundaries are typically associated with highly expressed genes longer than ˜2 kb.
Translating ribosomes do not contribute to domain boundary formation.
Transcription rate and transcript length contribute to an inhibition of supercoil diffusion, but this is not a primary factor in creating Hi‐C boundaries.
Long transcripts locally decompact DNA, which separates the flanking loci and creates the boundaries visible by Hi‐C.
Chromosome conformation capture in conjunction with deep sequencing (Hi‐C) and fluorescence microscopy identify mechanisms for topological domain in Caulobacter crescentus chromosomes.
In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-
system is widely employed for chromosome segregation in bacteria. A ...DNA-binding protein ParB nucleates on
sites and must associate with neighboring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at
sites recruit hundreds of further ParB to spread is not fully understood. Here, we reconstitute a
-dependent ParB spreading event using purified proteins from
and show that CTP is required for spreading. We further show that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-
with ParA and SMC.
Proper chromosome segregation is essential in all living organisms. The ParA-ParB-
system is widely employed for chromosome segregation in bacteria. Previously, we showed that
ParB requires cytidine ...triphosphate to escape the nucleation site
and spread by sliding to the neighboring DNA (Jalal et al., 2020). Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated
ParB with
and with a CTP analog. Nucleating ParB is an open clamp, in which
is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving
into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products are likely associated with reopening of the gates to release DNA and recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.
Bacterial Chromosome Organization and Segregation Badrinarayanan, Anjana; Le, Tung B.K; Laub, Michael T
Annual review of cell and developmental biology,
11/2015, Letnik:
31, Številka:
1
Journal Article
Recenzirano
Odprti dostop
If fully stretched out, a typical bacterial chromosome would be nearly 1 mm long, approximately 1,000 times the length of a cell. Not only must cells massively compact their genetic material, but ...they must also organize their DNA in a manner that is compatible with a range of cellular processes, including DNA replication, DNA repair, homologous recombination, and horizontal gene transfer. Recent work, driven in part by technological advances, has begun to reveal the general principles of chromosome organization in bacteria. Here, drawing on studies of many different organisms, we review the emerging picture of how bacterial chromosomes are structured at multiple length scales, highlighting the functions of various DNA-binding proteins and the impact of physical forces. Additionally, we discuss the spatial dynamics of chromosomes, particularly during their segregation to daughter cells. Although there has been tremendous progress, we also highlight gaps that remain in understanding chromosome organization and segregation.
Chromosomes must be highly compacted and organized within cells, but how this is achieved in vivo remains poorly understood. We report the use of chromosome conformation capture coupled with deep ...sequencing (Hi-C) to map the structure of bacterial chromosomes. Analysis of Hi-C data and polymer modeling indicates that the Caulobacter crescentus chromosome consists of multiple, largely independent spatial domains that are probably composed of supercoiled plectonemes arrayed into a bottle brush-like fiber. These domains are stable throughout the cell cycle and are reestablished concomitantly with DNA replication. We provide evidence that domain boundaries are established by highly expressed genes and the formation of plectoneme-free regions, whereas the histone-like protein HU and SMC (structural maintenance of chromosomes) promote short-range compaction and the colinearity of chromosomal arms, respectively. Collectively, our results reveal general principles for the organization and structure of chromosomes in vivo.
Structural maintenance of chromosomes (SMC) complexes play critical roles in chromosome dynamics in virtually all organisms, but how they function remains poorly understood. In the bacterium Bacillus ...subtilis, SMC-condensin complexes are topologically loaded at centromeric sites adjacent to the replication origin. Here we provide evidence that these ring-shaped assemblies tether the left and right chromosome arms together while traveling from the origin to the terminus (>2 megabases) at rates >50 kilobases per minute. Condensin movement scales linearly with time, providing evidence for an active transport mechanism. These data support a model in which SMC complexes function by processively enlarging DNA loops. Loop formation followed by processive enlargement provides a mechanism by which condensin complexes compact and resolve sister chromatids in mitosis and by which cohesin generates topologically associating domains during interphase.
SMC condensin complexes play a central role in compacting and resolving replicated chromosomes in virtually all organisms, yet how they accomplish this remains elusive. In Bacillus subtilis, ...condensin is loaded at centromeric parS sites, where it encircles DNA and individualizes newly replicated origins. Using chromosome conformation capture and cytological assays, we show that condensin recruitment to origin-proximal parS sites is required for the juxtaposition of the two chromosome arms. Recruitment to ectopic parS sites promotes alignment of large tracks of DNA flanking these sites. Importantly, insertion of parS sites on opposing arms indicates that these "zip-up" interactions only occur between adjacent DNA segments. Collectively, our data suggest that condensin resolves replicated origins by promoting the juxtaposition of DNA flanking parS sites, drawing sister origins in on themselves and away from each other. These results are consistent with a model in which condensin encircles the DNA flanking its loading site and then slides down, tethering the two arms together. Lengthwise condensation via loop extrusion could provide a generalizable mechanism by which condensin complexes act dynamically to individualize origins in B. subtilis and, when loaded along eukaryotic chromosomes, resolve them during mitosis.
Segregation of genetic material is a fundamental process in biology. In many bacterial species, segregation of chromosomes and low-copy plasmids is facilitated by the tripartite ParA–ParB–parS ...system. This system consists of a centromeric parS DNA site and interacting proteins ParA and ParB that are capable of hydrolyzing adenosine triphosphate and cytidine triphosphate (CTP), respectively. ParB first binds to parS before associating with adjacent DNA regions to spread outward from parS. These ParB–DNA complexes bind to ParA and, through repetitive cycles of ParA–ParB binding and unbinding, move the DNA cargo to each daughter cell. The recent discovery that ParB binds and hydrolyzes CTP as it cycles on and off the bacterial chromosome has dramatically changed our understanding of the molecular mechanism used by the ParABS system. Beyond bacterial chromosome segregation, CTP-dependent molecular switches are likely to be more widespread in biology than previously appreciated and represent an opportunity for new and unexpected avenues for future research and application.
Double-strand breaks (DSBs) can lead to the loss of genetic information and cell death. Although DSB repair via homologous recombination has been well characterized, the spatial organization of this ...process inside cells remains poorly understood, and the mechanisms used for chromosome resegregation after repair are unclear. In this paper, we introduced site-specific DSBs in Caulobacter crescentus and then used time-lapse microscopy to visualize the ensuing chromosome dynamics. Damaged loci rapidly mobilized after a DSB, pairing with their homologous partner to enable repair, before being resegregated to their original cellular locations, independent of DNA replication. Origin-proximal regions were resegregated by the ParABS system with the ParA structure needed for resegregation assembling dynamically in response to the DSB-induced movement of an origin-associated ParB away from one cell pole. Origin-distal regions were resegregated in a ParABS-independent manner and instead likely rely on a physical, spring-like force to segregate repaired loci. Collectively, our results provide a mechanistic basis for the resegregation of chromosomes after a DSB.
Highlights • Bacterial chromosomes are highly organized spatially and temporally. • Recent technical advances dramatically enhance studies of chromosome biology. • Microscopy approaches reveal ...chromosome organization at the single-cell level. • Genome-wide methods such as Hi-C generate high resolution maps of chromosomes.
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