Schizosaccharomyces pombe Cds1Chk2 regulates homologous recombination at stalled replication forks through the phosphorylation of recombination protein Rad60

ABSTRACT The Swi1 and Swi3 proteins are required for mat1 imprinting and mating-type switching in Schizosaccharomyces pombe, where they mediate a pause of leading-strand replication in response to a lagging-strand signal. In addition, Swi1 has been demonstrated to be involved in the checkpoint response to stalled replication forks, as was described for the Saccharomyces cerevisiae homologue Tof1. This study addresses the roles of Swi1 and Swi3 during a replication process perturbed by the presence of template bases alkylated by methyl methanesulfonate (MMS). Both the swi1 and swi3 mutations have additive effects on MMS sensitivity and on the MMS-induced damage checkpoint response when combined with chk1 and cds1, but they are nonadditive with hsk1. Cells with swi1, swi3, or hsk1 mutations are also defective in slowing progression through S phase in response to MMS damage. Moreover, swi1 and swi3 strains show increased levels of genomic instability even in the absence of exogenously induced DNA damage. Chromosome fragmentation, increased levels of single-stranded DNA, increased recombination, and instability of replication forks stalled in the presence of hydroxyurea are observed, consistent with the possibility that the replication process is affected in these mutants. In conclusion, Swi1, Swi3, and Hsk1 act in a novel S-phase checkpoint pathway that contributes to replication fork maintenance and to survival of alkylation damage.

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Transcription-Associated Recombination Is Dependent on Replication in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.00816-07 ◽

2007 ◽

Vol 28 (1) ◽

pp. 154-164 ◽

Cited By ~ 66

Author(s):

Ponnari Gottipati ◽

Tobias N. Cassel ◽

Linda Savolainen ◽

Thomas Helleday

Keyword(s):

Cell Cycle ◽

Homologous Recombination ◽

Mammalian Cells ◽

Strand Break ◽

Double Strand Break ◽

S Phase ◽

Replication Forks ◽

Higher Eukaryotes ◽

Stalled Replication Forks ◽

Gene Conversions

ABSTRACT Transcription can enhance recombination; this is a ubiquitous phenomenon from prokaryotes to higher eukaryotes. However, the mechanism of transcription-associated recombination in mammalian cells is poorly understood. Here we have developed a construct with a recombination substrate in which levels of recombination can be studied in the presence or absence of transcription. We observed a direct enhancement in recombination when transcription levels through the substrate were increased. This increase in homologous recombination following transcription is locus specific, since homologous recombination at the unrelated hprt gene is unaffected. In addition, we have shown that transcription-associated recombination involves both short-tract and long-tract gene conversions in mammalian cells, which are different from double-strand-break-induced recombination events caused by endonucleases. Transcription fails to enhance recombination in cells that are not in the S phase of the cell cycle. Furthermore, inhibition of transcription suppresses induction of recombination at stalled replication forks, suggesting that recombination may be involved in bypassing transcription during replication.

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Phenotypes of dnaXE145A Mutant Cells Indicate that the Escherichia coli Clamp Loader Has a Role in the Restart of Stalled Replication Forks

Journal of Bacteriology ◽

10.1128/jb.00412-17 ◽

2017 ◽

Vol 199 (24) ◽

Author(s):

Ingvild Flåtten ◽

Emily Helgesen ◽

Ida Benedikte Pedersen ◽

Torsten Waldminghaus ◽

Christiane Rothe ◽

...

Keyword(s):

Escherichia Coli ◽

Homologous Recombination ◽

Replication Fork ◽

Temperature Sensitive ◽

Replication Forks ◽

Clamp Loader ◽

Topoisomerase Iv ◽

Content Type ◽

Stalled Replication Forks ◽

Mutant Cells

ABSTRACT The Escherichia coli dnaXE145A mutation was discovered in connection with a screen for multicopy suppressors of the temperature-sensitive topoisomerase IV mutation parE10. The gene for the clamp loader subunits τ and γ, dnaX, but not the mutant dnaXE145A , was found to suppress parE10(Ts) when overexpressed. Purified mutant protein was found to be functional in vitro, and few phenotypes were found in vivo apart from problems with partitioning of DNA in rich medium. We show here that a large number of the replication forks that initiate at oriC never reach the terminus in dnaXE145A mutant cells. The SOS response was found to be induced, and a combination of the dnaXE145A mutation with recBC and recA mutations led to reduced viability. The mutant cells exhibited extensive chromosome fragmentation and degradation upon inactivation of recBC and recA, respectively. The results indicate that the dnaXE145A mutant cells suffer from broken replication forks and that these need to be repaired by homologous recombination. We suggest that the dnaX-encoded τ and γ subunits of the clamp loader, or the clamp loader complex itself, has a role in the restart of stalled replication forks without extensive homologous recombination. IMPORTANCE The E. coli clamp loader complex has a role in coordinating the activity of the replisome at the replication fork and loading β-clamps for lagging-strand synthesis. Replication forks frequently encounter obstacles, such as template lesions, secondary structures, and tightly bound protein complexes, which will lead to fork stalling. Some pathways of fork restart have been characterized, but much is still unknown about the actors and mechanisms involved. We have in this work characterized the dnaXE145A clamp loader mutant. We find that the naturally occurring obstacles encountered by a replication fork are not tackled in a proper way by the mutant clamp loader and suggest a role for the clamp loader in the restart of stalled replication forks.

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Phosphorylation of Rad55 on Serines 2, 8, and 14 Is Required for Efficient Homologous Recombination in the Recovery of Stalled Replication Forks

Molecular and Cellular Biology ◽

10.1128/mcb.01317-06 ◽

2006 ◽

Vol 26 (22) ◽

pp. 8396-8409 ◽

Cited By ~ 54

Author(s):

Kristina Herzberg ◽

Vladimir I. Bashkirov ◽

Michael Rolfsmeier ◽

Edwin Haghnazari ◽

W. Hayes McDonald ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Cellular Response ◽

Replication Fork ◽

Normal Growth ◽

Genotoxic Stress ◽

Replication Forks ◽

Replication Fork Stalling ◽

Fork Stalling ◽

Stalled Replication Forks

ABSTRACT DNA damage checkpoints coordinate the cellular response to genotoxic stress and arrest the cell cycle in response to DNA damage and replication fork stalling. Homologous recombination is a ubiquitous pathway for the repair of DNA double-stranded breaks and other checkpoint-inducing lesions. Moreover, homologous recombination is involved in postreplicative tolerance of DNA damage and the recovery of DNA replication after replication fork stalling. Here, we show that the phosphorylation on serines 2, 8, and 14 (S2,8,14) of the Rad55 protein is specifically required for survival as well as for normal growth under genome-wide genotoxic stress. Rad55 is a Rad51 paralog in Saccharomyces cerevisiae and functions in the assembly of the Rad51 filament, a central intermediate in recombinational DNA repair. Phosphorylation-defective rad55-S2,8,14A mutants display a very slow traversal of S phase under DNA-damaging conditions, which is likely due to the slower recovery of stalled replication forks or the slower repair of replication-associated DNA damage. These results suggest that Rad55-S2,8,14 phosphorylation activates recombinational repair, allowing for faster recovery after genotoxic stress.

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PIN1 isomerisation of BRCA1 promotes replication fork protection

10.1101/478511 ◽

2018 ◽

Author(s):

Manuel Daza-Martin ◽

Mohammed Jamshad ◽

Katarzyna Starowicz ◽

Anoop Singh Chauhan ◽

James F.J. Beesley ◽

...

Keyword(s):

Homologous Recombination ◽

Conformational Change ◽

Replication Fork ◽

Cancer Predisposition ◽

Replication Forks ◽

Prolyl Isomerase ◽

Missense Variants ◽

Brca1 Brca2 ◽

Stalled Replication Forks

AbstractBRCA1, BRCA2 and a subset of Fanconi’s Anaemia proteins act to promote RAD51-mediated protection of newly synthesised DNA at stalled replication forks from degradation by nucleases. How BRCA1 contributes, how it is regulated and whether replication fork protection relates to, or differs from, the roles BRCA1 has in homologous recombination is not clear. Here we show that the canonical BRCA1-PALB2 interaction is not required for fork protection and instead we demonstrate that the ability of BRCA1 to protect nascent DNA is regulated in an unexpected fashion through conformational change mediated by the phosphorylation-directed prolyl isomerase, PIN1. BRCA1 isomerisation enhances BRCA1-BARD1 interaction with RAD51 and consequently RAD51 presence at stalled replication structures. Our data suggest BRCA1-BARD1 promotes fork protection in part by enhancing the RAD51 synapse. Patient missense variants in the regulated BRCA1-BARD1 regions similarly show poor nascent strand protection but proficient homologous recombination, defining novel domains required for fork protection in BRCA1-BARD1 associated with cancer predisposition. Together these findings reveal a previously unrecognised pathway that governs BRCA1-mediated replication fork protection.

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Nucleotide proofreading functions by nematode RAD51 paralogs facilitate optimal RAD51 filament function

Nature Communications ◽

10.1038/s41467-021-25830-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Mário Špírek ◽

Martin R. G. Taylor ◽

Ondrej Belan ◽

Simon J. Boulton ◽

Lumir Krejci

Keyword(s):

Homologous Recombination ◽

Strand Exchange ◽

Atp Binding ◽

Replication Forks ◽

Nucleotide Content ◽

Rad51 Paralogs ◽

Strand Invasion ◽

Stalled Replication Forks ◽

Over Time ◽

Striking Contrast

AbstractThe RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that ‘nucleotide proofreading’ activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.

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