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Replication fork arrest and rDNA silencing are two independent and separable functions of the replication terminator protein Fob1 of Saccharomyces cerevisiae.

ABSTRACT: The replication terminator protein Fob1 of Saccharomyces cerevisiae is multifunctional, and it not only promotes polar replication fork arrest at the tandem Ter sites located in the intergenic spacer region of rDNA but also loads the NAD-dependent histone deacetylase Sir2 at Ter sites via a protein complex called RENT (regulator of nucleolar silencing and telophase exit). Sir2 is a component of the RENT complex, and its loading not only silences intrachromatid recombination in rDNA but also RNA polymerase II-catalyzed transcription. Here, we present three lines of evidence showing that the two aforementioned activities of Fob1 are independent of each other as well as functionally separable. First, a Fob1 ortholog of Saccharomyces bayanus expressed in a fob1Delta strain of S. cerevisiae restored polar fork arrest at Ter but not rDNA silencing. Second, a mutant form (I407T) of S. cerevisiae Fob1 retained normal fork arresting activity but was partially defective in rDNA silencing. We further show that the silencing defect of S. bayanus Fob1 and the Iota407Tau mutant of S. cerevisiae Fob1 were caused by the failure of the proteins to interact with two members of the S. cerevisiae RENT complex, namely S. cerevisiae Sir2 and S. cerevisiae Net1. Third, deletions of the intra-S phase checkpoint proteins Tof1 and Csm3 abolished fork arrest by Fob1 at Ter without causing loss of silencing. Taken together, the data support the conclusion that unlike some other functions of Fob1, rDNA silencing at Ter is independent of fork arrest.

SUBMITTER: Bairwa NK

PROVIDER: S-EPMC2857089 | biostudies-other | 2010 Apr

REPOSITORIES: biostudies-other

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Replication fork arrest and rDNA silencing are two independent and separable functions of the replication terminator protein Fob1 of Saccharomyces cerevisiae.

Bairwa Narendra K NK Zzaman Shamsu S Mohanty Bidyut K BK Bastia Deepak D

The Journal of biological chemistry 20100223 17

The replication terminator protein Fob1 of Saccharomyces cerevisiae is multifunctional, and it not only promotes polar replication fork arrest at the tandem Ter sites located in the intergenic spacer region of rDNA but also loads the NAD-dependent histone deacetylase Sir2 at Ter sites via a protein complex called RENT (regulator of nucleolar silencing and telophase exit). Sir2 is a component of the RENT complex, and its loading not only silences intrachromatid recombination in rDNA but also RNA ...[more]

PMID: 20179323

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Project description:The replication terminator protein (RTP) of Bacillus subtilis interacts with its cognate DNA terminators to cause replication fork arrest, thereby ensuring that the forks approaching one another at the conclusion of a round of replication meet within a restricted terminus region. A similar situation exists in Escherichia coli, but it appears that the fork-arrest systems in these two organisms have evolved independently of one another. In the present work, RTP homologs in four species closely related to B. subtilis (B. atrophaeus, B. amyloliquefaciens, B. mojavensis, and B. vallismortis) have been identified and characterized. An RTP homolog could not be identified in another closely related species, B. licheniformis. The nucleotide and amino acid changes from B. subtilis among the four homologs are consistent with the recently established phylogenetic tree for these species. The GC contents of the rtp genes raise the possibility that these organisms arose within this branch of the tree by horizontal transfer into a common ancestor after their divergence from B. licheniformis. Only 5 amino acid residue positions were changed among the four homologs, despite an up to 17.2% change in the nucleotide sequence, a finding that highlights the importance of the precise folded structure to the functioning of RTP. The absence of any significant change in the proposed DNA-binding region of RTP emphasizes the importance of its high affinity for the DNA terminator in its functioning. By coincidence, the single change (E30K) found in the B. mojavensis RTP corresponds exactly to that purposefully introduced by others into B. subtilis RTP to implicate a crucial role for E30 in the fork-arrest mechanism. The natural occurrence of this variant is difficult to reconcile with such an implication, and it was shown directly that RTP.E30K functions normally in fork arrest in B. subtilis in vivo. Additional DNA terminators were identified in the new RTP homolog-containing strains, allowing the definition of a Bacillus terminator consensus and identification of two more terminators in the B. subtilis 168 genome sequence to bring the total to nine.

Dataset Information

Replication fork arrest and rDNA silencing are two independent and separable functions of the replication terminator protein Fob1 of Saccharomyces cerevisiae.

Publications

Replication fork arrest and rDNA silencing are two independent and separable functions of the replication terminator protein Fob1 of Saccharomyces cerevisiae.

Similar Datasets

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