G-quadruplexes (G4s) are functional elements of the human genome, some of which inhibit DNA replication. We investigated replication of G4s within highly abundant microsatellite (GGGA, GGGT) and transposable element (L1 and SVA) sequences. We found that genome-wide, numerous motifs are located preferentially on the replication leading strand and the transcribed strand templates. We directly tested replicative polymerase ε and δ holoenzyme inhibition at these G4s, compared to low abundant motifs. For all G4s, DNA synthesis inhibition was higher on the G-rich than C-rich strand or control sequence. No single G4 was an absolute block for either holoenzyme; however, the inhibitory potential varied over an order of magnitude. Biophysical analyses showed the motifs form varying topologies, but replicative polymerase inhibition did not correlate with a specific G4 structure. Addition of the G4 stabilizer pyridostatin severely inhibited forward polymerase synthesis specifically on the G-rich strand, enhancing G/C strand asynchrony. Our results reveal that replicative polymerase inhibition at every G4 examined is distinct, causing complementary strand synthesis to become asynchronous, which could contribute to slowed fork elongation. Altogether, we provide critical information regarding how replicative eukaryotic holoenzymes navigate synthesis through G4s naturally occurring thousands of times in functional regions of the human genome.

• MeSH
• Aminoquinolines MeSH
• DNA Polymerase II * antagonists & inhibitors   metabolism   MeSH
• DNA Polymerase III * antagonists & inhibitors   metabolism   MeSH
• DNA chemistry   MeSH
• G-Quadruplexes * MeSH
• Genome, Human * MeSH
• Holoenzymes metabolism   MeSH
• Picolinic Acids pharmacology   MeSH
• Humans MeSH
• Microsatellite Repeats MeSH
• Poly-ADP-Ribose Binding Proteins MeSH
• DNA Replication * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Replication stress, particularly in hard-to-replicate regions such as telomeres and centromeres, leads to the accumulation of replication intermediates that must be processed to ensure proper chromosome segregation. In this study, we identify a critical role for the interaction between RECQ4 and MUS81 in managing such stress. We show that RECQ4 physically interacts with MUS81, targeting it to specific DNA substrates and enhancing its endonuclease activity. Loss of this interaction, results in significant chromosomal segregation defects, including the accumulation of micronuclei, anaphase bridges, and ultrafine bridges (UFBs). Our data further demonstrate that the RECQ4-MUS81 interaction plays an important role in ALT-positive cells, where MUS81 foci primarily colocalise with telomeres, highlighting its role in telomere maintenance. We also observe that a mutation associated with Rothmund-Thomson syndrome, which produces a truncated RECQ4 unable to interact with MUS81, recapitulates these chromosome instability phenotypes. This underscores the importance of RECQ4-MUS81 in safeguarding genome integrity and suggests potential implications for human disease. Our findings demonstrate the RECQ4-MUS81 interaction as a key mechanism in alleviating replication stress at hard-to-replicate regions and highlight its relevance in pathological conditions such as RTS.

• MeSH
• Chromosomal Instability MeSH
• DNA-Binding Proteins * metabolism   genetics   MeSH
• Endonucleases * metabolism   genetics   MeSH
• RecQ Helicases * metabolism   genetics   MeSH
• Telomere Homeostasis MeSH
• Humans MeSH
• Mutation MeSH
• DNA Replication * MeSH
• Rothmund-Thomson Syndrome * genetics   metabolism   MeSH
• Chromosome Segregation MeSH
• Telomere * metabolism   genetics   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Mismatched nucleobase uracil is commonly repaired through the base excision repair initiated by DNA uracil glycosylases. The data presented in this study strongly indicate that the nuclear uracil-N-glycosylase activity and nuclear protein content in human cell lines is highest in the S phase of the cell cycle and that its distribution kinetics partially reflect the DNA replication activity in replication foci. In this respect, the data demonstrate structural changes of the replication focus related to the uracil-N-glycosylase distribution several dozens of minutes before end of its replication. The analysis also showed that very popular synchronisation protocols based on the double thymidine block can result in changes in the UNG2 content and uracil excision rate. In response, we propose a new method for the description of the changes of the content and the activity of different cell components during cell cycle without the necessity to use synchronisation protocols.

• MeSH
• Cell Nucleus metabolism   MeSH
• Cell Cycle MeSH
• Kinetics MeSH
• Humans MeSH
• DNA Repair MeSH
• DNA Replication * MeSH
• S Phase MeSH
• Uracil-DNA Glycosidase * metabolism   MeSH
• Uracil metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Homologous recombination (HR) factors are crucial for DSB repair and processing stalled replication forks. RAD51 paralogs, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, have emerged as essential tumour suppressors, forming two subcomplexes, BCDX2 and CX3. Mutations in these genes are associated with cancer susceptibility and Fanconi anaemia, yet their biochemical activities remain unclear. This study reveals a linear arrangement of BCDX2 subunits compared to the RAD51 ring. BCDX2 shows a strong affinity towards single-stranded DNA (ssDNA) via unique binding mechanism compared to RAD51, and a contribution of DX2 subunits in binding branched DNA substrates. We demonstrate that BCDX2 facilitates RAD51 loading on ssDNA by suppressing the cooperative requirement of RAD51 binding to DNA and stabilizing the filament. Notably, BCDX2 also promotes RAD51 loading on short ssDNA and reversed replication fork substrates. Moreover, while mutants defective in ssDNA binding retain the ability to bind branched DNA substrates, they still facilitate RAD51 loading onto reversed replication forks. Our study provides mechanistic insights into how the BCDX2 complex stimulates the formation of BRCA2-independent RAD51 filaments on short stretches of ssDNA present at ssDNA gaps or stalled replication forks, highlighting its role in genome maintenance and DNA repair.

• MeSH
• DNA-Binding Proteins * metabolism   genetics   MeSH
• DNA, Single-Stranded * metabolism   genetics   MeSH
• Humans MeSH
• Multiprotein Complexes MeSH
• Mutation MeSH
• Rad51 Recombinase * metabolism   genetics   MeSH
• DNA Replication * genetics   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Replication forks stalled at co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage-religation cycles mediated by MUS81 endonuclease and DNA ligase IV (LIG4), which presumably relieve the topological barrier generated by the transcription-replication conflict (TRC) and facilitate ELL-dependent reactivation of transcription. Here, we report that the restart of R-loop-stalled replication forks via the MUS81-LIG4-ELL pathway requires senataxin (SETX), a helicase that can unwind RNA:DNA hybrids. We found that SETX promotes replication fork progression by preventing R-loop accumulation during S-phase. Interestingly, loss of SETX helicase activity leads to nascent DNA degradation upon induction of R-loop-mediated fork stalling by hydroxyurea. This fork degradation phenotype is independent of replication fork reversal and results from DNA2-mediated resection of MUS81-cleaved replication forks that accumulate due to defective replication restart. Finally, we demonstrate that SETX acts in a common pathway with the DEAD-box helicase DDX17 to suppress R-loop-mediated replication stress in human cells. A possible cooperation between these RNA/DNA helicases in R-loop unwinding at TRC sites is discussed.

• MeSH
• Flap Endonucleases metabolism   genetics   MeSH
• DEAD-box RNA Helicases * metabolism   genetics   MeSH
• DNA-Binding Proteins * metabolism   genetics   MeSH
• DNA Helicases * metabolism   genetics   MeSH
• DNA Ligase ATP metabolism   genetics   MeSH
• DNA metabolism   genetics   MeSH
• Endonucleases * metabolism   genetics   MeSH
• Transcription, Genetic MeSH
• Humans MeSH
• Multifunctional Enzymes * metabolism   genetics   MeSH
• R-Loop Structures * MeSH
• DNA Replication * MeSH
• RNA Helicases * metabolism   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Protein import and genome replication are essential processes for mitochondrial biogenesis and propagation. The J-domain proteins Pam16 and Pam18 regulate the presequence translocase of the mitochondrial inner membrane. In the protozoan Trypanosoma brucei, their counterparts are TbPam16 and TbPam18, which are essential for the procyclic form (PCF) of the parasite, though not involved in mitochondrial protein import. Here, we show that during evolution, the 2 proteins have been repurposed to regulate the replication of maxicircles within the intricate kDNA network, the most complex mitochondrial genome known. TbPam18 and TbPam16 have inactive J-domains suggesting a function independent of heat shock proteins. However, their single transmembrane domain is essential for function. Pulldown of TbPam16 identifies a putative client protein, termed MaRF11, the depletion of which causes the selective loss of maxicircles, akin to the effects observed for TbPam18 and TbPam16. Moreover, depletion of the mitochondrial proteasome results in increased levels of MaRF11. Thus, we have discovered a protein complex comprising TbPam18, TbPam16, and MaRF11, that controls maxicircle replication. We propose a working model in which the matrix protein MaRF11 functions downstream of the 2 integral inner membrane proteins TbPam18 and TbPam16. Moreover, we suggest that the levels of MaRF11 are controlled by the mitochondrial proteasome.

• MeSH
• DNA, Mitochondrial * genetics   metabolism   MeSH
• Mitochondrial Proteins metabolism   genetics   MeSH
• Mitochondria metabolism   genetics   MeSH
• Evolution, Molecular MeSH
• Protozoan Proteins * metabolism   genetics   MeSH
• DNA Replication * MeSH
• Trypanosoma brucei brucei * metabolism   genetics   MeSH
• Publication type
• Journal Article MeSH

RAD18 is an E3 ubiquitin ligase that prevents replication fork collapse by promoting DNA translesion synthesis and template switching. Besides this classical role, RAD18 has been implicated in homologous recombination; however, this function is incompletely understood. Here, we show that RAD18 is recruited to DNA lesions by monoubiquitination of histone H2A at K15 and counteracts accumulation of 53BP1. Super-resolution microscopy revealed that RAD18 localizes to the proximity of DNA double strand breaks and limits the distribution of 53BP1 to the peripheral chromatin nanodomains. Whereas auto-ubiquitination of RAD18 mediated by RAD6 inhibits its recruitment to DNA breaks, interaction with SLF1 promotes RAD18 accumulation at DNA breaks in the post-replicative chromatin by recognition of histone H4K20me0. Surprisingly, suppression of 53BP1 function by RAD18 is not involved in homologous recombination and rather leads to reduction of non-homologous end joining. Instead, we provide evidence that RAD18 promotes HR repair by recruiting the SMC5/6 complex to DNA breaks. Finally, we identified several new loss-of-function mutations in RAD18 in cancer patients suggesting that RAD18 could be involved in cancer development.

• MeSH
• Tumor Suppressor p53-Binding Protein 1 * metabolism   genetics   MeSH
• Chromatin * metabolism   genetics   MeSH
• DNA-Binding Proteins * metabolism   genetics   MeSH
• DNA Breaks, Double-Stranded * MeSH
• Histones * metabolism   MeSH
• Homologous Recombination genetics   MeSH
• Humans MeSH
• DNA End-Joining Repair MeSH
• DNA Repair MeSH
• Cell Cycle Proteins metabolism   genetics   MeSH
• Recombinational DNA Repair MeSH
• DNA Replication MeSH
• Ubiquitination * MeSH
• Ubiquitin-Protein Ligases * metabolism   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Increasing evidence has revealed that cellular senescence drives NDs, including Alzheimer's disease (AD) and Parkinson's disease. Different senescent cell populations secrete senescence-associated secretory phenotypes (SASP), including matrix metalloproteinase-3, interleukin (IL)-1α, IL-6, and IL-8, which can harm adjacent microglia. Moreover, these cells possess high expression levels of senescence hallmarks (p16 and p21) and elevated senescence-associated β-galactosidase activity in in vitro and in vivo ND models. These senescence phenotypes contribute to the deposition of β-amyloid and tau-protein tangles. Selective clearance of senescent cells and SASP regulation by inhibiting p38/mitogen-activated protein kinase and nuclear factor kappa B signaling attenuate β-amyloid load and prevent tau-protein tangle deposition, thereby improving cognitive performance in AD mouse models. In addition, telomere shortening, a cellular senescence biomarker, is associated with increased ND risks. Telomere dysfunction causes cellular senescence, stimulating IL-6, tumor necrosis factor-α, and IL-1β secretions. The forced expression of telomerase activators prevents cellular senescence, yielding considerable neuroprotective effects. This review elucidates the mechanism of cellular senescence in ND pathogenesis, suggesting strategies to eliminate or restore senescent cells to a normal phenotype for treating such diseases.

• MeSH
• Alzheimer Disease MeSH
• Amyloid beta-Peptides metabolism   MeSH
• Humans MeSH
• Neurodegenerative Diseases * MeSH
• Parkinson Disease metabolism   MeSH
• Senescence-Associated Secretory Phenotype MeSH
• Signal Transduction MeSH
• Cellular Senescence * drug effects   MeSH
• Telomere Shortening drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Reprogramming to pluripotency is associated with DNA damage and requires the functions of the BRCA1 tumor suppressor. Here, we leverage separation-of-function mutations in BRCA1/2 as well as the physical and/or genetic interactions between BRCA1 and its associated repair proteins to ascertain the relevance of homology-directed repair (HDR), stalled fork protection (SFP), and replication gap suppression (RGS) in somatic cell reprogramming. Surprisingly, loss of SFP and RGS is inconsequential for the transition to pluripotency. In contrast, cells deficient in HDR, but proficient in SFP and RGS, reprogram with reduced efficiency. Conversely, the restoration of HDR function through inactivation of 53bp1 rescues reprogramming in Brca1-deficient cells, and 53bp1 loss leads to elevated HDR and enhanced reprogramming in mouse and human cells. These results demonstrate that somatic cell reprogramming is especially dependent on repair of replication-associated double-strand breaks (DSBs) by the HDR activity of BRCA1 and BRCA2 and can be improved in the absence of 53BP1.

• MeSH
• Tumor Suppressor p53-Binding Protein 1 * metabolism   genetics   MeSH
• DNA Breaks, Double-Stranded * MeSH
• Humans MeSH
• Mice MeSH
• DNA Repair * MeSH
• Cellular Reprogramming * MeSH
• BRCA1 Protein * metabolism   genetics   MeSH
• Recombinational DNA Repair MeSH
• DNA Replication MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Accurate and complete replication of genetic information is a fundamental process of every cell division. The replication licensing is the first essential step that lays the foundation for error-free genome duplication. During licensing, minichromosome maintenance protein complexes, the molecular motors of DNA replication, are loaded to genomic sites called replication origins. The correct quantity and functioning of licensed origins are necessary to prevent genome instability associated with severe diseases, including cancer. Here, we delve into recent discoveries that shed light on the novel functions of licensed origins, the pathways necessary for their proper maintenance, and their implications for cancer therapies.

• MeSH
• Humans MeSH
• Minichromosome Maintenance Proteins genetics   metabolism   MeSH
• Neoplasms * genetics   MeSH
• DNA Replication * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH