Purification of replication factors using insect and mammalian cell expression systems

Uno, Shuji; You, Zhiying; Masai, Hisao

doi:10.1016/j.ymeth.2012.06.016

Cited by 22 publications

(38 citation statements)

References 38 publications

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“…1. The human Tim-Tipin complex was purified from mammalian cells transiently co-transfected with plasmids expressing His 6 -Tim-HA and His 6 -Tipin-3ϫFLAG, as recently described (28,29). A FLAG-tagged version of human Tim was produced separately in insect cells infected with a recombinant baculovirus, whereas human Tipin was expressed separately in bacterial cells as a fusion with GST at the N terminus (cleavable with the site-specific endoprotease thrombin) and a polyhistidine tag at the C terminus and purified as described under "Experimental Procedures."…”

Section: Resultsmentioning

confidence: 99%

“…The ORF sequence was confirmed by sequencing analysis (PRIMM; Milan, Italy). The plasmid expressing the truncated form of human Tim (FLAGTim5) was already described (28,29). The baculoviruses expressing the human DNA polymerase ⑀ subunits (p261, FLAG-p59, p17, and p12) were a generous gift from J. Hurwitz (Memorial Sloan Kettering Cancer Center, New York, NY).…”

Section: Plasmids and Baculoviruses-humanmentioning

confidence: 99%

“…Purification of the Human Tim-Tipin Complex and the Truncated Form Tim 5-The human Tim-Tipin complex was purified from HEK 293T cells transiently co-transfected with plasmids expressing His 6 -Tim-HA and His 6 -Tipin-3ϫFLAG, as recently described (28,29). FLAG-Tim 5 was produced and purified using the same procedure utilized for the Tim-Tipin complex.…”

Section: Plasmids and Baculoviruses-humanmentioning

confidence: 99%

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The Human Tim-Tipin Complex Interacts Directly with DNA Polymerase ϵ and Stimulates Its Synthetic Activity

Aria

Felice

Perna

et al. 2013

Journal of Biological Chemistry

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Background:The Tim-Tipin complex plays a critical role in the S phase checkpoint and replication fork stability by a molecular mechanism not yet elucidated. Results: The human Tim-Tipin complex specifically enhances the synthetic activity of DNA polymerase ⑀. Conclusion: The Tim-Tipin complex could modulate the DNA polymerase ⑀ function at the replication fork. Significance: These findings further our understanding of the replication fork dynamics in metazoans.

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Section: Resultsmentioning

confidence: 99%

Section: Plasmids and Baculoviruses-humanmentioning

confidence: 99%

Section: Plasmids and Baculoviruses-humanmentioning

confidence: 99%

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The Human Tim-Tipin Complex Interacts Directly with DNA Polymerase ϵ and Stimulates Its Synthetic Activity

Aria

Felice

Perna

et al. 2013

Journal of Biological Chemistry

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“…To determine whether Rif1 protein directly binds to G4-like structures, we expressed full-length fission yeast Rif1 protein, tagged at the C terminus with a FLAG epitope, in mammalian cells and purified it with two consecutive affinity columns and a conventional ionexchange column 30 (Supplementary Fig. 7c).…”

Section: Purified Rif Protein Binds To G-quadruplex Structuresmentioning

confidence: 99%

Rif1 binds to G quadruplexes and suppresses replication over long distances

Kanoh

Matsumoto

Fukatsu

et al. 2015

Nat Struct Mol Biol

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147

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Rif1 regulates replication timing and repair of double-strand DNA breaks. Using a chromatin immunoprecipitation-sequencing method, we identified 35 high-affinity Rif1-binding sites in fission yeast chromosomes. Binding sites tended to be located near dormant origins and to contain at least two copies of a conserved motif, CNWWGTGGGGG. Base substitution within these motifs resulted in complete loss of Rif1 binding and in activation of late-firing or dormant origins located up to 50 kb away. We show that Rif1-binding sites adopt G quadruplex-like structures in vitro, in a manner dependent on the conserved sequence and on other G tracts, and that purified Rif1 preferentially binds to this structure. These results suggest that Rif1 recognizes and binds G quadruplex-like structures at selected intergenic regions, thus generating local chromatin structures that may exert long-range suppressive effects on origin firing.

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“…The open reading frame of full-length Mrc1, Mrc1ΔHBS, or various Mrc1 segments (spanning positions 1 to 781, 225 to 781, 377 to 781, 396 to 781, or 536 to 781) was cloned at the BamHI site of the mammalian expression vector ver.3-4, resulting in the generation of N-terminally 6ϫHis-and C-terminally 3ϫFlag-tagged polypeptides (41). The expression plasmid was transfected into 293T cells, and Mrc1, Mrc1ΔHBS, or each Mrc1 deletion protein was purified by consecutive steps with anti-Flag antibody and nickel affinity columns.…”

Section: Purification Of Mrc1 Fragmentsmentioning

confidence: 99%

Checkpoint-Independent Regulation of Origin Firing by Mrc1 through Interaction with Hsk1 Kinase

Matsumoto

Kanoh

Shimmoto

et al. 2017

Molecular and Cellular Biology

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Mrc1 is a conserved checkpoint mediator protein that transduces the replication stress signal to the downstream effector kinase. The loss of mrc1 checkpoint activity results in the aberrant activation of late/dormant origins in the presence of hydroxyurea. Mrc1 was also suggested to regulate orders of early origin firing in a checkpoint-independent manner, but its mechanism was unknown. Here we identify HBS (Hsk1 bypass segment) on Mrc1. An ΔHBS mutant does not activate late/dormant origin firing in the presence of hydroxyurea but causes the precocious and enhanced activation of weak early-firing origins during normal S-phase progression and bypasses the requirement for Hsk1 for growth. This may be caused by the disruption of intramolecular binding between HBS and NTHBS (N-terminal target of HBS). Hsk1 binds to Mrc1 through HBS and phosphorylates a segment adjacent to NTHBS, disrupting the intramolecular interaction. We propose that Mrc1 exerts a "brake" on initiation (through intramolecular interactions) and that this brake can be released (upon the loss of intramolecular interactions) by either the Hsk1-mediated phosphorylation of Mrc1 or the deletion of HBS (or a phosphomimic mutation of putative Hsk1 target serine/threonine), which can bypass the function of Hsk1 for growth. The brake mechanism may explain the checkpoint-independent regulation of early origin firing in fission yeast.KEYWORDS DNA replication timing, Mrc1, Hsk1, intramolecular interaction, replication checkpoint I n eukaryotic cells, DNA replication is initiated from a number of sites (replication origins) (1, 2). In fission yeast, more than 1,000 prereplicative complexes (pre-RCs) are generated on chromosomes during G 1 phase, and only a subset of them are used for initiation (3-9). The choice of origins to be fired and the determination of the timing of their activation are influenced by many factors, including chromatin structures, local histone modifications, transcription factors, and even physiological conditions such as temperature (7,(10)(11)(12)(13)(14)(15)(16)(17).The DNA replication checkpoint (intra-S checkpoint) operates to delay the activation of late-firing/dormant origins in response to an inhibition of DNA synthesis. The conserved Mec1-Mrc1-Rad53/Rad3-Mrc1-Cds1/ATR-Claspin-Chk1 pathway plays a major role in mediating the replication checkpoint signal to the downstream pathway. Mrc1/Claspin plays a major role as a mediator for the activation of this pathway. We previously reported that Hsk1 is required for the activation of the replication checkpoint, probably through the phosphorylation of Mrc1 (18). On the other hand, Mrc1 was also shown to be required for the scaling of DNA replication timing in budding yeast. In most mutants that extend the S phase, the activation timings of most origins are delayed in proportion to the duration of S phase. In contrast, early-firing origins fire early, even in the extended S phase, in ΔMRC1 cells (19). Importantly, this requirement of Mrc1 for the scaling of origin activation could...

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Purification of replication factors using insect and mammalian cell expression systems

Cited by 22 publications

References 38 publications

The Human Tim-Tipin Complex Interacts Directly with DNA Polymerase ϵ and Stimulates Its Synthetic Activity

The Human Tim-Tipin Complex Interacts Directly with DNA Polymerase ϵ and Stimulates Its Synthetic Activity

Rif1 binds to G quadruplexes and suppresses replication over long distances

Checkpoint-Independent Regulation of Origin Firing by Mrc1 through Interaction with Hsk1 Kinase

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