The arrest of replication forks in the rDNA of yeast occurs independently of transcription

Brewer, Bonita J.; Lockshon, Daniel; Fangman, Walton L.

doi:10.1016/0092-8674(92)90355-g

Cited by 257 publications

(280 citation statements)

References 33 publications

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“…Preliminary work shows that the Y33A mutant of RTP fails to arrest RNAPs. 4 If protein-protein interaction is involved, future work will also be directed toward mapping physically the contact points between RNAPs and Tus and RTP by photo-cross-linking procedures (52,54 …”

Section: Discussionmentioning

confidence: 99%

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Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Mohanty

Sahoo

Bastia

1998

Journal of Biological Chemistry

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Since DNA replication and transcription often temporally and spatially overlap each other, the impact of one process on the other is of considerable interest. We have reported previously that transcription is impeded at the replication termini of Escherichia coli and Bacillus subtilis in a polar mode and that, when transcription is allowed to invade a replication terminus from the permissive direction, arrest of replication fork at the terminus is abrogated. In the present report, we have addressed four significant questions pertaining to the mechanism of transcription impedance by the replication terminator proteins. Is transcription arrested at the replication terminus or does RNA polymerase dissociate from the DNA causing authentic transcription termination? How does transcription cause abrogation of replication fork arrest at the terminus? Are the points of arrest of the replication fork and transcription the same or are these different? Are eukaryotic RNA polymerases also arrested at prokaryotic replication termini? Our results show that replication terminator proteins of E. coli and B. subtilis arrest but do not terminate transcription. Passage of an RNA transcript through the replication terminus causes the dissociation of the terminator protein from the terminus DNA, thus causing abrogation of replication fork arrest. DNA and RNA chain elongation are arrested at different locations on the terminator sites. Finally, although bacterial replication terminator proteins blocked yeast RNA polymerases in a polar fashion, a yeast transcription terminator protein (Reb1p) was unable to block T7 RNA polymerase and E. coli DnaB helicase.The chromosomes of Escherichia coli and Bacillus subtilis, and some plasmids like R6K, initiate replication from specific origins and the replication forks moving either bi-or unidirectionally, are arrested at sequence-specific replication termini (1-3). Sequence-specific replication fork barriers have also been observed in the nontranscribed spacer regions of ribosomal DNAs of Saccharomyces cerevisiae (4, 5), plants (6), Xenopus (7), and human (8).The replication termini are sequence-specific and bind to cognate terminator proteins, and the protein-DNA complex arrests replication forks in an orientation-dependent manner (9, 10). DnaB (11,12,19). Detailed biochemical and biophysical analyses have been made possible in both the systems by the determination of the crystal structures of RTP at 2.6 Å (21) and of Tus-Ter () complex at 2.7 Å (22). The DNA binding (23), dimer-dimer interaction (24), and DnaB interaction domains (25) of RTP have been determined by genetic and biochemical analyses that used the crystal structure as a guide. Similarly, the DNA binding domain of Tus has been determined with the help of crystal structure and genetic analysis (22).The impact of transcription on the initiation (26, 27) and elongation stages of DNA replication have been reported (28,29). We have reported previously that Tus and RTP can block RNA chain elongation catalyzed by several prokaryotic RN...

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Section: Discussionmentioning

confidence: 99%

“…Sequence-specific replication fork barriers have also been observed in the nontranscribed spacer regions of ribosomal DNAs of Saccharomyces cerevisiae (4,5), plants (6), Xenopus (7), and human (8).…”

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confidence: 92%

Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Mohanty

Sahoo

Bastia

1998

Journal of Biological Chemistry

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“…DNA was extracted from pooled S-phase cells of strain RM14-3a (MATa cdc7-1 bar1 ura3-52 trp1-289 leu2-3,112 his6) as previously described (Huberman et al 1987;Brewer et al 1992). Standard 2D gels were performed (see .…”

Section: Two-dimensional (2d) Agarose Gel Electrophoresismentioning

confidence: 99%

Replication profile of Saccharomyces cerevisiae chromosome VI

1997

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“…As for cis elements required for RFB activities, the work by Ward and coworkers (38) showed that some DNA elements are shared with those required for HOT1 activity, but others are required for one activity but not for the other. Although RFB activities were demonstrated using plasmids carrying cloned DNA containing the E element or the RFB region (3,13,38), the observed activities were reported to be very weak compared to the activities in the chromosomal context (3). This situation resembles that observed for transcription of a reporter gene; transcription of a Pol I reporter on a plasmid was reported to be much weaker relative to transcription of the same reporter integrated into the chromosomal rDNA repeats (16).…”

Section: Vol 21 2001mentioning

confidence: 99%

“…1). This 130-bp region overlaps the region containing the RFB site(s) mentioned above, which allows progression of DNA replication in the direction of 35S rDNA transcription, but not in the opposite direction (3,13). Because pausing of a replication fork is known, at least in bacterial systems, to stimulate both DNA double-strand breakage and genetic recombination (reviewed recently in reference 26), replication fork pausing at the RFB site could explain stimulation of recombination in the HOT1 system.…”

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confidence: 99%

Yeast RNA Polymerase I Enhancer Is Dispensable for Transcription of the Chromosomal rRNA Gene and Cell Growth, and Its Apparent Transcription Enhancement from Ectopic Promoters Requires Fob1 Protein

Wai

Johzuka

et al. 2001

Molecular and Cellular Biology

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At the end of the 35S rRNA gene within ribosomal DNA (rDNA) repeats in Saccharomyces cerevisiae lies an enhancer that has been shown to greatly stimulate rDNA transcription in ectopic reporter systems. We found, however, that the enhancer is not necessary for normal levels of rRNA synthesis from chromosomal rDNA or for cell growth. Yeast strains which have the entire enhancer from rDNA deleted did not show any defects in growth or rRNA synthesis. We found that the stimulatory activity of the enhancer for ectopic reporters is not observed in cells with disrupted nucleolar structures, suggesting that reporter genes are in general poorly accessible to RNA polymerase I (Pol I) machinery in the nucleolus and that the enhancer improves accessibility. We also found that a fob1 mutation abolishes transcription from the enhancer-dependent rDNA promoter integrated at the HIS4 locus without any effect on transcription from chromosomal rDNA. FOB1 is required for recombination hot spot (HOT1) activity, which also requires the enhancer region, and for recombination within rDNA repeats. We suggest that Fob1 protein stimulates interactions between rDNA repeats through the enhancer region, thus helping ectopic rDNA promoters to recruit the Pol I machinery normally present in the nucleolus.

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The arrest of replication forks in the rDNA of yeast occurs independently of transcription

Cited by 257 publications

References 33 publications

Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Replication profile of Saccharomyces cerevisiae chromosome VI

Yeast RNA Polymerase I Enhancer Is Dispensable for Transcription of the Chromosomal rRNA Gene and Cell Growth, and Its Apparent Transcription Enhancement from Ectopic Promoters Requires Fob1 Protein

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