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1.
Proc Natl Acad Sci U S A ; 121(12): e2316491121, 2024 Mar 19.
Article in English | MEDLINE | ID: mdl-38466836

ABSTRACT

Replication fork reversal is a fundamental process required for resolution of encounters with DNA damage. A key step in the stabilization and eventual resolution of reversed forks is formation of RAD51 nucleoprotein filaments on exposed single strand DNA (ssDNA). To avoid genome instability, RAD51 filaments are tightly controlled by a variety of positive and negative regulators. RADX (RPA-related RAD51-antagonist on the X chromosome) is a recently discovered negative regulator that binds tightly to ssDNA, directly interacts with RAD51, and regulates replication fork reversal and stabilization in a context-dependent manner. Here, we present a structure-based investigation of RADX's mechanism of action. Mass photometry experiments showed that RADX forms multiple oligomeric states in a concentration-dependent manner, with a predominance of trimers in the presence of ssDNA. The structure of RADX, which has no structurally characterized orthologs, was determined ab initio by cryo-electron microscopy (cryo-EM) from maps in the 2 to 4 Å range. The structure reveals the molecular basis for RADX oligomerization and the coupled multi-valent binding of ssDNA binding. The interaction of RADX with RAD51 filaments was imaged by negative stain EM, which showed a RADX oligomer at the end of filaments. Based on these results, we propose a model in which RADX functions by capping and restricting the end of RAD51 filaments.


Subject(s)
DNA-Binding Proteins , Rad51 Recombinase , DNA-Binding Proteins/metabolism , Rad51 Recombinase/metabolism , Cryoelectron Microscopy , Nucleoproteins/metabolism , DNA, Single-Stranded , DNA Replication
2.
bioRxiv ; 2023 Oct 27.
Article in English | MEDLINE | ID: mdl-37961428

ABSTRACT

G-quadruplexes (G4s) form throughout the genome and influence important cellular processes, but their deregulation can challenge DNA replication fork progression and threaten genome stability. Here, we demonstrate an unexpected, dual role for the dsDNA translocase HLTF in G4 metabolism. First, we find that HLTF is enriched at G4s in the human genome and suppresses G4 accumulation throughout the cell cycle using its ATPase activity. This function of HLTF affects telomere maintenance by restricting alternative lengthening of telomeres, a process stimulated by G4s. We also show that HLTF and MSH2, a mismatch repair factor that binds G4s, act in independent pathways to suppress G4s and to promote resistance to G4 stabilization. In a second, distinct role, HLTF restrains DNA synthesis upon G4 stabilization by suppressing PrimPol-dependent repriming. Together, the dual functions of HLTF in the G4 response prevent DNA damage and potentially mutagenic replication to safeguard genome stability.

3.
Cell Rep ; 42(11): 113427, 2023 11 28.
Article in English | MEDLINE | ID: mdl-37950866

ABSTRACT

Abasic sites are common DNA lesions stalling polymerases and threatening genome stability. When located in single-stranded DNA (ssDNA), they are shielded from aberrant processing by 5-hydroxymethyl cytosine, embryonic stem cell (ESC)-specific (HMCES) via a DNA-protein crosslink (DPC) that prevents double-strand breaks. Nevertheless, HMCES-DPCs must be removed to complete DNA repair. Here, we find that DNA polymerase α inhibition generates ssDNA abasic sites and HMCES-DPCs. These DPCs are resolved with a half-life of approximately 1.5 h. HMCES can catalyze its own DPC self-reversal reaction, which is dependent on glutamate 127 and is favored when the ssDNA is converted to duplex DNA. When the self-reversal mechanism is inactivated in cells, HMCES-DPC removal is delayed, cell proliferation is slowed, and cells become hypersensitive to DNA damage agents that increase AP (apurinic/apyrimidinic) site formation. In these circumstances, proteolysis may become an important mechanism of HMCES-DPC resolution. Thus, HMCES-DPC formation followed by self-reversal is an important mechanism for ssDNA AP site management.


Subject(s)
DNA Damage , Proteins , Proteins/genetics , DNA Replication , DNA Repair , DNA/genetics , DNA, Single-Stranded
4.
bioRxiv ; 2023 Sep 20.
Article in English | MEDLINE | ID: mdl-37786681

ABSTRACT

Replication fork reversal is a fundamental process required for resolution of encounters with DNA damage. A key step in the stabilization and eventual resolution of reversed forks is formation of RAD51 nucleoprotein filaments on exposed ssDNA. To avoid genome instability, RAD51 filaments are tightly controlled by a variety of positive and negative regulators. RADX is a recently discovered negative regulator that binds tightly to ssDNA, directly interacts with RAD51, and regulates replication fork reversal and stabilization in a context-dependent manner. Here we present a structure-based investigation of RADX's mechanism of action. Mass photometry experiments showed that RADX forms multiple oligomeric states in a concentration dependent manner, with a predominance of trimers in the presence of ssDNA. The structure of RADX, which has no structurally characterized orthologs, was determined ab initio by cryo-electron microscopy (EM) from maps in the 2-3 Å range. The structure reveals the molecular basis for RADX oligomerization and binding of ssDNA binding. The binding of RADX to RAD51 filaments was imaged by negative stain EM, which showed a RADX oligomer at the end of filaments. Based on these results, we propose a model in which RADX functions by capping and restricting the growing end of RAD51 filaments.

5.
J Mol Biol ; 435(19): 168236, 2023 10 01.
Article in English | MEDLINE | ID: mdl-37572935

ABSTRACT

RAD51 forms nucleoprotein filaments to promote homologous recombination, replication fork reversal, and fork protection. Numerous factors regulate the stability of these filaments and improper regulation leads to genomic instability and ultimately disease including cancer. RADX is a single stranded DNA binding protein that modulates RAD51 filament stability. Here, we utilize a CRISPR-dependent base editing screen to tile mutations across RADX to delineate motifs required for RADX function. We identified separation of function mutants of RADX that bind DNA and RAD51 but have a reduced ability to stimulate its ATP hydrolysis activity. Cells expressing these RADX mutants accumulate RAD51 on chromatin, exhibit replication defects, have reduced growth, accumulate DNA damage, and are hypersensitive to DNA damage and replication stress. These results indicate that RADX must promote RAD51 ATP turnover to regulate RAD51 and genome stability during DNA replication.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , RNA Editing , Rad51 Recombinase , Humans , Adenosine Triphosphate/metabolism , DNA Replication/genetics , DNA, Single-Stranded , Gene Editing , Genomic Instability/genetics , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism
6.
bioRxiv ; 2023 Jun 14.
Article in English | MEDLINE | ID: mdl-37398432

ABSTRACT

Abasic sites are common DNA lesions that stall polymerases and threaten genome stability. When located in single-stranded DNA (ssDNA), they are shielded from aberrant processing by HMCES via a DNA-protein crosslink (DPC) that prevents double-strand breaks. Nevertheless, the HMCES-DPC must be removed to complete DNA repair. Here, we found that DNA polymerase α inhibition generates ssDNA abasic sites and HMCES-DPCs. These DPCs are resolved with a half-life of approximately 1.5 hours. Resolution does not require the proteasome or SPRTN protease. Instead, HMCES-DPC self-reversal is important for resolution. Biochemically, self-reversal is favored when the ssDNA is converted to duplex DNA. When the self-reversal mechanism is inactivated, HMCES-DPC removal is delayed, cell proliferation is slowed, and cells become hypersensitive to DNA damage agents that increase AP site formation. Thus, HMCES-DPC formation followed by self-reversal is an important mechanism for ssDNA AP site management.

7.
Mol Cell ; 83(13): 2357-2366.e8, 2023 Jul 06.
Article in English | MEDLINE | ID: mdl-37295432

ABSTRACT

DNA replication preferentially initiates close to active transcription start sites (TSSs) in the human genome. Transcription proceeds discontinuously with an accumulation of RNA polymerase II (RNAPII) in a paused state near the TSS. Consequently, replication forks inevitably encounter paused RNAPII soon after replication initiates. Hence, dedicated machinery may be needed to remove RNAPII and facilitate unperturbed fork progression. In this study, we discovered that Integrator, a transcription termination machinery involved in the processing of RNAPII transcripts, interacts with the replicative helicase at active forks and promotes the removal of RNAPII from the path of the replication fork. Integrator-deficient cells have impaired replication fork progression and accumulate hallmarks of genome instability including chromosome breaks and micronuclei. The Integrator complex resolves co-directional transcription-replication conflicts to facilitate faithful DNA replication.


Subject(s)
DNA Replication , RNA Polymerase II , Humans , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Transcription, Genetic , DNA Helicases/genetics , DNA Helicases/metabolism , Genomic Instability
8.
Science ; 380(6643): 382-387, 2023 04 28.
Article in English | MEDLINE | ID: mdl-37104614

ABSTRACT

Replication fork reversal safeguards genome integrity as a replication stress response. DNA translocases and the RAD51 recombinase catalyze reversal. However, it remains unknown why RAD51 is required and what happens to the replication machinery during reversal. We find that RAD51 uses its strand exchange activity to circumvent the replicative helicase, which remains bound to the stalled fork. RAD51 is not required for fork reversal if the helicase is unloaded. Thus, we propose that RAD51 creates a parental DNA duplex behind the helicase that is used as a substrate by the DNA translocases for branch migration to create a reversed fork structure. Our data explain how fork reversal happens while maintaining the helicase in a position poised to restart DNA synthesis and complete genome duplication.


Subject(s)
DNA Replication , DNA-Binding Proteins , Rad51 Recombinase , Carrier Proteins/metabolism , DNA/genetics , DNA/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Humans , HCT116 Cells , Minichromosome Maintenance Complex Component 2/metabolism , Xenopus
9.
Nat Struct Mol Biol ; 30(1): 115-124, 2023 01.
Article in English | MEDLINE | ID: mdl-36593312

ABSTRACT

Genotoxins cause nascent strand degradation (NSD) and fork reversal during DNA replication. NSD and fork reversal are crucial for genome stability and are exploited by chemotherapeutic approaches. However, it is unclear how NSD and fork reversal are triggered. Additionally, the fate of the replicative helicase during these processes is unknown. We developed a biochemical approach to study synchronous, localized NSD and fork reversal using Xenopus egg extracts and validated this approach with experiments in human cells. We show that replication fork uncoupling stimulates NSD of both nascent strands and progressive conversion of uncoupled forks to reversed forks. Notably, the replicative helicase remains bound during NSD and fork reversal. Unexpectedly, NSD occurs before and after fork reversal, indicating that multiple degradation steps take place. Overall, our data show that uncoupling causes NSD and fork reversal and elucidate key events that precede fork reversal.


Subject(s)
DNA Replication , DNA-Binding Proteins , Animals , Humans , DNA-Binding Proteins/metabolism , DNA Helicases/metabolism , Genomic Instability , Xenopus laevis/metabolism
10.
J Biol Chem ; 298(9): 102307, 2022 09.
Article in English | MEDLINE | ID: mdl-35934051

ABSTRACT

Apurinic/apyrimidinic (AP, or abasic) sites in DNA are one of the most common forms of DNA damage. AP sites are reactive and form cross-links to both proteins and DNA, are prone to strand breakage, and inhibit DNA replication and transcription. The replication-associated AP site repair protein HMCES protects cells from strand breaks, inhibits mutagenic translesion synthesis, and participates in repair of interstrand DNA cross-links derived from AP sites by forming a stable thiazolidine DNA-protein cross-link (DPC) to AP sites in single-stranded DNA (ssDNA). Despite the importance of HMCES to genome maintenance and the evolutionary conservation of its catalytic SRAP (SOS Response Associated Peptidase) domain, the enzymatic mechanisms of DPC formation and resolution are unknown. Using the bacterial homolog YedK, we show that the SRAP domain catalyzes conversion of the AP site to its reactive, ring-opened aldehyde form, and we provide structural evidence for the Schiff base intermediate that forms prior to the more stable thiazolidine. We also report two new activities, whereby SRAP reacts with polyunsaturated aldehydes at DNA 3'-ends generated by bifunctional DNA glycosylases and catalyzes direct reversal of the DPC to regenerate the AP site, the latter of which we observe in both YedK and HMCES-SRAP proteins. Taken together, this work provides insights into possible mechanisms by which HMCES DPCs are resolved in cells.


Subject(s)
DNA Glycosylases , DNA, Single-Stranded , Aldehydes , DNA/metabolism , DNA Damage , DNA Glycosylases/metabolism , DNA Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Peptide Hydrolases/metabolism , Proteins/genetics , SOS Response, Genetics , Schiff Bases , Thiazolidines
11.
EMBO J ; 41(12): e110632, 2022 06 14.
Article in English | MEDLINE | ID: mdl-35578785

ABSTRACT

Topoisomerase II (TOP2) unlinks chromosomes during vertebrate DNA replication. TOP2 "poisons" are widely used chemotherapeutics that stabilize TOP2 complexes on DNA, leading to cytotoxic DNA breaks. However, it is unclear how these drugs affect DNA replication, which is a major target of TOP2 poisons. Using Xenopus egg extracts, we show that the TOP2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces TOP2-dependent DNA breaks and TOP2-dependent fork stalling by trapping TOP2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to stall replication forks independently of TOP2. In human cells, etoposide stalls forks in a TOP2-dependent manner, while doxorubicin stalls forks independently of TOP2. However, both drugs exhibit TOP2-dependent cytotoxicity. Thus, etoposide and doxorubicin inhibit DNA replication through distinct mechanisms despite shared genetic requirements for cytotoxicity.


Subject(s)
DNA Topoisomerases, Type II , Poisons , Animals , DNA , DNA Replication , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Doxorubicin/pharmacology , Etoposide/pharmacology , Humans , Vertebrates/genetics , Vertebrates/metabolism
12.
Sci Adv ; 8(13): eabm0314, 2022 04.
Article in English | MEDLINE | ID: mdl-35353580

ABSTRACT

Replication-coupled DNA repair and damage tolerance mechanisms overcome replication stress challenges and complete DNA synthesis. These pathways include fork reversal, translesion synthesis, and repriming by specialized polymerases such as PRIMPOL. Here, we investigated how these pathways are used and regulated in response to varying replication stresses. Blocking lagging-strand priming using a POLα inhibitor slows both leading- and lagging-strand synthesis due in part to RAD51-, HLTF-, and ZRANB3-mediated, but SMARCAL1-independent, fork reversal. ATR is activated, but CHK1 signaling is dampened compared to stalling both the leading and lagging strands with hydroxyurea. Increasing CHK1 activation by overexpressing CLASPIN in POLα-inhibited cells promotes replication elongation through PRIMPOL-dependent repriming. CHK1 phosphorylates PRIMPOL to promote repriming irrespective of the type of replication stress, and this phosphorylation is important for cellular resistance to DNA damage. However, PRIMPOL activation comes at the expense of single-strand gap formation, and constitutive PRIMPOL activity results in reduced cell fitness.


Subject(s)
DNA Replication , DNA-Directed DNA Polymerase , DNA Damage , DNA Repair , DNA-Directed DNA Polymerase/genetics , Phosphorylation
13.
J Biol Chem ; 298(3): 101672, 2022 03.
Article in English | MEDLINE | ID: mdl-35120927

ABSTRACT

Genome integrity requires complete and accurate DNA replication once per cell division cycle. Replication stress poses obstacles to this process that must be overcome to prevent replication fork collapse. An important regulator of replication fork stability is the RAD51 protein, which promotes replication fork reversal and protects nascent DNA strands from nuclease-mediated degradation. Many regulatory proteins control these RAD51 activities, including RADX, which binds both ssDNA and RAD51 at replication forks to ensure that fork reversal is confined to stalled forks. Many ssDNA-binding proteins function as hetero- or homo-oligomers. In this study, we addressed whether this is also the case for RADX. Using biochemical and genetic approaches, we found that RADX acts as a homo-oligomer to control replication fork stability. RADX oligomerizes using at least two different interaction surfaces, including one mapped to a C-terminal region. We demonstrate that mutations in this region prevent oligomerization and prevent RADX function in cells, and that addition of a heterologous dimerization domain to the oligomerization mutants restored their ability to regulate replication. Taken together, our results demonstrate that like many ssDNA-binding proteins, oligomerization is essential for RADX-mediated regulation of genome stability.


Subject(s)
DNA Replication , DNA-Binding Proteins , RNA-Binding Proteins , Rad51 Recombinase , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Genomic Instability , Humans , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Transcription Factors/genetics
14.
Mol Cell ; 81(14): 3007-3017.e5, 2021 07 15.
Article in English | MEDLINE | ID: mdl-34107305

ABSTRACT

RAD51 facilitates replication fork reversal and protects reversed forks from nuclease degradation. Although potentially a useful replication stress response mechanism, unregulated fork reversal can cause genome instability. Here we show that RADX, a single-strand DNA binding protein that binds to and destabilizes RAD51 nucleofilaments, can either inhibit or promote fork reversal depending on replication stress levels. RADX inhibits fork reversal at elongating forks, thereby preventing fork slowing and collapse. Paradoxically, in the presence of persistent replication stress, RADX localizes to stalled forks to generate reversed fork structures. Consequently, inactivating RADX prevents fork-reversal-dependent telomere dysfunction in the absence of RTEL1 and blocks nascent strand degradation when fork protection factors are inactivated. Addition of RADX increases SMARCAL1-dependent fork reversal in conditions in which pre-binding RAD51 to a model fork substrate is inhibitory. Thus, RADX directly interacts with RAD51 and single-strand DNA to confine fork reversal to persistently stalled forks.


Subject(s)
DNA Replication/genetics , DNA-Binding Proteins/genetics , Genomic Instability/genetics , Replication Origin/genetics , Cell Line , Cell Line, Tumor , DNA Breaks, Double-Stranded , DNA Helicases/genetics , DNA Repair/genetics , DNA, Single-Stranded/genetics , HEK293 Cells , HeLa Cells , Humans , Protein Binding/genetics , Rad51 Recombinase/genetics
15.
Front Med (Lausanne) ; 8: 616106, 2021.
Article in English | MEDLINE | ID: mdl-33748157

ABSTRACT

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, which has reached 28 million cases worldwide in 1 year. The serological detection of antibodies against the virus will play a pivotal role in complementing molecular tests to improve diagnostic accuracy, contact tracing, vaccine efficacy testing, and seroprevalence surveillance. Here, we aimed first to evaluate a lateral flow assay's ability to identify specific IgM and IgG antibodies against SARS-CoV-2 and second, to report the seroprevalence estimates of these antibodies among health care workers and healthy volunteer blood donors in Panama. We recruited study participants between April 30th and July 7th, 2020. For the test validation and performance evaluation, we analyzed serum samples from participants with clinical symptoms and confirmed positive RT-PCR for SARS-CoV-2, and a set of pre-pandemic serum samples. We used two by two table analysis to determine the test positive and negative percentage agreement as well as the Kappa agreement value with a 95% confidence interval. Then, we used the lateral flow assay to determine seroprevalence among serum samples from COVID-19 patients, potentially exposed health care workers, and healthy volunteer donors. Our results show this assay reached a positive percent agreement of 97.2% (95% CI 84.2-100.0%) for detecting both IgM and IgG. The assay showed a Kappa of 0.898 (95%CI 0.811-0.985) and 0.918 (95% CI 0.839-0.997) for IgM and IgG, respectively. The evaluation of serum samples from hospitalized COVID-19 patients indicates a correlation between test sensitivity and the number of days since symptom onset; the highest positive percent agreement [87% (95% CI 67.0-96.3%)] was observed at ≥15 days post-symptom onset (PSO). We found an overall antibody seroprevalence of 11.6% (95% CI 8.5-15.8%) among both health care workers and healthy blood donors. Our findings suggest this lateral flow assay could contribute significantly to implementing seroprevalence testing in locations with active community transmission of SARS-CoV-2.

16.
J Biol Chem ; 296: 100455, 2021.
Article in English | MEDLINE | ID: mdl-33636182

ABSTRACT

The checkpoint kinase ATR regulates DNA repair, cell cycle progression, and other DNA damage and replication stress responses. ATR signaling is stimulated by an ATR activating protein, and in metazoan cells, there are at least two ATR activators: TOPBP1 and ETAA1. Current evidence indicates TOPBP1 and ETAA1 activate ATR via the same biochemical mechanism, but several aspects of this mechanism remain undefined. For example, ATR and its obligate binding partner ATR interacting protein (ATRIP) form a tetrameric complex consisting of two ATR and two ATRIP molecules, but whether TOPBP1 or ETAA1 dimerization is similarly required for ATR function is unclear. Here, we show that fusion of the TOPBP1 and ETAA1 ATR activation domains (AADs) to dimeric tags makes them more potent activators of ATR in vitro. Furthermore, induced dimerization of both AADs using chemical dimerization of a modified FKBP tag enhances ATR kinase activation and signaling in cells. ETAA1 forms oligomeric complexes mediated by regions of the protein that are predicted to be intrinsically disordered. Induced dimerization of a "mini-ETAA1" protein that contains the AAD and Replication Protein A (RPA) interaction motifs enhances ATR signaling, rescues cellular hypersensitivity to DNA damaging agents, and suppresses micronuclei formation in ETAA1-deficient cells. Together, our results indicate that TOPBP1 and ETAA1 dimerization is important for optimal ATR signaling and genome stability.


Subject(s)
Antigens, Surface/metabolism , Ataxia Telangiectasia Mutated Proteins/metabolism , Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/physiology , Cell Cycle Proteins/metabolism , Cell Line, Tumor , DNA Damage/genetics , DNA Repair/genetics , DNA Replication/genetics , Dimerization , Humans , Phosphorylation , Protein Binding , Protein Domains/genetics , Protein Domains/physiology , Signal Transduction/physiology
17.
Mol Cell ; 81(5): 1074-1083.e5, 2021 03 04.
Article in English | MEDLINE | ID: mdl-33453169

ABSTRACT

The RAD51 recombinase forms nucleoprotein filaments to promote double-strand break repair, replication fork reversal, and fork stabilization. The stability of these filaments is highly regulated, as both too little and too much RAD51 activity can cause genome instability. RADX is a single-strand DNA (ssDNA) binding protein that regulates DNA replication. Here, we define its mechanism of action. We find that RADX inhibits RAD51 strand exchange and D-loop formation activities. RADX directly and selectively interacts with ATP-bound RAD51, stimulates ATP hydrolysis, and destabilizes RAD51 nucleofilaments. The RADX interaction with RAD51, in addition to its ssDNA binding capability, is required to maintain replication fork elongation rates and fork stability. Furthermore, BRCA2 can overcome the RADX-dependent RAD51 inhibition. Thus, RADX functions in opposition to BRCA2 in regulating RAD51 nucleofilament stability to ensure the right level of RAD51 function during DNA replication.


Subject(s)
BRCA2 Protein/genetics , DNA Replication , DNA, Single-Stranded/genetics , DNA-Binding Proteins/genetics , RNA-Binding Proteins/genetics , Rad51 Recombinase/genetics , Adenosine Triphosphate/metabolism , BRCA2 Protein/metabolism , Cell Line, Tumor , DNA/genetics , DNA/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Regulation , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Hydrolysis , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , RNA-Binding Proteins/metabolism , Rad51 Recombinase/metabolism , Signal Transduction , Single Molecule Imaging , Red Fluorescent Protein
18.
Nat Rev Mol Cell Biol ; 21(10): 633-651, 2020 10.
Article in English | MEDLINE | ID: mdl-32612242

ABSTRACT

Complete and accurate DNA replication requires the progression of replication forks through DNA damage, actively transcribed regions, structured DNA and compact chromatin. Recent studies have revealed a remarkable plasticity of the replication process in dealing with these obstacles, which includes modulation of replication origin firing, of the architecture of replication forks, and of the functional organization of the replication machinery in response to replication stress. However, these specialized mechanisms also expose cells to potentially dangerous transactions while replicating DNA. In this Review, we discuss how replication forks are actively stalled, remodelled, processed, protected and restarted in response to specific types of stress. We also discuss adaptations of the replication machinery and the role of chromatin modifications during these transactions. Finally, we discuss interesting recent data on the relevance of replication fork plasticity to human health, covering its role in tumorigenesis, its crosstalk with innate immunity responses and its potential as an effective cancer therapy target.


Subject(s)
DNA Damage/genetics , DNA Replication/genetics , DNA/genetics , Replication Origin/genetics , Animals , Carcinogenesis/genetics , Chromatin/genetics , Humans , Immunity, Innate/genetics
19.
Cell Rep ; 31(9): 107705, 2020 06 02.
Article in English | MEDLINE | ID: mdl-32492421

ABSTRACT

5-Hydroxymethylcytosine (5hmC) binding, ES-cell-specific (HMCES) crosslinks to apurinic or apyrimidinic (AP, abasic) sites in single-strand DNA (ssDNA). To determine whether HMCES responds to the ssDNA abasic site in cells, we exploited the activity of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3A (APOBEC3A). APOBEC3A preferentially deaminates cytosines to uracils in ssDNA, which are then converted to abasic sites by uracil DNA glycosylase. We find that HMCES-deficient cells are hypersensitive to nuclear APOBEC3A localization. HMCES relocalizes to chromatin in response to nuclear APOBEC3A and protects abasic sites from processing into double-strand breaks (DSBs). Abasic sites induced by APOBEC3A slow both leading and lagging strand synthesis, and HMCES prevents further slowing of the replication fork by translesion synthesis (TLS) polymerases zeta (Polζ) and kappa (Polκ). Thus, our study provides direct evidence that HMCES responds to ssDNA abasic sites in cells to prevent DNA cleavage and balance the engagement of TLS polymerases.


Subject(s)
Cytidine Deaminase/metabolism , DNA Breaks, Double-Stranded , DNA-Binding Proteins/metabolism , Proteins/metabolism , 5-Methylcytosine/analogs & derivatives , 5-Methylcytosine/metabolism , Cell Line , Cell Nucleus/metabolism , Chromatin/metabolism , Cytidine Deaminase/genetics , DNA Replication , DNA, Single-Stranded/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/antagonists & inhibitors , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Deamination , Endonucleases/antagonists & inhibitors , Endonucleases/genetics , Endonucleases/metabolism , Humans , Multifunctional Enzymes/antagonists & inhibitors , Multifunctional Enzymes/genetics , Multifunctional Enzymes/metabolism , Proteins/genetics , RNA Interference , RNA, Small Interfering/metabolism , Uracil/metabolism , Uracil-DNA Glycosidase/metabolism
20.
DNA Repair (Amst) ; 90: 102866, 2020 06.
Article in English | MEDLINE | ID: mdl-32417669

ABSTRACT

Thousands of apurinic/apyrimidinic (AP or abasic) sites form in each cell, each day. This simple DNA lesion can have profound consequences to cellular function, genome stability, and disease. As potent blocks to polymerases, they interfere with the reading and copying of the genome. Since they provide no coding information, they are potent sources of mutation. Due to their reactive chemistry, they are intermediates in the formation of lesions that are more challenging to repair including double-strand breaks, interstrand crosslinks, and DNA protein crosslinks. Given their prevalence and deleterious consequences, cells have multiple mechanisms of repairing and tolerating these lesions. While base excision repair of abasic sites in double-strand DNA has been studied for decades, new interest in abasic site processing has come from more recent insights into how they are processed in single-strand DNA. In this review, we discuss the source of abasic sites, their biological consequences, tolerance mechanisms, and how they are repaired in double and single-stranded DNA.


Subject(s)
DNA Damage , DNA Repair , Animals , Bacteria/genetics , Bacteria/metabolism , DNA/metabolism , DNA, Single-Stranded/metabolism , Eukaryota/genetics , Eukaryota/metabolism , Genomic Instability , Humans
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