Smc5/6 functions with Sgs1-Top3-Rmi1 to complete chromosome replication at natural pause sites

AbstractSmc5/6 is essential for genome structural integrity by yet unknown mechanisms. Here we find that Smc5/6 co-localizes with the DNA crossed-strand processing complex Sgs1-Top3-Rmi1 (STR) at genomic regions known as natural pausing sites (NPSs) where it facilitates Top3 retention. Individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (JMs) composed of reversed forks, double Holliday Junctions and hemicatenanes, indicative of Smc5/6 regulating Sgs1 and Top3 DNA processing activities. We isolate an intra-allelic suppressor of smc6-56 proficient in Top3 retention but affected in pathways that act complementarily with Sgs1 and Top3 to resolve JMs arising at replication termination. Upon replication stress, the smc6-56 suppressor requires STR and Mus81-Mms4 functions for recovery, but not Srs2 and Mph1 helicases that prevent maturation of recombination intermediates. Thus, Smc5/6 functions jointly with Top3 and STR to mediate replication completion and influences the function of other DNA crossed-strand processing enzymes at NPSs.

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

Nature Communications ◽

10.1038/s41467-020-20818-5 ◽

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.

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Translesion DNA Synthesis Across Lesions Induced by Oxidative Products of Pyrimidines: An Insight into the Mechanism by Microscale Thermophoresis

International Journal of Molecular Sciences ◽

10.3390/ijms20205012 ◽

2019 ◽

Vol 20 (20) ◽

pp. 5012 ◽

Cited By ~ 1

Author(s):

Ondrej Hrabina ◽

Viktor Brabec ◽

Olga Novakova

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Dna Polymerases ◽

Dna Duplexes ◽

Translesion Dna Synthesis ◽

Processing Enzymes ◽

Microscale Thermophoresis ◽

Oxidative Products ◽

Dna Processing ◽

Damaged Dna

Oxidative stress in cells can lead to the accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized nucleotides such as 2’-deoxyribo-5-hydroxyuridin (HdU) and 2’-deoxyribo-5-hydroxymethyluridin (HMdU) can be inserted into DNA during replication and repair. HdU and HMdU have attracted particular interest because they have different effects on damaged-DNA processing enzymes that control the downstream effects of the lesions. Herein, we studied the chemically simulated translesion DNA synthesis (TLS) across the lesions formed by HdU or HMdU using microscale thermophoresis (MST). The thermodynamic changes associated with replication across HdU or HMdU show that the HdU paired with the mismatched deoxyribonucleoside triphosphates disturbs DNA duplexes considerably less than thymidine (dT) or HMdU. Moreover, we also demonstrate that TLS by DNA polymerases across the lesion derived from HdU was markedly less extensive and potentially more mutagenic than that across the lesion formed by HMdU. Thus, DNA polymerization by DNA polymerase η (polη), the exonuclease-deficient Klenow fragment of DNA polymerase I (KF–), and reverse transcriptase from human immunodeficiency virus type 1 (HIV-1 RT) across these pyrimidine lesions correlated with the different stabilization effects of the HdU and HMdU in DNA duplexes revealed by MST. The equilibrium thermodynamic data obtained by MST can explain the influence of the thermodynamic alterations on the ability of DNA polymerases to bypass lesions induced by oxidative products of pyrimidines. The results also highlighted the usefulness of MST in evaluating the impact of oxidative products of pyrimidines on the processing of these lesions by damaged DNA processing enzymes.

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Untwisting and Unzipping: Magnetic Tweezers Based Measurements of DNA Processing Enzymes

Optics in the Life Sciences ◽

10.1364/ota.2015.otw3e.1 ◽

2015 ◽

Cited By ~ 1

Author(s):

Keir C. Neuman ◽

Yeonee Seol

Keyword(s):

Magnetic Tweezers ◽

Processing Enzymes ◽

Dna Processing

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FANCD 2 protects genome stability by recruiting RNA processing enzymes to resolve R‐loops during mild replication stress

FEBS Journal ◽

10.1111/febs.14700 ◽

2018 ◽

Vol 286 (1) ◽

pp. 139-150 ◽

Cited By ~ 13

Author(s):

Yusuke Okamoto ◽

Masako Abe ◽

Akiko Itaya ◽

Junya Tomida ◽

Masamichi Ishiai ◽

...

Keyword(s):

Rna Processing ◽

Genome Stability ◽

Replication Stress ◽

Processing Enzymes

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MUS81 nuclease activity is essential for replication stress tolerance and chromosome segregation in BRCA2-deficient cells

Nature Communications ◽

10.1038/ncomms15983 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 39

Author(s):

Xianning Lai ◽

Ronan Broderick ◽

Valérie Bergoglio ◽

Jutta Zimmer ◽

Sophie Badie ◽

...

Keyword(s):

Stress Tolerance ◽

Chromosome Segregation ◽

Replication Fork ◽

Replication Stress ◽

Nuclease Activity ◽

Dna Lesions ◽

Genome Replication ◽

Replication Fork Progression ◽

Nucleolytic Activity ◽

Genomic Regions

AbstractFailure to restart replication forks stalled at genomic regions that are difficult to replicate or contain endogenous DNA lesions is a hallmark of BRCA2 deficiency. The nucleolytic activity of MUS81 endonuclease is required for replication fork restart under replication stress elicited by exogenous treatments. Here we investigate whether MUS81 could similarly facilitate DNA replication in the context of BRCA2 abrogation. Our results demonstrate that replication fork progression in BRCA2-deficient cells requires MUS81. Failure to complete genome replication and defective checkpoint surveillance enables BRCA2-deficient cells to progress through mitosis with under-replicated DNA, which elicits severe chromosome interlinking in anaphase. MUS81 nucleolytic activity is required to activate compensatory DNA synthesis during mitosis and to resolve mitotic interlinks, thus facilitating chromosome segregation. We propose that MUS81 provides a mechanism of replication stress tolerance, which sustains survival of BRCA2-deficient cells and can be exploited therapeutically through development of specific inhibitors of MUS81 nuclease activity.

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Replication Stress Shapes a Protective Chromatin Environment across Fragile Genomic Regions

Molecular Cell ◽

10.1016/j.molcel.2017.11.021 ◽

2018 ◽

Vol 69 (1) ◽

pp. 36-47.e7 ◽

Cited By ~ 31

Author(s):

Jeongkyu Kim ◽

David Sturgill ◽

Robin Sebastian ◽

Simran Khurana ◽

Andy D. Tran ◽

...

Keyword(s):

Replication Stress ◽

Genomic Regions

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Karyotypic Flexibility of the Complex Cancer Genome and the Role of Polyploidization in Maintenance of Structural Integrity of Cancer Chromosomes

Cancers ◽

10.3390/cancers12030591 ◽

2020 ◽

Vol 12 (3) ◽

pp. 591

Author(s):

Christina Raftopoulou ◽

Fani-Marlen Roumelioti ◽

Eleni Dragona ◽

Stefanie Gimelli ◽

Frédérique Sloan-Béna ◽

...

Keyword(s):

Structural Integrity ◽

Replication Stress ◽

Therapy Resistance ◽

Cancer Genome ◽

Therapeutic Interventions ◽

Molecular Karyotyping ◽

Comparative Genomic ◽

Tumor Evolution ◽

Osteosarcoma Cell ◽

High Tendency

Ongoing chromosomal instability in neoplasia (CIN) generates intratumor genomic heterogeneity and limits the efficiency of oncotherapeutics. Neoplastic human cells utilizing the alternative lengthening of telomeres (ALT)-pathway, display extensive structural and numerical CIN. To unravel patterns of genome evolution driven by oncogene-replication stress, telomere dysfunction, or genotoxic therapeutic interventions, we examined by comparative genomic hybridization five karyotypically-diverse outcomes of the ALT osteosarcoma cell line U2-OS. These results demonstrate a high tendency of the complex cancer genome to perpetuate specific genomic imbalances despite the karyotypic evolution, indicating an ongoing process of genome dosage maintenance. Molecular karyotyping in four ALT human cell lines showed that mitotic cells with low levels of random structural CIN display frequent evidence of whole genome doubling (WGD), suggesting that WGD may protect clonal chromosome aberrations from hypermutation. We tested this longstanding hypothesis in ALT cells exposed to gamma irradiation or to inducible DNA replication stress under overexpression of p21. Single-cell cytogenomic analyses revealed that although polyploidization promotes genomic heterogeneity, it also protects the complex cancer genome and hence confers genotoxic therapy resistance by generating identical extra copies of driver chromosomal aberrations, which can be spared in the process of tumor evolution if they undergo unstable or unfit rearrangements.

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The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1909017116 ◽

2019 ◽

Vol 116 (50) ◽

pp. 25068-25077 ◽

Cited By ~ 7

Author(s):

Aishwarya Devaraj ◽

John R. Buzzo ◽

Lauren Mashburn-Warren ◽

Erin S. Gloag ◽

Laura A. Novotny ◽

...

Keyword(s):

Structural Integrity ◽

Bacterial Species ◽

Holliday Junctions ◽

Extracellular Dna ◽

Bacterial Biofilms ◽

Human Pathogens ◽

E Coli ◽

Functional Equivalent ◽

Biofilm Disruption ◽

Biofilm Structure

Extracellular DNA (eDNA) is a critical component of the extracellular matrix of bacterial biofilms that protects the resident bacteria from environmental hazards, which includes imparting significantly greater resistance to antibiotics and host immune effectors. eDNA is organized into a lattice-like structure, stabilized by the DNABII family of proteins, known to have high affinity and specificity for Holliday junctions (HJs). Accordingly, we demonstrated that the branched eDNA structures present within the biofilms formed by NTHI in the middle ear of the chinchilla in an experimental otitis media model, and in sputum samples recovered from cystic fibrosis patients that contain multiple mixed bacterial species, possess an HJ-like configuration. Next, we showed that the prototypic Escherichia coli HJ-specific DNA-binding protein RuvA could be functionally exchanged for DNABII proteins in the stabilization of biofilms formed by 3 diverse human pathogens, uropathogenic E. coli, nontypeable Haemophilus influenzae, and Staphylococcus epidermidis. Importantly, while replacement of DNABII proteins within the NTHI biofilm matrix with RuvA was shown to retain similar mechanical properties when compared to the control NTHI biofilm structure, we also demonstrated that biofilm eDNA matrices stabilized by RuvA could be subsequently undermined upon addition of the HJ resolvase complex, RuvABC, which resulted in significant biofilm disruption. Collectively, our data suggested that nature has recapitulated a functional equivalent of the HJ recombination intermediate to maintain the structural integrity of bacterial biofilms.

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