Rtf1-Mediated Eukaryotic Site-Specific Replication Termination

Eydmann, Trevor; Sommariva, Elena; Inagawa, Takabumi; Mian, Saira; Klar, Amar J. S.; Dalgaard, Jacob Z.

doi:10.1534/genetics.108.089243

Cited by 36 publications

(33 citation statements)

References 51 publications

(75 reference statements)

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“…In budding yeast, a single replication terminator protein called Fob1 binds to the Ter sites (RFBs) located in nontranscribed spacer 1 (NTS1) of rDNA to cause polar fork arrest (10,11), which prevents transcription-replication collision, fork stalling, and genome instability. Fission yeast has three known terminator proteins: Rtf1, which functions at the mating-type switch locus (12,13), and Reb1 (Sp. Reb1) and Sap1, which act at the Ter sites of rDNA spacers (14)(15)(16)(17)(18).…”

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confidence: 99%

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Bastia

Srivastava

Zaman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1–Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2–7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2–7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2–7 had to be phosphorylated for binding to phospho-Tof1–Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2–7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1–Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1–Ter complex.

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mentioning

confidence: 99%

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Bastia

Srivastava

Zaman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…This composite DBD arrangement appears to be conserved in all of the different members of the terminator protein family. Secondary structure prediction and domain mapping studies of TTF-I and Rtf1 showed that protein constructs with impaired DNA binding properties have deletions in regions that we now predict to have the first MybAD (27,38). The alignment shows structural differences among family members in the composite DBD.…”

Section: Structure Of Reb1 Defines the Prototypic Architecture Of A Ementioning

confidence: 87%

“…1 region that may be involved in oligomerization (27,(37)(38)(39)(40) and two or more Myb-like sequence-specific DNA binding domains at the C terminus. However, additional regions located immediately N-terminal to the predicted Myb domains are also needed for full binding affinity (37,39).…”

Section: Significancementioning

confidence: 99%

Functional architecture of the Reb1-Ter complex of Schizosaccharomyces pombe

Jaiswal

Choudhury

Zaman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Reb1 of Schizosaccharomyces pombe represents a family of multifunctional proteins that bind to specific terminator sites (Ter) and cause polar termination of transcription catalyzed by RNA polymerase I (pol I) and arrest of replication forks approaching the Ter sites from the opposite direction. However, it remains to be investigated whether the same mechanism causes arrest of both DNA transactions. Here, we present the structure of Reb1 as a complex with a Ter site at a resolution of 2.7 Å. Structure-guided molecular genetic analyses revealed that it has distinct and well-defined DNA binding and transcription termination (TTD) domains. The region of the protein involved in replication termination is distinct from the TTD. Mechanistically, the data support the conclusion that transcription termination is not caused by just high affinity Reb1-Ter protein-DNA interactions. Rather, protein-protein interactions between the TTD with the Rpa12 subunit of RNA pol I seem to be an integral part of the mechanism. This conclusion is further supported by the observation that double mutations in TTD that abolished its interaction with Rpa12 also greatly reduced transcription termination thereby revealing a conduit for functional communications between RNA pol I and the terminator protein.crystal structure | RNA polymerase I | transcription termination | protein-DNA interaction | replication termination E ukaryotic rDNAs are found as multiple tandem copies encoding pre-rRNA and upstream and downstream regulatory elements (1-3) including DNA sequences (Ter) that promote site-specific termination of transcription catalyzed by RNA polymerase I (pol I) from yeast to humans (4-14). Specialized transcription terminator proteins bind to Ter sites and not only arrest transcription by RNA polymerase I (pol I) in a polar mode but also replication forks approaching from the opposite direction. Several studies have suggested that pol I transcription termination is a multistep process that requires (i) pausing of chain elongation by the terminator protein and (ii) dissociation and release of pol I and the primary transcript from the template. In mice, dissociation of pol I and release of the transcript require the release factor PTRF, a 44-kDa protein that interacts with the largest subunit of pol I (15). In yeast, additional factors for the processing of the end include the endonuclease Rnt1, the 5′-3′ Rat1 exonuclease, Sen1 helicase, and the kinase Grc3. They are part of an alternate pathway for termination by transcriptional coprocessing (4,(16)(17)(18)(19). In addition, in vivo analyses of Saccharomyces cerevisiae have shown a requirement for Rpa12, a component of pol I necessary for transcription termination (20). Whether the polar arrest of transcription is caused by an interaction between Rpa12 with the terminator protein is unknown.The terminator proteins that mediate transcription termination have been identified in multiple organisms and include Reb1 and Nsi1 [also called yeast transcription terminator (Ytt1)] of S. cerevisiae (...

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“…Interestingly, S. pombe RTS1, another natural RFB located in the mating-type locus, determines the appropriate direction of replication of this region required for efficient mating-type switching (Dalgaard and Klar, 2001). Rtf1 is a DNA-binding protein responsible for the function of RTS1 that shows a high similarity to Reb1 (Eydmann et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

The fission yeast rDNA-binding protein Reb1 regulates G1 phase under nutritional stress

Rodríguez-Sánchez

Rodríguez-López

García

et al. 2011

Journal of Cell Science

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SummaryYeast Reb1 and its mammalian ortholog TTF1 are conserved Myb-type DNA-binding proteins that bind to specific sites near the 3Ј-end of rRNA genes (rDNA). Here, they participate in the termination of transcription driven by RNA polymerase I and block DNA replication forks approaching in the opposite direction. We found that Schizosaccharomyces pombe Reb1 also upregulates transcription of the ste9 + gene that is required for nitrogen-starvation-induced growth arrest with a G1 DNA content and sexual differentiation. Ste9 activates the anaphase-promoting complex or cyclosome ('APC/C') in G1, targeting B-cyclin for proteasomal degradation in response to nutritional stress. Reb1 binds in vivo and in vitro to a specific DNA sequence at the promoter of ste9 + , similar to the sequence recognized in the rDNA, and this binding is required for ste9 + transcriptional activation and G1 arrest. This suggests that Reb1 acts as a link between rDNA metabolism and cell cycle control in response to nutritional stress. In agreement with this new role for Reb1 in the regulation of the G1-S transition, reb1⌬ and wee1 ts mutations are synthetically lethal owing to the inability of these cells to lengthen G1 before entering S phase. Similarly, reb1⌬ cdc10 ts cells are unable to arrest in G1 and die at the semi-permissive temperature.

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Rtf1-Mediated Eukaryotic Site-Specific Replication Termination

Cited by 36 publications

References 51 publications

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Functional architecture of the Reb1-Ter complex of Schizosaccharomyces pombe

The fission yeast rDNA-binding protein Reb1 regulates G1 phase under nutritional stress

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