Biology subjects

Cortez, D.

Publications and source records attributed to Cortez, D..

13 recordsLinked to original sources

bioRxiv2025

FET proteins and PARylation-dependent condensates promote replication fork reversal and genome stability

Targeting replication-associated DNA repair mechanisms, including those regulated by PARP1/2 and PARG control of ADP-ribosylation is a powerful cancer therapeutic approach. However, the mechanisms by which PARG inhibition impacts DNA replication remain unclear. Here we combined isolation of proteins on nascent DNA (iPOND) with quantitative proteomics and functional assays to investigate replication fork dynamics upon acute PARG inhibition. We found that FET family proteins (FUS, EWS, and TAF15) are recruited to replication forks in a PAR-dependent manner, forming condensates that slow fork progression and promote fork reversal. FET proteins control fork dynamics in response to some, but not all, replication stresses. FUS inactivation leads to unrestrained fork progression via RECQ1 and PRIMPOL, increased single-stranded DNA gaps, genome instability, and synthetic lethality with BRCA1 deficiency. These findings reveal that FET protein condensates modulate replication stress responses, influencing genome stability and the cellular response to cancer therapeutics targeting PARylation pathways.

molecular biology↗

bioRxiv2025

Structural basis for fork reversal and RAD51 regulation by the SCF ubiquitin ligase complex of F-box helicase 1

Replication fork reversal helps maintain genomic stability during replication stress. F-box helicase 1 (FBH1) catalyzes fork reversal and is an SCF (SKP-CUL1-F-box) E3 ubiquitin ligase that limits RAD51 association with chromatin. Here, we show that preferential binding of SCFFBH1 to the lagging strand template at DNA fork structures stimulates helicase activity and is required for fork reversal. A cryo-EM structure of SCFFBH1 bound to DNA representing a stalled fork reveals an intimate interaction between FBH1 and the fork junction. Disruption of this interface severely curtails fork reversal in vitro and replication progression in cells, providing a model for how ssDNA translocation by FBH1 facilitates annealing of parental DNA by a fundamentally different mechanism than the fork remodelers SMARCAL, HLTF, and ZRANB3. The structure also provides a model for SCFFBH1 disassembly of RAD51 filaments through translocation and ubiquitination, and implies that RAD51 is associated with the lagging strand at stalled forks.

biochemistry↗

bioRxiv2025

Resolution of collapsed forks is separate from completion of DNA synthesis

Replication fork collapse at single-strand DNA breaks (SSBs) poses a serious threat to genome stability. Using Xenopus egg extracts, we show that a replication fork encountering an SSB on either the leading- or lagging-strand template produces a single-ended double-strand break (seDSB). These broken ends are efficiently resolved by homologous recombination to yield D-loops and erroneous end-to-end fusions. Surprisingly, DNA synthesis downstream of an seDSB is highly inefficient. In contrast, when two forks converge at an SSB, they generate a double-ended DSB (deDSB) that efficiently completes DNA synthesis through double-strand break repair that is not dependent on homologous recombination. Leading, but not lagging, seDSBs can undergo extensive nucleolytic degradation that disassembles the divergent fork. These secondary collapse events efficiently resolve seDSBs but without completion of DNA synthesis. Moreover, PARP inhibition can enhance fork collapse at unmodified SSBs but not at abasic site SSBs, contrary to expectations. Our findings distinguish end resolution from replication completion and demonstrate flexibility in how PARP inhibition affects fork collapse.

biochemistry↗

bioRxiv2025

Leading and lagging strand abasic sites differentially affect vertebrate replisome progression but involve analogous bypass mechanisms

Abasic sites are one of the most frequent forms of DNA damage that interfere with DNA replication. However, abasic sites exhibit complex effects because they can be processed into other types of DNA damage. Thus, it remains poorly understood how abasic sites affect replisome progression, which replication-coupled repair pathways they elicit, and whether this is affected by the template strand that is damaged. Using Xenopus egg extracts, we developed an approach to analyze replication of DNA containing a site-specific, stable abasic site on the leading or lagging strand template. We show that abasic sites robustly stall synthesis of nascent DNA strands but exert different effects when encountered on the leading or lagging strand template. At a leading strand AP site, replisomes stall [~]100 bp from the lesion until it is bypassed or a converging fork triggers termination. At a lagging strand abasic site, replisome progression is unaffected and lagging strands are reprimed downstream, generating a post-replicative gap, which is then bypassed. Despite different effects on replisome progression, both leading and lagging strand abasic sites rely on translesion DNA synthesis for bypass. Our results detail similarities and differences between how leading and lagging strand AP sites affect vertebrate DNA replication.

biochemistry↗

bioRxiv2024

Strand dependent bypass of DNA lesions during fork reversal by ATP-dependent translocases SMARCAL1, ZRANB3, and HLTF

Withdrawal StatementThe authors have withdrawn this manuscript because they identified problems with how some figure panels were processed. Those experiments will be repeated before deposition of a new manuscript. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding authors.

biochemistry↗

bioRxiv2024

Hierarchical determinants of the oxidation-induced mutational landscape in human cells.

8-oxoguanine (8-oxoG) is a common oxidative DNA lesion, which causes G>T substitutions that compose COSMIC single base substitution signature 18 (SBS18) in human cancers. Determinants of local and regional differences in 8-oxoG-induced mutability are currently unknown. To uncover factors influencing the topology of 8-oxoG-induced mutations, we assessed spontaneous and KBrO3-induced 8-oxoG mutagenesis in human cell lines. KBrO3 exposure produced a SBS18-like substitution spectrum and a distinct never-before reported INDEL signature that we also observed in human cancers. KBrO3-induced 8-oxoG lesions occurred with similar sequence preference as KBrO3-induced substitutions, indicating that the reactivity of specific reactive oxygen species (ROS) dictates the trinucleotide motif specificity for 8-oxoG-induced mutagenesis. While 8-oxoG lesions occurred relatively uniformly across chromatin states and nucleosomes, 8-oxoG-induced mutations occurred more frequently in more compact regions of the genome, within nucleosomal DNA, and at inward facing guanines within strongly positioned nucleosomes. Cryo-EM structures of OGG1 bound to nucleosomes indicate that these effects originate from OGG1s ability to flip outward positioned 8-oxoG lesions into the catalytic pocket with only minor alterations to nucleosome structure, while inward facing lesions occluded by the histone octamer are unrecognized. Mutation spectra from cells with DNA repair deficiencies revealed a hierarchical DNA repair network limiting 8-oxoG mutagenesis in human cells, where OGG1- and MUTY-mediated BER is supplemented by replication-associated factors participating in tolerance of 8-oxoG or derived repair intermediates (i.e. Pol {eta} and HMCES). Surprisingly, analysis of transcriptional asymmetry of KBrO3-induced mutations demonstrated transcription-coupled repair of 8-oxoG in Pol {eta}-deficient cells. Thus, radical chemistry, chromatin structures, and DNA repair processes combine to dictate the oxidative mutational landscape in human genomes.

cancer biology↗

bioRxiv2023

HLTF Prevents G4 Accumulation and Promotes G4-induced Fork Slowing to Maintain Genome Stability

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.

molecular biology↗

bioRxiv2023

Structure of RADX and mechanism for regulation of RAD51 nucleofilaments

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 RADXs 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 [A] 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. SignificanceDespite the central role of RAD51 in DNA replication and repair processes, the mechanisms of action of its many modulators are poorly understood. Here we combine structural and biophysical data to determine how the negative regulator RADX functions. We show that RADX oligomerizes upon binding DNA, and caps RAD51 filaments at the ends to prevent extension. This work advances knowledge of how RAD51 filaments can be modulated to regulate replication fork reversal and maintain genomic stability.

biophysics↗

bioRxiv2023

CRISPR-dependent base editing screens identify separation of function mutants of RADX with altered RAD51 regulatory activity

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 bind RAD51 and promote RAD51 ATP turnover to regulate RAD51 and genome stability during DNA replication.

molecular biology↗

bioRxiv2023

Self-reversal facilitates the resolution of HMCES-DNA protein crosslinks in cells

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 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.

molecular biology↗

bioRxiv2022

Abasic site ring opening and DNA-protein crosslink reversal by the SRAP protein YedK

Apuirinic/apyrimidinic (AP, or abasic) sites in DNA are one of the most common forms of DNA damage. AP sites are reactive and form crosslinks to both proteins and DNA, are prone to strand breakage, and inhibit DNA replication and transcription. The protein HMCES protects cells from strand breaks, inhibits mutagenic translesion synthesis, and participates in repair of interstrand DNA crosslinks derived from AP sites by forming a stable thiazolidine DNA-protein crosslink (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 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, which provide insight into possible mechanisms by which HMCES DPCs are resolved in cells.

biochemistry↗

bioRxiv2022

A study of thermally-induced sex reversal in casque-headed lizards

Non-avian reptiles, unlike mammals and birds, have undergone numerous sex determination turnovers. For example, casque-headed lizards replaced the ancestral XY system shared across pleurodonts with a new pair of XY chromosomes. However, the evolutionary forces that triggered this transition have remained unclear. An interesting evolutionary hypothesis suggests that species with intermediate states, with sex chromosomes but also thermal-induced sex reversal at specific incubation temperatures, could be more susceptible to sex determination turnovers. We contrasted genotypic data (presence/absence of the Y chromosome) against the histology of gonads of embryos from stages 35-37 incubated at various temperatures, including typical male-producing (26{degrees}C) and female-producing (32{degrees}C) temperatures. We observed perfect concordance between genotype and phenotype at all temperatures. However, analysis of transcriptomic data from embryos incubated at 26{degrees}C and 32{degrees}C identified transcript variants of the chromatin modifiers JARID2 and KDM6B that have been linked to temperature-dependent sex determination in other reptiles. Besides, our work reports for the first time to our knowledge the histology of gonads, including morphological changes, from stages 35-37 of development in the Corytophanidae family. We also observed that all embryos developed hemipenes, suggesting sex-linked developmental heterochrony.Our work tested the validity of a mixed sex determination system in the Corytophanidae family. We showed that XY chromosomes are dominant, however, our work supports the hypothesis of a conserved transcriptional response to incubation temperatures across non-avian reptiles that could be the reminiscence of an ancestral sex determination system.

developmental biology↗

bioRxiv2021

Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms

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 induces 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 inhibit replication fork progression. In human cells, etoposide stalls replication forks in a Top2-dependent manner, while doxorubicin stalls forks independently of Top2. However, both drugs exhibit Top2-dependent cytotoxicity. Thus, despite shared genetic requirements for cytotoxicity etoposide and doxorubicin inhibit DNA replication through distinct mechanisms.

biochemistry↗