scholarly journals Swi1 and Swi3 Are Components of a Replication Fork Protection Complex in Fission Yeast

2004 ◽  
Vol 24 (19) ◽  
pp. 8342-8355 ◽  
Author(s):  
Eishi Noguchi ◽  
Chiaki Noguchi ◽  
W. Hayes McDonald ◽  
John R. Yates ◽  
Paul Russell
Keyword(s):  
Fission Yeast ◽  
Replication Fork ◽  
Replication Forks ◽  
Replication Checkpoint ◽  
Recombination Proteins ◽  
Acid Protein ◽  
Fork Protection Complex ◽  
Stalled Replication Forks ◽  
Lagging Strand ◽  
Amino Acid Protein

ABSTRACT Swi1 is required for programmed pausing of replication forks near the mat1 locus in the fission yeast Schizosaccharomyces pombe. This fork pausing is required to initiate a recombination event that switches mating type. Swi1 is also needed for the replication checkpoint that arrests division in response to fork arrest. How Swi1 accomplishes these tasks is unknown. Here we report that Swi1 copurifies with a 181-amino-acid protein encoded by swi3 +. The Swi1-Swi3 complex is required for survival of fork arrest and for activation of the replication checkpoint kinase Cds1. Association of Swi1 and Swi3 with chromatin during DNA replication correlated with movement of the replication fork. swi1Δ and swi3Δ mutants accumulated Rad22 (Rad52 homolog) DNA repair foci during replication. These foci correlated with the Rad22-dependent appearance of Holliday junction (HJ)-like structures in cells lacking Mus81-Eme1 HJ resolvase. Rhp51 and Rhp54 homologous recombination proteins were not required for viability in swi1Δ or swi3Δ cells, indicating that the HJ-like structures arise from single-strand DNA gaps or rearranged forks instead of broken forks. We propose that Swi1 and Swi3 define a fork protection complex that coordinates leading- and lagging-strand synthesis and stabilizes stalled replication forks.

scholarly journals Fork Pausing Complex Engages Topoisomerases at the Replisome

2019 ◽  
Author(s):  
Maksym Shyian ◽  
Benjamin Albert ◽  
Andreja Moset Zupan ◽  
Vitalii Ivanitsa ◽  
Gabriel Charbonnet ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Topoisomerase I ◽  
Replication Fork ◽  
Topoisomerase Ii ◽  
Replication Forks ◽  
Replication Checkpoint ◽  
Slowing Down ◽  
C Terminus ◽  
Dna Replication Checkpoint ◽  
Molecular Brake

ABSTRACTReplication forks temporarily or terminally pause at hundreds of hard-to-replicate regions around the genome. A conserved pair of budding yeast replisome components Tof1-Csm3 (fission yeast Swi1-Swi3 and human TIMELESS-TIPIN) acts as a ‘molecular brake’ and promotes fork slowdown at proteinaceous replication fork barriers (RFBs), while the accessory helicase Rrm3 assists the replisome in removing protein obstacles. Here we show that Tof1-Csm3 complex promotes fork pausing independently of Rrm3 helicase by recruiting topoisomerase I (Top1) to the replisome. Topoisomerase II (Top2) partially compensates for the pausing decrease in cells when Top1 is lost from the replisome. The C-terminus of Tof1 is specifically required for Top1 recruitment to the replisome and fork pausing but not for DNA replication checkpoint (DRC) activation. We propose that forks pause at proteinaceous RFBs through a ‘sTOP’ mechanism (‘slowing down with TOPoisomerases I-II’), which we show also contributes to protecting cells from topoisomerase-blocking agents.

scholarly journals SDE2 Integrates into the TIMELESS-TIPIN Complex to Protect Stalled Replication Forks

2020 ◽  
Author(s):  
Julie Rageul ◽  
Jennifer J. Park ◽  
Ping Ping Zeng ◽  
Eun-A Lee ◽  
Jihyeon Yang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Stability ◽  
Essential Role ◽  
Replication Stress ◽  
Replication Forks ◽  
Genomic Integrity ◽  
Fork Protection Complex ◽  
Stalled Replication Forks ◽  
Stalled Fork

ABSTRACTProtecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances TIM stability and its localization to replication forks, thereby aiding the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

scholarly journals SDE2 integrates into the TIMELESS-TIPIN complex to protect stalled replication forks

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Julie Rageul ◽  
Jennifer J. Park ◽  
Ping Ping Zeng ◽  
Eun-A Lee ◽  
Jihyeon Yang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Stability ◽  
Essential Role ◽  
Replication Stress ◽  
Replication Forks ◽  
Genomic Integrity ◽  
Fork Protection Complex ◽  
Stalled Replication Forks ◽  
Stalled Fork

Abstract Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances its stability, thereby aiding TIM localization to replication forks and the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

scholarly journals Replication Checkpoint Protein Mrc1 Is Regulated by Rad3 and Tel1 in Fission Yeast

2003 ◽  
Vol 23 (22) ◽  
pp. 8395-8403 ◽  
Author(s):  
Hui Zhao ◽  
Katsunori Tanaka ◽  
Eishi Nogochi ◽  
Chiaki Nogochi ◽  
Paul Russell
Keyword(s):  
Fission Yeast ◽  
Phosphorylation Sites ◽  
Replication Forks ◽  
Yeast Two Hybrid ◽  
Fha Domain ◽  
Replication Checkpoint ◽  
Checkpoint Protein ◽  
Replication Arrest ◽  
Differential Phosphorylation ◽  
Two Hybrid

ABSTRACT Fission yeast Mrc1 (mediator of replication checkpoint 1) is an adaptor checkpoint protein required for Rad3-dependent activation of the checkpoint kinase Cds1 in response to arrest of replication forks. Here we report studies on the regulation of Mrc1 by phosphorylation. Replication arrest induced by hydroxyurea (HU) induces Mrc1 phosphorylation that is detected by a change in Mrc1 electrophoretic mobility. Phosphorylation is maintained in cds1Δ, rad3Δ, and tel1Δ single mutants but eliminated in a rad3Δ tel1Δ double mutant. Mrc1 has two clusters of S/TQ motifs that are potential Rad3/Tel1 phosphorylation sites. Mutation of six S/TQ motifs in these two clusters strongly impairs Mrc1 phosphorylation. Two motifs located at S604 and T645 are vital for HU resistance. The T645A mutation strongly impairs a Cds1-Mrc1 yeast two-hybrid interaction that is dependent on a functional forkhead-associated (FHA) domain in Cds1, indicating that phosphorylation of T645 mediates Mrc1's association with Cds1. Consistent with this model, the T645 region of Mrc1 effectively substitutes for the T11 region of Cds1 that is thought to be phosphorylated by Rad3 and to mediate FHA-dependent oligomerization of Cds1. The S/TQ cluster that includes S604 is needed for Mrc1's increased association with chromatin in replication-arrested cells. These data indicate that Rad3 and Tel1 regulate Mrc1 through differential phosphorylation to control Cds1.

scholarly journals Escherichia coli Cells with Increased Levels of DnaA and Deficient in Recombinational Repair Have Decreased Viability

2003 ◽  
Vol 185 (2) ◽  
pp. 630-644 ◽  
Author(s):  
Aline V. Grigorian ◽  
Rachel B. Lustig ◽  
Elena C. Guzmán ◽  
Joseph M. Mahaffy ◽  
Judith W. Zyskind
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Replication Initiation ◽  
Recombinational Repair ◽  
Replication Forks ◽  
Dna Replication Initiation ◽  
Escherichia Coli Cells ◽  
Repair Pathways ◽  
Stalled Replication Forks

ABSTRACT The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, β clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, β clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the β clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 μm) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional ∼100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.

scholarly journals Mms1 and Mms22 stabilize the replisome during replication stress

2011 ◽  
Vol 22 (13) ◽  
pp. 2396-2408 ◽  
Author(s):  
Jessica A. Vaisica ◽  
Anastasija Baryshnikova ◽  
Michael Costanzo ◽  
Charles Boone ◽  
Grant W. Brown
Keyword(s):  
Dna Replication ◽  
Ubiquitin Ligase ◽  
Replication Fork ◽  
E3 Ubiquitin Ligase ◽  
Replication Stress ◽  
Replication Forks ◽  
Replication Fork Progression ◽  
Binding Partners ◽  
Efficient Recovery ◽  
Stalled Replication Forks

Mms1 and Mms22 form a Cul4Ddb1-like E3 ubiquitin ligase with the cullin Rtt101. In this complex, Rtt101 is bound to the substrate-specific adaptor Mms22 through a linker protein, Mms1. Although the Rtt101Mms1/Mms22ubiquitin ligase is important in promoting replication through damaged templates, how it does so has yet to be determined. Here we show that mms1Δ and mms22Δ cells fail to properly regulate DNA replication fork progression when replication stress is present and are defective in recovery from replication fork stress. Consistent with a role in promoting DNA replication, we find that Mms1 is enriched at sites where replication forks have stalled and that this localization requires the known binding partners of Mms1—Rtt101 and Mms22. Mms1 and Mms22 stabilize the replisome during replication stress, as binding of the fork-pausing complex components Mrc1 and Csm3, and DNA polymerase ε, at stalled replication forks is decreased in mms1Δ and mms22Δ. Taken together, these data indicate that Mms1 and Mms22 are important for maintaining the integrity of the replisome when DNA replication forks are slowed by hydroxyurea and thereby promote efficient recovery from replication stress.

scholarly journals Chromatin remodeler Fft3 plays a dual role at blocked DNA replication forks

2019 ◽  
Vol 2 (5) ◽  
pp. e201900433 ◽  
Author(s):  
Anissia Ait-Saada ◽  
Olga Khorosjutina ◽  
Jiang Chen ◽  
Karol Kramarz ◽  
Vladimir Maksimov ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Fission Yeast ◽  
Chromatin Remodeling ◽  
Replication Fork ◽  
Strand Break ◽  
Replication Forks ◽  
Damage Repair ◽  
Single Strand Annealing ◽  
Strand Annealing

Here, we investigate the function of fission yeast Fun30/Smarcad1 family of SNF2 ATPase-dependent chromatin remodeling enzymes in DNA damage repair. There are three Fun30 homologues in fission yeast, Fft1, Fft2, and Fft3. We find that only Fft3 has a function in DNA repair and it is needed for single-strand annealing of an induced double-strand break. Furthermore, we use an inducible replication fork barrier system to show that Fft3 has two distinct roles at blocked DNA replication forks. First, Fft3 is needed for the resection of nascent strands, and second, it is required to restart the blocked forks. The latter function is independent of its ATPase activity.

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