Genome control by SMC complexes

Hoencamp, Claire; Rowland, Benjamin D.

doi:10.1038/s41580-023-00609-8

Cited by 52 publications

(30 citation statements)

References 285 publications

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“…When a relatively low concentration of M-CDK (10 nM) was added to the assay (Table 1), phosphorylation of H1. Condensin I is a pentameric protein complex that plays an indispensable role in mitotic chromosome assembly [37,38]. A native condensin I comeplex purified from egg extracts has been shown to act as one of the six protein factors required for chromosome reconstitution in vitro, but it does so only when it is phosphorylated by cyclin B-Cdk1 [25].…”

Section: Resultsmentioning

confidence: 99%

Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylationin vitro

Shintomi,

Masahara-Negishi,

Shima

et al. 2023

Preprint

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Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B phosphorylates multiple sites on hundreds of proteins during mitosis. However, it is not fully understood how multi-site mitotic phosphorylation by cyclin B-Cdk1 controls the structures and functions of individual substrates. Here we develop an easy-to-use protocol to express recombinant vertebrate cyclin B and Cdk1 in insect cells from a single baculovirus vector and to purify their complexes with excellent homogeneity. A series of in-vitro assays demonstrate that the recombinant cyclin B-Cdk1 can efficiently and specifically phosphorylate the SP and TP motifs in substrates. The addition of Suc1 (a Cks1 homolog in fission yeast) accelerates multi-site phosphorylation of an artificial substrate containing TP motifs. Importantly, we show that mitosis-specific multi-subunit and multi-site phosphorylation of the condensin I complex can be recapitulated in vitro using recombinant cyclin B-Cdk1-Suc1. The materials and protocols described here will pave the way for dissecting the biochemical basis of critical mitotic processes that accompany Cdk1-mediated large-scale phosphorylation.

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Section: Resultsmentioning

confidence: 99%

Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylationin vitro

Shintomi,

Masahara-Negishi,

Shima

et al. 2023

Preprint

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“…In addition to the effects of LeishIF4E2 on histone expression either direct or indirect, we observed major changes in expression of proteins involved in genome organization, stability, and integrity. Proteins involved in these processes include the structural maintenance of chromosome complexes (SMC) that are responsible for DNA replication, genome organization, chromosomal segregation, and repair of damaged DNA 61–63 . In addition, the minichromosome maintenance proteins (MCM) generate a heteromeric DNA helicase that is involved in initiation and elongation of DNA replication as well as in DNA repair 64 .…”

Section: Discussionmentioning

confidence: 99%

“…Proteins involved in these processes include the structural maintenance of chromosome complexes (SMC) that are responsible for DNA replication, genome organization, chromosomal segregation, and repair of damaged DNA. [61][62][63] In addition, the minichromosome maintenance proteins (MCM) generate a heteromeric DNA helicase that is involved in initiation and elongation of DNA replication as well as in DNA repair. 64 We observed an increase in the SMC and MCM proteins as a result of LeishIF4E2 overexpression in transgenic lines.…”

Section: Discussionmentioning

confidence: 99%

LeishIF4E2 is a cap‐binding protein that plays a role in Leishmania cell cycle progression

Baron,

Purushotham,

Pullaiahgari

et al. 2023

The FASEB Journal

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Leishmania encode six paralogs of the cap‐binding protein eIF4E and five eIF4G candidates, forming unique complexes. Two cap‐binding proteins, LeishIF4E1 and LeishIF4E2, do not bind any identified LeishIF4Gs, thus their roles are intriguing. Here, we combine structural prediction, proteomic analysis, and interaction assays to shed light on LeishIF4E2 function. A nonconserved C‐terminal extension was identified through structure prediction and sequence alignment. m7GTP‐binding assays involving both recombinant and transgenic LeishIF4E2 with and without the C‐terminal extension revealed that this extension functions as a regulatory gate, modulating the cap‐binding activity of LeishIF4E2. The interactomes of the two LeishIF4E2 versions were investigated, highlighting the role of the C‐terminal extension in binding to SLBP2. SLBP2 is known to interact with a stem‐loop structure in the 3′ UTRs of histone mRNAs. Consistent with the predicted inhibitory effect of SLBP2 on histone expression in Xenopus laevis, a hemizygous deletion mutant of LeishIF4E2, exhibited an upregulation of several histones. We therefore propose that LeishIF4E2 is involved in histone expression, possibly through its interaction between SLBP2 and LeishIF4E2, thus affecting cell cycle progression. In addition, cell synchronization showed that LeishIF4E2 expression decreased during the S‐phase, when histones are known to be synthesized. Previous studies in T. brucei also highlighted an association between TbEIF4E2 and SLBP2, and further reported on an interaction between TbIF4E2 and S‐phase‐abundant mRNAs. Our results show that overexpression of LeishIF4E2 correlates with upregulation of cell cycle and chromosome maintenance proteins. Along with its effect on histone expression, we propose that LeishIF4E2 is involved in cell cycle progression.

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“…The SMC protein complexes cohesin and condensin are key determinants of chromatin organization in interphase and in mitosis (1)(2)(3)(4). During prophase, a cohesin-dominated interphase organization with topologically associating domains (TADs) and compartments transitions into a condensin-dominated mitotic loop array (5,6).…”

Section: Introductionmentioning

confidence: 99%

Rules of engagement for condensins and cohesins guide mitotic chromosome formation

Samejima,

Gibcus,

Abraham

et al. 2024

Preprint

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During mitosis, interphase chromatin is rapidly converted into rod-shaped mitotic chromosomes. Using Hi-C, imaging, proteomics and polymer modeling, we determine how the activity and interplay between loop-extruding SMC motors accomplishes this dramatic transition. Our work reveals rules of engagement for SMC complexes that are critical for allowing cells to refold interphase chromatin into mitotic chromosomes. We find that condensin disassembles interphase chromatin loop organization by evicting or displacing extrusive cohesin. In contrast, condensin bypasses cohesive cohesins, thereby maintaining sister chromatid cohesion while separating the sisters. Studies of mitotic chromosomes formed by cohesin, condensin II and condensin I alone or in combination allow us to develop new models of mitotic chromosome conformation. In these models, loops are consecutive and not overlapping, implying that condensins do not freely pass one another but stall upon encountering each other. The dynamics of Hi-C interactions and chromosome morphology reveal that during prophase loops are extruded in vivo at ~1-3 kb/sec by condensins as they form a disordered discontinuous helical scaffold within individual chromatids.

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Genome control by SMC complexes

Cited by 52 publications

References 285 publications

Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylationin vitro

Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylationin vitro

LeishIF4E2 is a cap‐binding protein that plays a role in Leishmania cell cycle progression

Rules of engagement for condensins and cohesins guide mitotic chromosome formation

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