scholarly journals SIRF: Quantitative in situ analysis of protein interactions at DNA replication forks

2018 ◽  
Vol 217 (4) ◽  
pp. 1521-1536 ◽  
Author(s):  
Sunetra Roy ◽  
Jessica W. Luzwick ◽  
Katharina Schlacher
Keyword(s):  
Quantitative Analysis ◽  
Dna Replication ◽  
Protein Interactions ◽  
Accelerated Aging ◽  
In Situ Analysis ◽  
Replication Forks ◽  
Cancer Etiology ◽  
Validation Data ◽  
Stalled Replication Forks

DNA replication reactions are central to diverse cellular processes including development, cancer etiology, drug treatment, and resistance. Many proteins and pathways exist to ensure DNA replication fidelity and protection of stalled or damaged replication forks. Consistently, mutations in proteins involved in DNA replication are implicated in diverse diseases that include defects during embryonic development and immunity, accelerated aging, increased inflammation, blood disease, and cancer. Thus, tools for efficient quantitative analysis of protein interactions at active and stalled replication forks are key for advanced and accurate biological understanding. Here we describe a sensitive single-cell–level assay system for the quantitative analysis of protein interactions with nascent DNA. Specifically, we achieve robust in situ analysis of protein interactions at DNA replication forks (SIRF) using proximity ligation coupled with 5′-ethylene-2′-deoxyuridine click chemistry suitable for multiparameter analysis in heterogeneous cell populations. We provide validation data for sensitivity, accuracy, proximity, and quantitation. Using SIRF, we obtained new insight on the regulation of pathway choice by 53BP1 at transiently stalled replication forks.

scholarly journals SIRFing the replication fork: Assessing protein interactions with nascent DNA

2018 ◽  
Vol 217 (4) ◽  
pp. 1177-1179
Author(s):  
Dana Branzei ◽  
Michele Giannattasio
Keyword(s):  
Quantitative Analysis ◽  
Dna Replication ◽  
Single Cell ◽  
Protein Interactions ◽  
Replication Fork ◽  
Cell Populations ◽  
Replication Forks ◽  
Multiparameter Measurements ◽  
Stalled Replication Forks

Roy et al. (2018. J. Cell. Biol. https://doi.org/10.1083/jcb.201709121) describe an ingenious single-cell assay system, in situ analysis of protein interactions at DNA replication forks (SIRF), for the quantitative analysis of protein interactions with nascent DNA at active and stalled replication forks. The sensitive and accurate SIRF methodology is suitable for multiparameter measurements in cell populations.

scholarly journals SIRF: Quantitative in situ analysis of protein interactions at DNA replication forks

2018 ◽  
Vol 217 (4) ◽  
pp. 1553-1553 ◽  
Author(s):  
Sunetra Roy ◽  
Jessica W. Luzwick ◽  
Katharina Schlacher
Keyword(s):  
Dna Replication ◽  
Protein Interactions ◽  
In Situ Analysis ◽  
Replication Forks

KISS: A Mammalian Two-Hybrid Method for In Situ Analysis of Protein–Protein Interactions

2018 ◽  
pp. 269-278 ◽  
Author(s):  
Delphine Masschaele ◽  
Sarah Gerlo ◽  
Irma Lemmens ◽  
Sam Lievens ◽  
Jan Tavernier
Keyword(s):  
Protein Interactions ◽  
Hybrid Method ◽  
In Situ Analysis ◽  
Protein Protein Interactions ◽  
Two Hybrid

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 Rescuing stalled replication forks: MMS22L-TONSL, a novel complex for DNA replication fork repair in human cells

Cell Cycle ◽  
2011 ◽  
Vol 10 (11) ◽  
pp. 1703-1705 ◽  
Author(s):  
Wojciech Piwko ◽  
Raymond Buser ◽  
Matthias Peter
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Human Cells ◽  
Replication Forks ◽  
Novel Complex ◽  
Stalled Replication Forks

Fine Structural in Situ Analysis of Nascent DNA Movement Following DNA Replication

2000 ◽  
Vol 260 (2) ◽  
pp. 313-323 ◽  
Author(s):  
Françoise Jaunin ◽  
Astrid E. Visser ◽  
Dusan Cmarko ◽  
Jacob A. Aten ◽  
Stanislav Fakan
Keyword(s):  
Dna Replication ◽  
In Situ Analysis

scholarly journals DNA Replication: Pif1 Pulls the Plug on Stalled Replication Forks

2012 ◽  
Vol 22 (10) ◽  
pp. R404-R405 ◽  
Author(s):  
Kenji Shimada ◽  
Susan M. Gasser
Keyword(s):  
Dna Replication ◽  
Replication Forks ◽  
Stalled Replication Forks

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.

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