scholarly journals TopBP1 and DNA polymerase alpha-mediated recruitment of the 9-1-1 complex to stalled replication forks: Implications for a replication restart-based mechanism for ATR checkpoint activation

Cell Cycle ◽  
2009 ◽  
Vol 8 (18) ◽  
pp. 2877-2884 ◽  
Author(s):  
Shan Yan ◽  
W. Matthew Michael
Keyword(s):  
Dna Polymerase ◽  
Replication Forks ◽  
Checkpoint Activation ◽  
Dna Polymerase Alpha ◽  
Replication Restart ◽  
Stalled Replication Forks

scholarly journals Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

2015 ◽  
Vol 197 (17) ◽  
pp. 2792-2809 ◽  
Author(s):  
Sarita Mallik ◽  
Ellen M. Popodi ◽  
Andrew J. Hanson ◽  
Patricia L. Foster
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Replication Forks ◽  
Content Type ◽  
Pol Iv ◽  
Dna Polymerase Iv ◽  
Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

scholarly journals TopBP1 and DNA polymerase-α directly recruit the 9-1-1 complex to stalled DNA replication forks

2009 ◽  
Vol 184 (6) ◽  
pp. 793-804 ◽  
Author(s):  
Shan Yan ◽  
W. Matthew Michael
Keyword(s):  
Dna Polymerase ◽  
Replication Stress ◽  
Replication Forks ◽  
Ataxia Telangiectasia Mutated ◽  
Interacting Protein ◽  
Checkpoint Signaling ◽  
Binding Partner ◽  
Assembly Pathway ◽  
Egg Extracts ◽  
Stalled Replication Forks

TopBP1 and the Rad9–Rad1–Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR–ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-α to sites of replication stress. Furthermore, we show that pol-α is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-α, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress.

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 Colocalization of Mec1 and Mrc1 is sufficient for Rad53 phosphorylation in vivo

2012 ◽  
Vol 23 (6) ◽  
pp. 1058-1067 ◽  
Author(s):  
Theresa J. Berens ◽  
David P. Toczyski
Keyword(s):  
Dna Replication ◽  
Double Strand Breaks ◽  
Sensor Kinase ◽  
Replication Forks ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Strand Breaks ◽  
Crucial Step ◽  
Stalled Replication Forks

When DNA is damaged or DNA replication goes awry, cells activate checkpoints to allow time for damage to be repaired and replication to complete. In Saccharomyces cerevisiae, the DNA damage checkpoint, which responds to lesions such as double-strand breaks, is activated when the lesion promotes the association of the sensor kinase Mec1 and its targeting subunit Ddc2 with its activators Ddc1 (a member of the 9-1-1 complex) and Dpb11. It has been more difficult to determine what role these Mec1 activators play in the replication checkpoint, which recognizes stalled replication forks, since Dpb11 has a separate role in DNA replication itself. Therefore we constructed an in vivo replication-checkpoint mimic that recapitulates Mec1-dependent phosphorylation of the effector kinase Rad53, a crucial step in checkpoint activation. In the endogenous replication checkpoint, Mec1 phosphorylation of Rad53 requires Mrc1, a replisome component. The replication-checkpoint mimic requires colocalization of Mrc1-LacI and Ddc2-LacI and is independent of both Ddc1 and Dpb11. We show that these activators are also dispensable for Mec1 activity and cell survival in the endogenous replication checkpoint but that Ddc1 is absolutely required in the absence of Mrc1. We propose that colocalization of Mrc1 and Mec1 is the minimal signal required to activate the replication checkpoint.

scholarly journals PrimPol is required for replication reinitiation after mtDNA damage

2017 ◽  
Vol 114 (43) ◽  
pp. 11398-11403 ◽  
Author(s):  
Rubén Torregrosa-Muñumer ◽  
Josefin M. E. Forslund ◽  
Steffi Goffart ◽  
Annika Pfeiffer ◽  
Gorazd Stojkovič ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Translesion Synthesis ◽  
Replication Forks ◽  
Mtdna Damage ◽  
Replicative Stress ◽  
Mtdna Replication ◽  
Stalled Replication Forks ◽  
Lagging Strand

Eukaryotic PrimPol is a recently discovered DNA-dependent DNA primase and translesion synthesis DNA polymerase found in the nucleus and mitochondria. Although PrimPol has been shown to be required for repriming of stalled replication forks in the nucleus, its role in mitochondria has remained unresolved. Here we demonstrate in vivo and in vitro that PrimPol can reinitiate stalled mtDNA replication and can prime mtDNA replication from nonconventional origins. Our results not only help in the understanding of how mitochondria cope with replicative stress but can also explain some controversial features of the lagging-strand replication.

scholarly journals Human HEL308 Localizes to Damaged Replication Forks and Unwinds Lagging Strand Structures

2011 ◽  
Vol 286 (18) ◽  
pp. 15832-15840 ◽  
Author(s):  
Agnieszka A. Tafel ◽  
Leonard Wu ◽  
Peter J. McHugh
Keyword(s):  
Protein A ◽  
Dna Helicase ◽  
Replication Forks ◽  
Double Stranded Dna ◽  
Replication Restart ◽  
Two Factors ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Stalled Replication Forks ◽  
Lagging Strand

HEL308 is a superfamily II DNA helicase, conserved from archaea through to humans. HEL308 family members were originally isolated by their similarity to the Drosophila melanogaster Mus308 protein, which contributes to the repair of replication-blocking lesions such as DNA interstrand cross-links. Biochemical studies have established that human HEL308 is an ATP-dependent enzyme that unwinds DNA with a 3′ to 5′ polarity, but little else is know about its mechanism. Here, we show that GFP-tagged HEL308 localizes to replication forks following camptothecin treatment. Moreover, HEL308 colocalizes with two factors involved in the repair of damaged forks by homologous recombination, Rad51 and FANCD2. Purified HEL308 requires a 3′ single-stranded DNA region to load and unwind duplex DNA structures. When incubated with substrates that model stalled replication forks, HEL308 preferentially unwinds the parental strands of a structure that models a fork with a nascent lagging strand, and the unwinding action of HEL308 is specifically stimulated by human replication protein A. Finally, we show that HEL308 appears to target and unwind from the junction between single-stranded to double-stranded DNA on model fork structures. Together, our results suggest that one role for HEL308 at sites of blocked replication might be to open up the parental strands to facilitate the loading of subsequent factors required for replication restart.

scholarly journals DNA polymerase κ-dependent DNA synthesis at stalled replication forks is important for CHK1 activation

2013 ◽  
Vol 32 (15) ◽  
pp. 2172-2185 ◽  
Author(s):  
Rémy Bétous ◽  
Marie-Jeanne Pillaire ◽  
Laura Pierini ◽  
Siem van der Laan ◽  
Bénédicte Recolin ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Replication Forks ◽  
Stalled Replication Forks

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.

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