scholarly journals Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Claudia Lancey ◽  
Muhammad Tehseen ◽  
Souvika Bakshi ◽  
Matthew Percival ◽  
Masateru Takahashi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Zinc Fingers ◽  
Structural Basis ◽  
Replication Forks ◽  
Catalytic Core ◽  
Dna Templates ◽  
Secondary Interaction ◽  
Ubiquitin Binding ◽  
Y Family ◽  
Stalled Replication Forks

AbstractY-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.

scholarly journals Structural basis of HMCES interactions with DNA reveals multivalent substrate recognition

2019 ◽  
Author(s):  
Levon Halabelian ◽  
Mani Ravichandran ◽  
Yanjun Li ◽  
Hong Zheng ◽  
L. Aravind ◽  
...  
Keyword(s):  
Dna Damage ◽  
Crystal Structures ◽  
Genome Instability ◽  
Substrate Recognition ◽  
Catalytic Triad ◽  
Structural Basis ◽  
Replication Forks ◽  
Abasic Sites ◽  
Stalled Replication Forks ◽  
Gapped Dna

ABSTRACTHMCES can covalently crosslink to abasic sites in single-stranded DNA at stalled replication forks to prevent genome instability. Here, we report crystal structures of the HMCES SRAP domain in complex with DNA-damage substrates, revealing interactions with both single-stranded and duplex segments of 3’ overhang DNA. HMCES may also bind gapped DNA and 5’ overhang structures to align single stranded abasic sites for crosslinking to the conserved Cys2 of its catalytic triad.

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 DNA Damage Discrimination at Stalled Replication Forks by the Rad5 Homologs HLTF and SHPRH

2011 ◽  
Vol 42 (2) ◽  
pp. 141-143 ◽  
Author(s):  
George-Lucian Moldovan ◽  
Alan D. D'Andrea
Keyword(s):  
Dna Damage ◽  
Replication Forks ◽  
Stalled Replication Forks

scholarly journals Regulation of E2F1 by BRCT Domain-Containing Protein TopBP1

2003 ◽  
Vol 23 (9) ◽  
pp. 3287-3304 ◽  
Author(s):  
Kang Liu ◽  
Fang-Tsyr Lin ◽  
J. Michael Ruppert ◽  
Weei-Chin Lin
Keyword(s):  
Dna Damage ◽  
Cell Cycle Progression ◽  
Specific Interaction ◽  
Dna Damage Checkpoint ◽  
Brct Domain ◽  
Replication Forks ◽  
Direct Role ◽  
Dna Topoisomerase ◽  
E2f Transcription Factor ◽  
Stalled Replication Forks

ABSTRACT The E2F transcription factor integrates cellular signals and coordinates cell cycle progression. Our prior studies demonstrated selective induction and stabilization of E2F1 through ATM-dependent phosphorylation in response to DNA damage. Here we report that DNA topoisomerase IIβ binding protein 1 (TopBP1) regulates E2F1 during DNA damage. TopBP1 contains eight BRCT (BRCA1 carboxyl-terminal) motifs and upon DNA damage is recruited to stalled replication forks, where it participates in a DNA damage checkpoint. Here we demonstrated an interaction between TopBP1 and E2F1. The interaction depended on the amino terminus of E2F1 and the sixth BRCT domain of TopBP1. It was specific to E2F1 and was not observed in E2F2, E2F3, or E2F4. This interaction was induced by DNA damage and phosphorylation of E2F1 by ATM. Through this interaction, TopBP1 repressed multiple activities of E2F1, including transcriptional activity, induction of S-phase entry, and apoptosis. Furthermore, TopBP1 relocalized E2F1 from diffuse nuclear distribution to discrete punctate nuclear foci, where E2F1 colocalized with TopBP1 and BRCA1. Thus, the specific interaction between TopBP1 and E2F1 during DNA damage inhibits the known E2F1 activities but recruits E2F1 to a BRCA1-containing repair complex, suggesting a direct role of E2F1 in DNA damage checkpoint/repair at stalled replication forks.

DNA damage-induced accumulation of Rad18 protein at stalled replication forks in mammalian cells involves upstream protein phosphorylation

2004 ◽  
Vol 323 (3) ◽  
pp. 831-837 ◽  
Author(s):  
Andrey Nikiforov ◽  
Maria Svetlova ◽  
Lioudmila Solovjeva ◽  
Lioudmila Sasina ◽  
Joseph Siino ◽  
...  
Keyword(s):  
Dna Damage ◽  
Protein Phosphorylation ◽  
Mammalian Cells ◽  
Replication Forks ◽  
Stalled Replication Forks

scholarly journals Regulation of Rtt107 Recruitment to Stalled DNA Replication Forks by the Cullin Rtt101 and the Rtt109 Acetyltransferase

2008 ◽  
Vol 19 (1) ◽  
pp. 171-180 ◽  
Author(s):  
Tania M. Roberts ◽  
Iram Waris Zaidi ◽  
Jessica A. Vaisica ◽  
Matthias Peter ◽  
Grant W. Brown
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Replication Forks ◽  
Chromatin Binding ◽  
Direct Role ◽  
Repair Enzymes ◽  
Damage Response ◽  
Stalled Replication Forks

RTT107 (ESC4, YHR154W) encodes a BRCA1 C-terminal domain protein that is important for recovery from DNA damage during S phase. Rtt107 is a substrate of the checkpoint kinase Mec1, and it forms complexes with DNA repair enzymes, including the nuclease subunit Slx4, but the role of Rtt107 in the DNA damage response remains unclear. We find that Rtt107 interacts with chromatin when cells are treated with compounds that cause replication forks to arrest. This damage-dependent chromatin binding requires the acetyltransferase Rtt109, but it does not require acetylation of the known Rtt109 target, histone H3-K56. Chromatin binding of Rtt107 also requires the cullin Rtt101, which seems to play a direct role in Rtt107 recruitment, because the two proteins are found in complex with each other. Finally, we provide evidence that Rtt107 is bound at or near stalled replication forks in vivo. Together, these results indicate that Rtt109, Rtt101, and Rtt107, which genetic evidence suggests are functionally related, form a DNA damage response pathway that recruits Rtt107 complexes to damaged or stalled replication forks.

scholarly journals The RAD51 recombinase protects mitotic chromatin in human cells

2020 ◽  
Author(s):  
Isabel E. Wassing ◽  
Xanita Saayman ◽  
Lucia Rampazzo ◽  
Christine Ralf ◽  
Andrew Bassett ◽  
...  
Keyword(s):  
Dna Damage ◽  
Spindle Assembly Checkpoint ◽  
De Novo ◽  
Human Cells ◽  
Replication Forks ◽  
Chromosomal Replication ◽  
Anaphase Onset ◽  
The Stability ◽  
Stalled Replication Forks ◽  
Mitotic Chromatin

AbstractThe RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in repairing broken DNA and protecting stalled replication forks are well characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. MiDAS was globally detectable irrespective of DNA damage and was promoted by de novo RAD51 recruitment, RAD51-mediated fork protection, and RAD51 phosphorylation by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of RAD51-promoted MiDAS led to mitotic DNA damage, delayed anaphase onset and induced centromere fragility, revealing a mechanism that prevents the satisfaction of the spindle assembly checkpoint when chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting the stability of mitotic chromatin.

Chemical strategies for development of ATR inhibitors

2014 ◽  
Vol 16 ◽  
Author(s):  
Sabin Llona-Minguez ◽  
Andreas Höglund ◽  
Sylvain A. Jacques ◽  
Tobias Koolmeister ◽  
Thomas Helleday
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Checkpoint Inhibitors ◽  
Genome Integrity ◽  
Signalling Pathways ◽  
Replication Forks ◽  
Damage Response ◽  
Key Players ◽  
Chemical Strategies ◽  
Stalled Replication Forks

ATR protein kinase is one of the key players in maintaining genome integrity and coordinating of the DNA damage response and repair signalling pathways. Inhibition of ATR prevents signalling from stalled replication forks and enhances the formation of DNA damage, particularly under conditions of replication stress present in cancers. For this reason ATR/CHK1 checkpoint inhibitors can potentiate the effect of DNA cross-linking agents, as evidenced by ATR inhibitors recently entering human clinical trials. This review aims to compile the existing literature on small molecule inhibitors of ATR, both from academia and the pharmaceutical industry, and will provide the reader with a comprehensive summary of this promising oncology target.

scholarly journals Structural basis of the 3′-end recognition of a leading strand in stalled replication forks by PriA

2007 ◽  
Vol 26 (10) ◽  
pp. 2584-2593 ◽  
Author(s):  
Kaori Sasaki ◽  
Toyoyuki Ose ◽  
Naoaki Okamoto ◽  
Katsumi Maenaka ◽  
Taku Tanaka ◽  
...  
Keyword(s):  
Structural Basis ◽  
Replication Forks ◽  
Leading Strand ◽  
Stalled Replication Forks

scholarly journals Checkpoint regulation of replication forks: global or local?

2013 ◽  
Vol 41 (6) ◽  
pp. 1701-1705 ◽  
Author(s):  
Divya Ramalingam Iyer ◽  
Nicholas Rhind
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
S Phase ◽  
Cell Cycle Checkpoints ◽  
Dna Damage Checkpoint ◽  
Replication Forks ◽  
Origin Firing ◽  
Replication Fork Progression ◽  
Late Origin ◽  
Stalled Replication Forks

Cell-cycle checkpoints are generally global in nature: one unattached kinetochore prevents the segregation of all chromosomes; stalled replication forks inhibit late origin firing throughout the genome. A potential exception to this rule is the regulation of replication fork progression by the S-phase DNA damage checkpoint. In this case, it is possible that the checkpoint is global, and it slows all replication forks in the genome. However, it is also possible that the checkpoint acts locally at sites of DNA damage, and only slows those forks that encounter DNA damage. Whether the checkpoint regulates forks globally or locally has important mechanistic implications for how replication forks deal with damaged DNA during S-phase.

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