bims-replis Biomed News
on Replisome
Issue of 2025–05–11
eleven papers selected by
Anna Zawada, International Centre for Translational Eye Research
  1. Most human DNA replication initiation is dispersed throughout the genome with only a minority within previously identified initiation zones.
  2. Human DNA replication initiation sites are specified epigenetically by oxidation of 5-methyl-deoxycytidine.
  3. Selective interactions at pre-replication complexes categorize baseline and dormant origins.
  4. R-loop control and mitochondrial genome stability require the 5'-3' exonuclease/flap endonuclease OEX1.
  5. Structure and function of histone chaperones in replication-coupled chromatin assembly.
  6. Distinct roles of the two BRCA2 DNA-binding domains in DNA damage repair and replication fork preservation.
  7. DNA2 and MSH2 activity collectively mediate chemically stabilized G4 for efficient telomere replication.
  8. Control of replication and gene expression by ADP-ribosylation of DNA in Mycobacterium tuberculosis.
  9. Vulnerable Nucleotide Pools and Genomic Instability in Yeast Strains with Deletion of the ADE12 Gene Encoding for Adenylosuccinate Synthetase.
  10. Reversible association of ubiquitin with PCNA is important for template switching in S. cerevisiae.
  11. Distinct mechanisms underlying extrachromosomal telomere DNA generation in ALT cancers.
  1. Genome Biol. 2025 May 09. 26(1): 122
    Most human DNA replication initiation is dispersed throughout the genome with only a minority within previously identified initiation zones.
    Jamie T Carrington, Rosemary H C Wilson, Eduardo de La Vega, Sathish Thiyagarajan, Tom Barker, Leah Catchpole, Alex Durrant, Vanda Knitlhoffer, Chris Watkins, Karim Gharbi, Conrad A Nieduszynski.
       BACKGROUND: The identification of sites of DNA replication initiation in mammalian cells has been challenging. Here, we present unbiased detection of replication initiation events in human cells using BrdU incorporation and single-molecule nanopore sequencing.
    RESULTS: Increases in BrdU incorporation allow us to measure DNA replication dynamics, including identification of replication initiation, fork direction, and termination on individual nanopore sequencing reads. Importantly, initiation and termination events are identified on single molecules with high resolution, throughout S-phase, genome-wide, and at high coverage at specific loci using targeted enrichment. We find a significant enrichment of initiation sites within the broad initiation zones identified by population-level studies. However, these focused initiation sites only account for ~ 20% of all identified replication initiation events. Most initiation events are dispersed throughout the genome and are missed by cell population approaches. This indicates that most initiation occurs at sites that, individually, are rarely used. These dispersed initiation sites contrast with the focused sites identified by population studies, in that they do not show a strong relationship to transcription or a particular epigenetic signature.
    CONCLUSIONS: We show here that single-molecule sequencing enables unbiased detection and characterization of DNA replication initiation events, including the numerous dispersed initiation events that replicate most of the human genome.
    Keywords:  DNAscent; Origin mapping; Replication origin; Ultra-long; nCATS
    DOI:  https://doi.org/10.1186/s13059-025-03591-w
  2. Nucleic Acids Res. 2025 Apr 22. pii: gkaf362. [Epub ahead of print]53(8):
    Human DNA replication initiation sites are specified epigenetically by oxidation of 5-methyl-deoxycytidine.
    Torsten Krude, Jiaming Bi, Rachel Doran, Rebecca A Jones, James C Smith.
      DNA replication initiates at tens of thousands of sites on the human genome during each S phase. However, no consensus DNA sequence has been found that specifies the locations of these replication origins. Here, we investigate modifications of human genomic DNA by density equilibrium centrifugation and DNA sequencing. We identified short discrete sites with increased density during quiescence and G1 phase that overlap with DNA replication origins before their activation in S phase. The increased density is due to the oxidation of 5-methyl-deoxycytidines by ten-eleven-translocation DNA dioxygenase (TET) enzymes at GC-rich domains. Reversible inhibition of de novo methylation and of subsequent oxidation of deoxycytidines results in a reversible inhibition of DNA replication and of cell proliferation. Our findings suggest a mechanism for the epigenetic specification and semiconservative inheritance of DNA replication origin sites in human cells that also provides a stable integral DNA replication licence to support once-per-cell cycle control of origin activation.
    DOI:  https://doi.org/10.1093/nar/gkaf362
  3. Nat Commun. 2025 May 03. 16(1): 4140
    Selective interactions at pre-replication complexes categorize baseline and dormant origins.
    Bhushan L Thakur, Christophe E Redon, Haiqing Fu, Robin Sebastian, Nana A Kusi, Sophie Z Zhuang, Lorinc S Pongor, Vilhelm A Bohr, Mirit I Aladjem.
      DNA synthesis in metazoans initiates within a select group of replication origins (baseline origins), whereas other (dormant) origins do not initiate replication despite recruiting apparently indistinguishable pre-replication complexes. Dormant origins are activated as backups when DNA synthesis stalls, allowing for complete genome duplication, yet it is unclear how cells selectively differentiate between baseline and dormant origins. We report here that during unperturbed cell proliferation, dormant origins selectively bind phosphorylated RecQL4 (pRecQL4), a member of the RecQ helicase family mutated in Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes. Origin-bound pRecQL4 prevents the binding of an essential replication initiation complex, MTBP-TICRR/TRESLIN, to dormant origins, thus restricting replication initiation to baseline origins. When cells encounter replication stress, pRecQL4 is required for the dissociation of the MTBP-TICRR/TRESLIN complex from chromatin, which, in turn, facilitates the subsequent redistribution of MTBP-TICRR/TRESLIN to both baseline and dormant origins and allows recovery from replication inhibition. Thus, the interactions between the MTBP-TICRR/TRESLIN complex and pRecQL4 at replication origins are critical for replication origin choice and facilitate recovery from replication stress.
    DOI:  https://doi.org/10.1038/s41467-025-59509-4
  4. Plant Cell. 2025 May 05. pii: koaf104. [Epub ahead of print]
    R-loop control and mitochondrial genome stability require the 5'-3' exonuclease/flap endonuclease OEX1.
    Déborah Schatz, Anaïs Le Blevenec, Fabio G Moratti, Kin Pan Chung, Pierre Mercier, Rana Khalid Iqbal, Elody Vallet, André Dietrich, Ralph Bock, Frédérique Weber-Lotfi, José M Gualberto.
      Maintenance of the plant organelle genomes involves factors mostly inherited from their symbiotic ancestors. In bacteria, DNA Polymerase I (Pol I) performs multiple replication and repair functions through its 5'-3'-exonuclease/flap-endonuclease domain. Plant organelles possess two DNA polymerases that are evolutionarily derived from Pol I but lack this key domain. ORGANELLAR EXONUCLEASES 1 and 2 (OEX1 and OEX2) compensate for this missing function and are targeted to mitochondria and chloroplasts, respectively, in Arabidopsis (Arabidopsis thaliana). Loss of OEX1 causes developmental and fertility defects that increase with increasing differential segregation of mitochondrial DNA (mtDNA) subgenomes generated by recombination. OEX1 activity is modulated by alternative splicing, which generates two isoforms that variably affect mtDNA stability and repair. OEX1 has 5'-3'-exonuclease and flap endonuclease activities, with a high affinity for RNA-DNA hybrids. It rapidly degrades RNA in Okazaki-like structures and R-loops. Consistent with a role in suppressing R-loops, oex1 mutant plants accumulate RNA-DNA hybrids in highly transcribed mtDNA regions. Taken together, our results identify OEX1 as an important factor that compensates for the missing activity of plant organellar polymerases, playing multiple important roles in the processing of replication and recombination intermediates, such as replication primers and R-loops, whose accumulation can lead to genome instability.
    DOI:  https://doi.org/10.1093/plcell/koaf104
  5. Curr Opin Struct Biol. 2025 May 07. pii: S0959-440X(25)00077-6. [Epub ahead of print]92 103059
    Structure and function of histone chaperones in replication-coupled chromatin assembly.
    Chao-Pei Liu, Rui-Ming Xu.
      Eukaryotic cell divisions pass on genetic and epigenetic information from parental to daughter cells through replication of the chromatin, which needs to be reestablished following DNA replication, as its building block, the nucleosome, is disrupted by the passage of the DNA replication fork. This replication-coupled (RC) nucleosome assembly process takes place in distinct pathways depending on whether newly synthesized or parental histones are used. This review highlights recent progress in structural and biochemical studies of RC nucleosome assembly, focusing on the roles of histone chaperones in both de novo assembly of nucleosomes from newly synthesized histones and the recycling of parental histones. We also discuss the interactions between histone chaperones and replisome components that govern the coupling of nucleosome assembly to chromatin replication. Finally, we offer our perspective on future efforts in advancing this important research direction.
    DOI:  https://doi.org/10.1016/j.sbi.2025.103059
  6. Cell Rep. 2025 May 03. pii: S2211-1247(25)00425-5. [Epub ahead of print]44(5): 115654
    Distinct roles of the two BRCA2 DNA-binding domains in DNA damage repair and replication fork preservation.
    Francisco E Neal, Wenjing Li, Mollie E Uhrig, Jeffrey N Katz, Shahrez Syed, Neelam Sharma, Arijit Dutta, Sandeep Burma, Robert Hromas, Alexander V Mazin, Eloise Dray, David S Libich, Shaun K Olsen, Elizabeth V Wasmuth, Weixing Zhao, Claus S Sørensen, Claudia Wiese, Youngho Kwon, Patrick Sung.
      Homologous recombination (HR) removes DNA double-strand breaks (DSBs) and preserves stressed DNA replication forks. Successful HR execution requires the tumor suppressor BRCA2, which harbors distinct DNA-binding domains (DBDs): one that possesses three oligonucleotide/oligosaccharide-binding (OB) folds (OB-DBD) and another residing in the C-terminal recombinase binding domain (CTRB-DBD). Here, we employ multi-faceted approaches to delineate the contributions of these domains toward HR and replication fork maintenance. We show that OB-DBD and CTRB-DBD confer single-strand DNA (ssDNA)- and dsDNA-binding capabilities, respectively, and that BRCA2 variants mutated in either domain are impaired in their ability to load the recombinase RAD51 onto ssDNA pre-occupied by RPA. While the CTRB-DBD mutant is modestly affected by DNA break repair, it exhibits a strong defect in the protection of stressed replication forks. In contrast, the OB-DBD is indispensable for both BRCA2 functions. Our study thus defines the unique contributions of the two BRCA2 DBDs in genome maintenance.
    Keywords:  BRCA2; CP: Molecular biology; DNA; DNA repair; DNA replication; RAD51 recombinase; homologous recombination; replication fork
    DOI:  https://doi.org/10.1016/j.celrep.2025.115654
  7. bioRxiv. 2025 Apr 06. pii: 2025.04.04.647332. [Epub ahead of print]
    DNA2 and MSH2 activity collectively mediate chemically stabilized G4 for efficient telomere replication.
    Anthony Fernandez, Tingting Zhou, Steven Esworthy, Changxian Shen, Helen Liu, Jessica D Hess, Hang Yuan, Nian Liu, Guojun Shi, Mian Zhou, Settapong Kosiyatrakul, Vikas Gaur, Joshua Sommers, Winfried Edelman, Guo-Min Li, Robert Brosh, Weihang Chai, Marietta Y W T Lee, Dong Zhang, Carl Schildkraut, Li Zheng, Binghui Shen.
      G-quadruplexes (G4s) are widely existing stable DNA secondary structures in mammalian cells. A long-standing hypothesis is that timely resolution of G4s is needed for efficient and faithful DNA replication. In vitro , G4s may be unwound by helicases or alternatively resolved via DNA2 nuclease mediated G4 cleavage. However, little is known about the biological significance and regulatory mechanism of the DNA2-mediated G4 removal pathway. Here, we report that DNA2 deficiency or its chemical inhibition leads to a significant accumulation of G4s and stalled replication forks at telomeres, which is demonstrated by a high-resolution technology: Single molecular analysis of replicating DNA (SMARD). We further identify that the DNA repair complex MutSα (MSH2-MSH6) binds G4s and stimulates G4 resolution via DNA2-mediated G4 excision. MSH2 deficiency, like DNA2 deficiency or inhibition, causes G4 accumulation and defective telomere replication. Meanwhile, G4-stabilizing environmental compounds block G4 unwinding by helicases but not G4 cleavage by DNA2. Consequently, G4 stabilizers impair telomere replication and cause telomere instabilities, especially in cells deficient in DNA2 or MSH2.
    DOI:  https://doi.org/10.1101/2025.04.04.647332
  8. EMBO J. 2025 May 08.
    Control of replication and gene expression by ADP-ribosylation of DNA in Mycobacterium tuberculosis.
    Rachel E Butler, Marion Schuller, Ritu Jaiswal, Jayanta Mukhopadhyay, Jim Barber, Suzie Hingley-Wilson, Emily Wasson, Alex Couto Alves, Ivan Ahel, Graham R Stewart.
      Mycobacterium tuberculosis maintains long-term infections characterised by the need to regulate growth and adapt to contrasting in vivo environments. Here we show that M. tuberculosis complex bacteria utilise reversible ADP-ribosylation of single-stranded DNA as a mechanism to coordinate stationary phase growth with transcriptional adaptation. The DNA modification is controlled by DarT, an ADP-ribosyltransferase, which adds ADP-ribose to thymidine, and DarG, which enzymatically removes this base modification. Using darG-knockdown M. bovis BCG, we map the first DNA ADP-ribosylome from any organism. We show that inhibition of replication by DarT is reversible and accompanied by extensive ADP-ribosylation at the origin of replication (OriC). In addition, we observe ADP-ribosylation across the genome and demonstrate that ADP-ribose-thymidine alters the transcriptional activity of M. tuberculosis RNA polymerase. Furthermore, we demonstrate that during stationary phase, DarT-dependent ADP-ribosylation of M. tuberculosis DNA is required to optimally induce expression of the Zur regulon, including the ESX-3 secretion system and multiple alternative ribosome proteins. Thus, ADP-ribosylation of DNA can provide a mechanistic link through every aspect of DNA biology from replication to transcription to translation.
    Keywords:  ADP-ribosylation; ADPr-Seq; DNA Modification; PARP; Transcription Regulation
    DOI:  https://doi.org/10.1038/s44318-025-00451-y
  9. Int J Mol Sci. 2025 Apr 08. pii: 3458. [Epub ahead of print]26(8):
    Vulnerable Nucleotide Pools and Genomic Instability in Yeast Strains with Deletion of the ADE12 Gene Encoding for Adenylosuccinate Synthetase.
    Elena R Tarakhovskaya, Yulia V Andreychuk, Tatiana E Bilova, Claudia Wiesner, Youri I Pavlov, Elena I Stepchenkova.
      Adenylosuccinate synthetase (AdSS), encoded by the ADE12 gene in yeast Saccharomyces cerevisiae, plays a critical role in purine biosynthesis, catalyzing the conversion of inosine 5'-monophosphate (IMP) and aspartic acid to adenylosuccinate, a substrate for the following adenosine 5'-monophosphate (AMP) synthesis step. Mutants lacking AdSS activity exhibit a range of pleiotropic phenotypes: slow growth, poor spore germination, accumulation, and secretion of inosine and hypoxanthine. We report new phenotypes of ade12 mutants and explain their molecular mechanisms. A GC-MS analysis showed that ade12 mutants have highly altered metabolite profiles: the accumulation of IMP leads to an impaired cellular energy metabolism, resulting in a dysregulation of key processes-the metabolism of nucleotides, carbohydrates, and amino acids. These metabolic perturbations explain the cell division arrest observed in ade12 yeast strains. A slowed replication in ade12 mutants, because of the insufficient availability of energy, nucleotides, and proteins, leads to the error-prone DNA polymerase ζ-dependent elevation of spontaneous mutagenesis, connecting multiple roles of AdSS in metabolism with genome stability control.
    Keywords:  Saccharomyces cerevisiae; adenylosuccinate synthetase (AdSS); dNTP pool; metabolome; mutagenesis; purine biosynthesis
    DOI:  https://doi.org/10.3390/ijms26083458
  10. DNA Repair (Amst). 2025 Apr 29. pii: S1568-7864(25)00038-2. [Epub ahead of print]149 103842
    Reversible association of ubiquitin with PCNA is important for template switching in S. cerevisiae.
    Cindy Meister, Ronald P Wong, Zhi-Hoon Park, Helle D Ulrich.
      Polyubiquitylation of the replication factor PCNA activates the replicative bypass of DNA lesions via an error-free pathway involving template switching. However, the mechanism by which the K63-linked polyubiquitin chains facilitate damage bypass is poorly understood. Intriguingly, stable fusions of linear ubiquitin oligomers to PCNA, designed as mimics of the native K63-linked chains, are not functional, while enzymatic modification of PCNA with linear chains supports template switching in budding yeast. To investigate the cause of this discrepancy, we have taken an alternative approach to identify the features of polyubiquitylated PCNA essential for activating damage bypass. We designed linear, non-cleavable ubiquitin constructs that can be recruited non-covalently to PCNA via a PIP motif. We found that these partially suppress the damage sensitivity and elevated spontaneous mutation rates of yeast strains defective in PCNA ubiquitylation. Genetic analysis confirms that this rescue is due to an activation of the template switching pathway. Surprisingly, even the recruitment of monoubiquitin units promotes activity in this setting. These observations suggest that the reversibility of ubiquitin's association with PCNA is more important than the actual linkage of the polyubiquitin chain. Thus, our study highlights the dynamic nature of ubiquitin signaling in the context of DNA damage bypass.
    Keywords:  DNA damage bypass; DNA damage tolerance; PCNA; Template switching; Translesion synthesis; Ubiquitin
    DOI:  https://doi.org/10.1016/j.dnarep.2025.103842
  11. bioRxiv. 2025 Apr 19. pii: 2025.04.15.648952. [Epub ahead of print]
    Distinct mechanisms underlying extrachromosomal telomere DNA generation in ALT cancers.
    Junyeop Lee, Eric J Sohn, Jina Lee, Angelo Taglialatela, Alberto Ciccia, Jaewon Min.
      Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism observed in 15% of human cancers. A hallmark of ALT cancers is the presence of C-circles, circular single-stranded DNAs (ssDNAs) enriched with cytosine-rich telomere (C-rich, CCCTAA) sequences. G-circles, containing guanosine-rich telomere (G-rich, GGGTTA) ssDNAs, also exist but are much less abundant. Recent studies indicate that excessive displacement of Okazaki fragments during lagging-strand synthesis is a unique feature of ALT telomeres and responsible for generating C-circles/C-rich ssDNAs. However, the distinct characteristics of C-circles compared to G-circles remain unclear. Here, we demonstrate that co-deficiency of the DNA translocases SMARCAL1 and FANCM leads to abundant generation of G-circle/G-rich ssDNAs. These G-rich ssDNAs mainly exist in linear form, ranging in size from 500 to 3000 nucleotides, which differs significantly from the structure and size of C-circle/C-rich ssDNAs. Mechanistically, both C-rich and G-rich ssDNAs originate from BLM/POLD-mediated excessive strand displacement; however, they differ in their origins and initiation mechanisms. Specifically, C-rich ssDNAs arise from lagging daughter strands initiated by the CST complex, whereas G-rich ssDNAs originate from leading daughter strands through RAD51-dependent G-strand synthesis. Our findings propose two distinct mechanisms for generating two different extrachromosomal telomere DNAs, C-and G-circles, during ALT-mediated telomere elongation.
    DOI:  https://doi.org/10.1101/2025.04.15.648952
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