scholarly journals Bloom syndrome complex promotes FANCM recruitment to stalled replication forks and facilitates both repair and traverse of DNA interstrand crosslinks

2016 ◽  
Vol 2 (1) ◽  
Author(s):  
Chen Ling ◽  
Jing Huang ◽  
Zhijiang Yan ◽  
Yongjiang Li ◽  
Mioko Ohzeki ◽  
...  
Keyword(s):  
Bloom Syndrome ◽  
Replication Forks ◽  
Interstrand Crosslinks ◽  
Dna Interstrand Crosslinks ◽  
Stalled Replication Forks

scholarly journals Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

2019 ◽  
Author(s):  
Kimberly A. Rickman ◽  
Ray Noonan ◽  
Francis P. Lach ◽  
Sunandini Sridhar ◽  
Anderson T. Wang ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Essential Gene ◽  
Dna Lesions ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Interstrand Crosslinks ◽  
Pathogenic Variants ◽  
Dna Interstrand Crosslinks ◽  
Stalled Replication Forks

SummaryDNA interstrand crosslinks (ICLs) are a form of DNA damage that requires the interplay of a number of repair proteins including those of the Fanconi anemia (FA) and the homologous recombination (HR) pathways. Pathogenic variants in the essential gene BRCA2/FANCD1, when monoallelic, predispose to breast and ovarian cancer, and when biallelic, results in a severe subtype of Fanconi anemia. BRCA2 function in the FA pathway is attributed to its role as a mediator of the RAD51 recombinase in HR repair of the programmed DNA double strand breaks (DSB). BRCA2 and RAD51 functions are also required to protect stalled replication forks from nucleolytic degradation during response to hydroxyurea (HU). While RAD51 has been shown to be necessary in the early steps of ICL repair to prevent aberrant nuclease resection, the role of BRCA2 in this process has not been described. Here, based on the analysis of BRCA2 DNA binding domain (DBD) mutants discovered in FA patients presenting with atypical FA-like phenotypes, we establish that BRCA2 is necessary for protection of DNA at an ICL. Cells carrying DBD BRCA2 mutations are sensitive to ICL inducing agents but resistant to HU treatment consistent with relatively high HR repair in these cells. BRCA2 function at an ICL protects against DNA2-WRN nuclease-helicase complex and not the MRE11 nuclease implicated in the resection of HU-stalled replication forks. Our results also indicate that unlike the processing at HU-stalled forks, function of the SNF2 translocases (SMARCAL1, ZRANB3, or HLTF), implicated in fork reversal, are not an integral component of the ICL repair, pointing to a different mechanism of fork protection at different DNA lesions.

scholarly journals The Bloom syndrome complex senses RPA-coated single-stranded DNA to restart stalled replication forks

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ann-Marie K. Shorrocks ◽  
Samuel E. Jones ◽  
Kaima Tsukada ◽  
Carl A. Morrow ◽  
Zoulikha Belblidia ◽  
...  
Keyword(s):  
Replication Stress ◽  
Bloom Syndrome ◽  
Holliday Junctions ◽  
Replication Forks ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Ssdna Binding Protein ◽  
Dna Replication Stress ◽  
Dna End Resection ◽  
Stalled Replication Forks

AbstractThe Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.

scholarly journals DNA clamp function of the mono-ubiquitinated Fanconi Anemia FANCI-FANCD2 complex

2019 ◽  
Author(s):  
Renjing Wang ◽  
Shengliu Wang ◽  
Ankita Dhar ◽  
Christopher Peralta ◽  
Nikola P. Pavletich
Keyword(s):  
Fanconi Anemia ◽  
Translesion Synthesis ◽  
The Other ◽  
Replication Forks ◽  
Dna Structures ◽  
Cancer Predisposition Syndrome ◽  
Interstrand Crosslinks ◽  
Branched Dna ◽  
Closed Ring ◽  
Dna Interstrand Crosslinks

ABSTRACTThe FANCI-FANCD2 (ID) complex, mutated in the Fanconi Anemia (FA) cancer predisposition syndrome, is required for the repair of replication forks stalled at DNA interstrand crosslinks (ICL) and related lesions1. The FA pathway is activated when two replication forks converge onto an ICL2, triggering the mono-ubiquitination of the ID complex. ID mono-ubiquitination is essential for ICL repair by excision, translesion synthesis and homologous recombination, but its function was hitherto unknown1,3. Here, the 3.48 Å cryo-EM structure of mono-ubiquitinated ID (IDUb) bound to DNA reveals that it forms a closed ring that encircles the DNA. Compared to the cryo-EM structure of the non-ubiquitinated ID complex bound to ICL DNA, described here as well, mono-ubiquitination triggers a complete re-arrangement of the open, trough-like ID structure through the ubiquitin of one protomer binding to the other protomer in a reciprocal fashion. The structures, in conjunction with biochemical data, indicate the mono-ubiquitinated ID complex looses its preference for ICL and related branched DNA structures, becoming a sliding DNA clamp that can coordinate the subsequent repair reactions. Our findings also reveal how mono-ubiquitination in general can induce an alternate structure with a new function.

scholarly journals Bloom syndrome cells undergo p53-dependent apoptosis and delayed assembly of BRCA1 and NBS1 repair complexes at stalled replication forks

2003 ◽  
Vol 162 (7) ◽  
pp. 1197-1209 ◽  
Author(s):  
Albert R. Davalos ◽  
Judith Campisi
Keyword(s):  
Human Fibroblasts ◽  
Postnatal Growth ◽  
Bloom Syndrome ◽  
Dna Helicase ◽  
Dominant Negative ◽  
Replication Forks ◽  
Dna Breaks ◽  
Early Responder ◽  
Dominant Negative Activity ◽  
Stalled Replication Forks

Bloom syndrome (BS) is a hereditary disorder characterized by pre- and postnatal growth retardation, genomic instability, and cancer. BLM, the gene defective in BS, encodes a DNA helicase thought to participate in genomic maintenance. We show that BS human fibroblasts undergo extensive apoptosis after DNA damage specifically when DNA replication forks are stalled. Damage during S, but not G1, caused BLM to rapidly form foci with γH2AX at replication forks that develop DNA breaks. These BLM foci recruited BRCA1 and NBS1. Damaged BS cells formed BRCA1/NBS1 foci with markedly delayed kinetics. Helicase-defective BLM showed dominant-negative activity with respect to apoptosis, but not BRCA1/NBS1 recruitment, suggesting catalytic and structural roles for BLM. Strikingly, inactivation of p53 prevented the death of damaged BS cells and delayed recruitment of BRCA1/NBS1. These findings suggest that BLM is an early responder to damaged replication forks. Moreover, p53 eliminates cells that rapidly assemble BRCA1/NBS1 without BLM, suggesting that BLM is essential for timely BRCA1/NBS1 function.

scholarly journals Single Molecular Analysis of the Encounter of Replication Forks with DNA Interstrand Crosslinks

2013 ◽  
Vol 27 (S1) ◽  
Author(s):  
Jing Huang ◽  
Shuo Liu ◽  
Yinsheng Wang ◽  
Arun Kalliat Thazhathveetil ◽  
Weidong Wang ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Replication Forks ◽  
Interstrand Crosslinks ◽  
Dna Interstrand Crosslinks

scholarly journals PrimPol primase mediates replication traverse of DNA interstrand crosslinks

2020 ◽  
Author(s):  
Daniel González-Acosta ◽  
Elena Blanco-Romero ◽  
Karun Mutreja ◽  
Susana Llanos ◽  
Samuel Míguez ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Dna Lesions ◽  
Replication Forks ◽  
Tolerance Mechanism ◽  
Dna Damage Tolerance ◽  
Genetic Ablation ◽  
Interstrand Crosslinks ◽  
Dna Interstrand Crosslinks ◽  
Dna Crosslinking ◽  
Crosslinking Agents

ABSTRACTInterstrand crosslinks (ICLs) are DNA lesions frequently induced by chemotherapy that interfere with essential processes such as replication and transcription. ICL repair may be initiated by the convergence of two replication forks at the crosslink, which results in a termination-like DNA structure recognized and processed by the Fanconi Anemia (FA) pathway. An alternative possibility to generate a suitable substrate for ICL repair involves “ICL traverse”, a DNA damage tolerance mechanism in which a single fork arriving at the ICL can skip the lesion and restart DNA synthesis from a downstream point. This reaction requires FANCM translocase, the BLM/TOP3A/RMI1-2 (BTR) complex and other factors. Here we report that PrimPol, the second primase-polymerase identified in mammalian cells after Polα/Primase, interacts with BTR and participates in the ICL traverse reaction. A functional complementation assay reveals that the primase activity of PrimPol is required, confirming the need for re-priming events during ICL traverse. Genetic ablation of PRIMPOL strongly impaired this tolerance mechanism, making cells more dependent on fork convergence to initiate ICL repair. PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs, opening the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.

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