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

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.

scholarly journals The homologous recombination complex of the Rad51 paralogs Rad55-Rad57 avoids translesion DNA polymerase recruitment and counterbalances mutagenesis induced by UV radiation

2021 ◽  
Author(s):  
Laurent G Maloisel ◽  
Emilie Ma ◽  
Eric Coic
Keyword(s):  
Homologous Recombination ◽  
Uv Radiation ◽  
Dna Lesions ◽  
Template Switching ◽  
Dna Double Strand Breaks ◽  
Dna Damage Tolerance ◽  
Strand Breaks ◽  
Rad51 Paralogs ◽  
Replicative Polymerases ◽  
Stalled Replication Forks

Bypass of DNA lesions that block replicative polymerases during DNA replication relies on several DNA damage tolerance pathways. The error-prone translesion synthesis (TLS) pathway involves specialized DNA polymerases that incorporate nucleotides in front of base lesions. The template switching and the homologous recombination (HR) pathways are mostly error-free because the bypass is performed by using typically the sister chromatid as a template. This is promoted by the Rad51 recombinase that forms nucleoprotein filaments on single-strand DNA (ssDNA). The balance between error-prone and error-free pathways controls the level of mutagenesis. In yeast, the Rad55-Rad57 complex of Rad51 paralogs is required for Rad51 filament formation and stability, notably by counteracting the Srs2 antirecombinase. Several reports showed that Rad55-Rad57 promotes HR at stalled replication forks more than at DNA double-strand breaks (DSB), suggesting that this complex is more efficient at ssDNA gaps and thus, could control the recruitment of TLS polymerases. To address this point, we studied the interplay between Rad55-Rad57 and the TLS polymerases Polζ and Polη following UV radiation. We confirmed that Rad55-Rad57 protects Rad51 filaments from Srs2 dismantling activity but we found that it is also essential for the promotion of UV-induced HR independently of Srs2. In addition, we observed that cell UV sensitivity, but not DSB sensitivity, is synergistically increased when Rad55 and Polζ deletions are combined. Moreover, we found that mutagenesis and HR frequency were increased in rad55∆ mutants and in TLS-deficient cells, respectively. Finally, UV-induced HR was partially restored in Rad55-deficient cells with mutated Polζ or Polη. Overall, our data suggest that the HR and TLS pathways compete for the same ssDNA substrates and that the Rad55-Rad57 complex of Rad51 paralogs prevents the recruitment of TLS polymerases and counterbalances mutagenesis.

scholarly journals Non-Recombinogenic Functions of Rad51, BRCA2, and Rad52 in DNA Damage Tolerance

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1550
Author(s):  
Félix Prado
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Damage Tolerance ◽  
Molecular Switches ◽  
Strand Exchange ◽  
Template Switching ◽  
Replication Forks ◽  
Lesion Bypass ◽  
Dna Damage Tolerance ◽  
Exchange Activity

The DNA damage tolerance (DDT) response is aimed to timely and safely complete DNA replication by facilitating the advance of replication forks through blocking lesions. This process is associated with an accumulation of single-strand DNA (ssDNA), both at the fork and behind the fork. Lesion bypass and ssDNA filling can be performed by translation synthesis (TLS) and template switching mechanisms. TLS uses low-fidelity polymerases to incorporate a dNTP opposite the blocking lesion, whereas template switching uses a Rad51/ssDNA nucleofilament and the sister chromatid to bypass the lesion. Rad51 is loaded at this nucleofilament by two mediator proteins, BRCA2 and Rad52, and these three factors are critical for homologous recombination (HR). Here, we review recent advances showing that Rad51, BRCA2, and Rad52 perform some of these functions through mechanisms that do not require the strand exchange activity of Rad51: the formation and protection of reversed fork structures aimed to bypass blocking lesions, and the promotion of TLS. These findings point to the central HR proteins as potential molecular switches in the choice of the mechanism of DDT.

scholarly journals BRCA2 regulates DMC1-mediated recombination through the BRC repeats

2016 ◽  
Vol 113 (13) ◽  
pp. 3515-3520 ◽  
Author(s):  
Juan S. Martinez ◽  
Catharina von Nicolai ◽  
Taeho Kim ◽  
Åsa Ehlén ◽  
Alexander V. Mazin ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Spatial Organization ◽  
Strand Exchange ◽  
Molecule Formation ◽  
Brca2 Protein ◽  
Dna Strand Exchange ◽  
Functional Consequences ◽  
Strand Invasion ◽  
Dna Strand ◽  
Stimulation Of

In somatic cells, BRCA2 is needed for RAD51-mediated homologous recombination. The meiosis-specific DNA strand exchange protein, DMC1, promotes the formation of DNA strand invasion products (joint molecules) between homologous molecules in a fashion similar to RAD51. BRCA2 interacts directly with both human RAD51 and DMC1; in the case of RAD51, this interaction results in stimulation of RAD51-promoted DNA strand exchange. However, for DMC1, little is known regarding the basis and functional consequences of its interaction with BRCA2. Here we report that human DMC1 interacts directly with each of the BRC repeats of BRCA2, albeit most tightly with repeats 1–3 and 6–8. However, BRC1–3 bind with higher affinity to RAD51 than to DMC1, whereas BRC6–8 bind with higher affinity to DMC1, providing potential spatial organization to nascent filament formation. With the exception of BRC4, each BRC repeat stimulates joint molecule formation by DMC1. The basis for this stimulation is an enhancement of DMC1–ssDNA complex formation by the stimulatory BRC repeats. Lastly, we demonstrate that full-length BRCA2 protein stimulates DMC1-mediated DNA strand exchange between RPA–ssDNA complexes and duplex DNA, thus identifying BRCA2 as a mediator of DMC1 recombination function. Collectively, our results suggest unique and specialized functions for the BRC motifs of BRCA2 in promoting homologous recombination in meiotic and mitotic cells.

scholarly journals Non-enzymatic roles of human RAD51 at stalled replication forks

2018 ◽  
Author(s):  
Jennifer M. Mason ◽  
Yuen-Ling Chan ◽  
Ralph W. Weichselbaum ◽  
Douglas K. Bishop
Keyword(s):  
Dna Binding ◽  
Replication Fork ◽  
Replication Stress ◽  
Strand Exchange ◽  
Replication Forks ◽  
Enzymatic Function ◽  
Replication Restart ◽  
Stalled Replication Forks ◽  
Exchange Activity ◽  
Human Rad51

ABSTRACTThe central recombination enzyme RAD51 has been implicated in replication fork processing and restart in response to replication stress. Here, we use a separation-of-function allele of RAD51 that retains DNA binding, but not strand exchange activity, to reveal mechanistic aspects of RAD51’s roles in the response to replication stress. We find that cells lacking RAD51 strand exchange activity protect replication forks from MRE11-dependent degradation, as expected from previous studies. Unexpectedly we find that RAD51’s strand exchange activity is not required to convert stalled forks to a form that can be degraded by DNA2. Such conversion was shown previously to require replication fork reversal, supporting a model in which fork reversal depends on a non-enzymatic function of RAD51. We also show RAD51 promotes replication restart by both strand exchange-dependent and strand exchange-independent mechanisms.

scholarly journals Transcription-Associated Recombination Is Dependent on Replication in Mammalian Cells

2007 ◽  
Vol 28 (1) ◽  
pp. 154-164 ◽  
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.

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

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.

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

2006 ◽  
Vol 26 (22) ◽  
pp. 8396-8409 ◽  
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.

scholarly journals FANCD2 directly inhibits DNA2 nuclease at stalled replication forks and acts as a RAD51 mediator in strand exchange

2021 ◽  
Author(s):  
Wenpeng Liu ◽  
Ivan Roubal ◽  
Piotr Polaczek ◽  
Yuan Meng ◽  
Won-chae Choe ◽  
...  
Keyword(s):  
Protein A ◽  
Nuclease Activity ◽  
Strand Exchange ◽  
Replication Forks ◽  
Ssdna Binding ◽  
Interstrand Crosslink ◽  
Interstrand Crosslink Repair ◽  
Stalled Replication Forks ◽  
Exchange Activity

FANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea induced stalled forks. The mechanisms of fork protection are not well studied. Here, we purified FANCD2 to study how FANCD2 regulates DNA resection at stalled forks. In vitro, we showed that FANCD2 inhibits fork degradation in two ways: 1) it inhibits DNA2 nuclease activity by directly binding to DNA2. 2) independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit various nucleases, including DNA2. More unexpectedly, FANCD2 acts as a RAD51 mediator to stimulate the strand exchange activity of RAD51, and does so by enhancing ssDNA binding of RAD51. Our work biochemically explains mechanisms by which FANCD2 protects stalled forks and further provides a simple molecular explanation for genetic interactions between FANCD2 and the BRCA2 mediator.

scholarly journals Homologous recombination induced by a replication fork barrier requires cooperation between strand invasion and strand annealing activities

2021 ◽  
Author(s):  
Lea Marie ◽  
Lorraine S Symington
Keyword(s):  
Replication Fork ◽  
Repetitive Sequences ◽  
Replication Stress ◽  
Yeast Genome ◽  
Physical Analysis ◽  
Replication Forks ◽  
Strand Invasion ◽  
Strand Annealing ◽  
Recombination Pathway ◽  
Stalled Replication Forks

Replication stress and abundant repetitive sequences have emerged as primary conditions underlying genomic instability in eukaryotes. Elucidating the mechanism of recombination between repeated sequences in the context of replication stress is essential to understanding how genome rearrangements occur. To gain insight into this process, we used a prokaryotic Tus/Ter barrier designed to induce transient replication fork stalling near inverted repeats in the budding yeast genome. Remarkably, we show that the replication fork block stimulates a unique recombination pathway dependent on Rad51 strand invasion and Rad52-Rad59 strand annealing activities, as well as Mph1/Rad5 fork remodelers, Mre11/Exo1 short and long-range resection machineries, Rad1-Rad10 nuclease and DNA polymerase δ. Furthermore, we show recombination at stalled replication forks is limited by the Srs2 helicase and Mus81-Mms4/Yen1 structure-selective nucleases. Physical analysis of replication-associated recombinants revealed that half are associated with an inversion of sequence between the repeats. Based on our extensive genetic characterization, we propose a model for recombination of closely linked repeats at stalled replication forks that can actively contribute to genomic rearrangements.

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