bims-replis 2025-04-20 papers

bims-replis

Biomed News

on Replisome

Issue of 2025–04–20
eleven papers selected by
Anna Zawada, International Centre for Translational Eye Research

Structural basis of human replisome progression into a nucleosome.
Structure of the Saccharolobus solfataricus GINS tetramer.
Chromatin as a Coevolutionary Graph: Modeling the Interplay of Replication with Chromatin Dynamics.
The POLγ Y951N patient mutation disrupts the switch between DNA synthesis and proofreading, triggering mitochondrial DNA instability.
PCNA encircling primer/template junctions is eliminated by exchange of RPA for Rad51: Implications for the interplay between human DNA damage tolerance pathways.
Functional asymmetry in processivity clamp proteins.
The Yeast HMGB Protein Hmo1 Is a Multifaceted Regulator of DNA Damage Tolerance.
The yeast CST and Polα/primase complexes act in concert to ensure proper telomere maintenance and protection.
ATM priming and end resection-coupled phosphorylation of MRE11 is important for fork protection and replication restart.
Mechanistic insights into direct DNA and RNA strand transfer and dynamic protein exchange of SSB and RPA.
Mechanism of DNA replication fork breakage and PARP1 hyperactivation during replication catastrophe.

bioRxiv. 2025 Apr 05. pii: 2025.04.04.647053. [Epub ahead of print]

Structural basis of human replisome progression into a nucleosome.

Felix Steinruecke, Jonathan W Markert, Lucas Farnung.

Epigenetic inheritance requires the transfer of parental histones to newly synthesized DNA during eukaryotic chromosome replication, yet the structural mechanisms underlying replisome engagement with nucleosomes remain unclear. Here we establish an in vitro chromatin replication system and report four cryo-EM structures of the human replisome in complex with a parental nucleosome. The structures capture distinct states of nucleosomal DNA unwrapping and nucleosome integrity during nucleosome disassembly by the encroaching replisome.

DOI: https://doi.org/10.1101/2025.04.04.647053
Acta Crystallogr F Struct Biol Commun. 2025 May 01.

Structure of the Saccharolobus solfataricus GINS tetramer.

Srihari Shankar, Eric J Enemark.

  DNA replication is tightly regulated to ensure genomic stability and prevent several diseases, including cancers. Eukaryotes and archaea partly achieve this regulation by strictly controlling the activation of hexameric minichromosome maintenance (MCM) helicase rings that unwind DNA during its replication. In eukaryotes, MCM activation critically relies on the sequential recruitment of the essential factors Cdc45 and a tetrameric GINS complex at the onset of the S-phase to generate a larger CMG complex. We present the crystal structure of the tetrameric GINS complex from the archaeal organism Saccharolobus solfataricus (Sso) to reveal a core structure that is highly similar to the previously determined GINS core structures of other eukaryotes and archaea. Using molecular modeling, we illustrate that a subdomain of SsoGINS would need to move to accommodate known interactions of the archaeal GINS complex and to generate a SsoCMG complex analogous to that of eukaryotes.

Keywords:  DNA replication; GINS; archaea; minichromosome maintenance

DOI:  https://doi.org/10.1107/S2053230X25003085
bioRxiv. 2025 Apr 04. pii: 2025.03.31.646315. [Epub ahead of print]

Chromatin as a Coevolutionary Graph: Modeling the Interplay of Replication with Chromatin Dynamics.

Sevastianos Korsak, Krzysztof H Banecki, Karolina Buka, Piotr J Górski, Dariusz Plewczynski.

Modeling DNA replication poses significant challenges due to the intricate interplay of biophysical processes and the need for precise parameter optimization. In this study, we explore the interactions among three key biophysical factors that influence chromatin folding: replication, loop extrusion, and compartmentalization. Replication forks, known to act as barriers to the motion of loop extrusion factors, also correlate with the phase separation of chromatin into A and B compartments. Our approach integrates three components: (1) a numerical model that takes into advantage single-cell replication timing data to simulate replication fork propagation; (2) a stochastic Monte Carlo simulation that captures the interplay between the biophysical factors, with loop extrusion factors binding, unbinding, and extruding dynamically, while CTCF barriers and replication forks act as static and moving barriers, and a Potts Hamiltonian governs the spreading of epigenetic states driving chromatin compartmentalization; and (3) a 3D OpenMM simulation that reconstructs the chromatin's 3D structure based on the states generated by the stochastic model. To our knowledge, this is the first framework to dynamically integrate and simulate these three biophysical factors, enabling insights into chromatin behavior during replication. Furthermore, we investigate how replication stress alters these dynamics and affects chromatin structure.

DOI: https://doi.org/10.1101/2025.03.31.646315
Proc Natl Acad Sci U S A. 2025 Apr 22. 122(16): e2417477122

The POLγ Y951N patient mutation disrupts the switch between DNA synthesis and proofreading, triggering mitochondrial DNA instability.

Josefin M E Forslund, Tran V H Nguyen, Vimal Parkash, Andreas Berner, Steffi Goffart, Jaakko L O Pohjoismäki, Paulina H Wanrooij, Erik Johansson, Sjoerd Wanrooij.

  Mitochondrial DNA (mtDNA) stability, essential for cellular energy production, relies on DNA polymerase gamma (POLγ). Here, we show that the POLγ Y951N disease-causing mutation induces replication stalling and severe mtDNA depletion. However, unlike other POLγ disease-causing mutations, Y951N does not directly impair exonuclease activity and only mildly affects polymerase activity. Instead, we found that Y951N compromises the enzyme's ability to efficiently toggle between DNA synthesis and degradation, and is thus a patient-derived mutation with impaired polymerase-exonuclease switching. These findings provide insights into the intramolecular switch when POLγ proofreads the newly synthesized DNA strand and reveal a new mechanism for causing mitochondrial DNA instability.

Keywords:  DNA polymerases; mitochondria; mitochondrial disease; mtDNA; mtDNA replication

DOI:  https://doi.org/10.1073/pnas.2417477122
bioRxiv. 2025 Apr 02. pii: 2025.03.27.645792. [Epub ahead of print]

PCNA encircling primer/template junctions is eliminated by exchange of RPA for Rad51: Implications for the interplay between human DNA damage tolerance pathways.

Jessica L Norris, Lindsey O Rogers, Grace Young, Kara G Pytko, Rachel L Dannenberg, Samuel Perreault, Vikas Kaushik, Edwin Antony, Mark Hedglin.

The DNA genome is constantly exposed to agents, such as ultraviolet radiation (UVR), that can alter or eliminate its coding properties through covalent modifications of the template bases. Many of these damaging modifications (i.e., lesions) persist into S-phase of the cell cycle where they may stall the canonical DNA replication machinery. In humans, these stalling events are circumvented by one of at least three interconnected DNA damage tolerance (DDT) pathways; translesion DNA synthesis (TLS), Template Switching (TS), and Homology-dependent Recombination (HDR). Currently, the functional interplay between these pathways is unclear, leaving wide gaps in our fundamental understanding of human DDT. To gain insights, we focus on the activation mechanisms of the DDT pathways. PCNA sliding clamps encircling primer/template (P/T) junctions of stalled replication sites are central to the activation of both TLS and TS whereas exchange of RPA for Rad51 filaments on the single strand DNA (ssDNA) sequences of stalled replication sites is central to HDR activation. Utilizing direct, ensemble FRET approaches developed by our lab, we independently monitor and directly compare PCNA occupancy and RPA/Rad51 exchange at P/T junctions under a variety of conditions that mimic in vivo scenarios. Collectively, the results reveal that assembly of stable Rad51 filaments at P/T junctions via RPA/Rad51 exchange causes complete and irreversible unloading of the resident PCNA, both in the presence and absence of an abundant PCNA-binding protein complex. Further investigations decipher the mechanism of RPA/Rad51 exchange-dependent unloading of PCNA. Collectively, these studies provide critical insights into the interplay between human DDT pathways and direction for future studies.

DOI: https://doi.org/10.1101/2025.03.27.645792
Biophys J. 2025 Apr 16. pii: S0006-3495(25)00241-3. [Epub ahead of print]

Functional asymmetry in processivity clamp proteins.

Sam Mahdi, Penny J Beuning, Dmitry M Korzhnev.

Symmetric homo-oligomeric proteins comprising multiple copies of identical subunits are abundant in all domains of life. To fulfill their biological function, these complexes undergo conformational changes, binding events, or post-translational modifications leading to loss of symmetry. Processivity clamp proteins that encircle DNA and play multiple roles in DNA replication and repair are archetypical homo-oligomeric symmetric protein complexes. The symmetrical nature of processivity clamps enables simultaneous interactions with multiple protein binding partners; such interactions result in asymmetric changes that facilitate the transition between clamp loading and DNA replication, and between DNA replication and repair. The ring-shaped processivity clamps are opened and loaded onto DNA by clamp-loader complexes via asymmetric intermediates with one of the intermonomer interfaces disrupted, undergo spontaneous opening events, and bind heterogeneous partners. Eukaryotic clamp proteins are subject to ubiquitylation, SUMOylation, and acetylation affecting their biological functions. There is increasing evidence of the functional asymmetry of the processivity clamp proteins from structural, biophysical, and computational studies. Here, we review the symmetry and asymmetry of processivity clamps and their roles in regulating the various functions of the clamps.

DOI: https://doi.org/10.1016/j.bpj.2025.04.014
Int J Mol Sci. 2025 Apr 01. pii: 3255. [Epub ahead of print]26(7):

The Yeast HMGB Protein Hmo1 Is a Multifaceted Regulator of DNA Damage Tolerance.

Jinlong Huo, Anhui Wei, Na Guo, Ruotong Wang, Xin Bi.

  The Saccharomyces cerevisiae chromosomal architectural protein Hmo1 is categorized as an HMGB protein, as it contains two HMGB motifs that bind DNA in a structure-specific manner. However, Hmo1 has a basic C-terminal domain (CTD) that promotes DNA bending instead of an acidic one found in a canonical HMGB protein. Hmo1 has diverse functions in genome maintenance and gene regulation. It is implicated in DNA damage tolerance (DDT) that enables DNA replication to bypass lesions on the template. Hmo1 is believed to direct DNA lesions to the error-free template switching (TS) pathway of DDT and to aid in the formation of the key TS intermediate sister chromatid junction (SCJ), but the underlying mechanisms have yet to be resolved. In this work, we used genetic and molecular biology approaches to further investigate the role of Hmo1 in DDT. We found extensive functional interactions of Hmo1 with components of the genome integrity network in cellular response to the genotoxin methyl methanesulfonate (MMS), implicating Hmo1 in the execution or regulation of homology-directed DNA repair, replication-coupled chromatin assembly, and the DNA damage checkpoint. Notably, our data pointed to a role for Hmo1 in directing SCJ to the nuclease-mediated resolution pathway instead of the helicase/topoisomerase mediated dissolution pathway for processing/removal. They also suggested that Hmo1 modulates both the recycling of parental histones and the deposition of newly synthesized histones on nascent DNA at the replication fork to ensure proper chromatin formation. We found evidence that Hmo1 counteracts the function of histone H2A variant H2A.Z (Htz1 in yeast) in DDT possibly due to their opposing effects on DNA resection. We showed that Hmo1 promotes DNA negative supercoiling as a proxy of chromatin structure and MMS-induced DNA damage checkpoint signaling, which is independent of the CTD of Hmo1. Moreover, we obtained evidence indicating that whether the CTD of Hmo1 contributes to its function in DDT is dependent on the host's genetic background. Taken together, our findings demonstrated that Hmo1 can contribute to, or regulate, multiple processes of DDT via different mechanisms.

Keywords:  DNA damage checkpoint; DNA damage tolerance; DNA replication; HMGB protein; Hmo1; Htz1; chromatin

DOI:  https://doi.org/10.3390/ijms26073255
Nucleic Acids Res. 2025 Apr 10. pii: gkaf245. [Epub ahead of print]53(7):

The yeast CST and Polα/primase complexes act in concert to ensure proper telomere maintenance and protection.

Kimberly Calugaru, Eun Young Yu, Sophie Huang, Nayim González-Rodríguez, Javier Coloma, Neal F Lue.

Polα/primase (PP), the polymerase that initiates DNA synthesis at replication origins, also completes the task of genome duplication by synthesizing the telomere C-strand under the control of the CTC1/CDC13-STN1-TEN1 (CST) complex. Using cryo-electron microscopy (cryo-EM) structures of the human CST-Polα/primase-DNA complex as guides in conjunction with AlphaFold modeling, we identified structural elements in yeast CST and PP that promote complex formation. Mutating these structures in Candida glabrata Stn1, Ten1, Pri1, and Pri2 abrogated the stimulatory activity of CST on PP in vitro, supporting the functional relevance of the physical contacts in cryo-EM structures as well as the conservation of mechanisms between yeast and humans. Introducing these mutations into C. glabrata yielded two distinct groups of mutants. One group exhibited progressive, telomerase-dependent telomere elongation without evidence of DNA damage. The other manifested slow growth, telomere length heterogeneity, single-stranded DNA accumulation and elevated C-circles, which are indicative of telomere deprotection. These telomere deprotection phenotypes are altered or suppressed by mutations in multiple DNA damage response (DDR) and DNA repair factors. We conclude that in yeast, the telomerase inhibition and telomere protection function previously ascribed to the CST complex are mediated jointly by both CST and Polα/primase, highlighting the critical importance of a replicative DNA polymerase in telomere regulation.

DOI: https://doi.org/10.1093/nar/gkaf245
Proc Natl Acad Sci U S A. 2025 Apr 22. 122(16): e2422720122

ATM priming and end resection-coupled phosphorylation of MRE11 is important for fork protection and replication restart.

Huimin Zhang, Youhang Li, Sameer Bikram Shah, Shibo Li, Qingrong Li, Joshua Oaks, Tinghong Lv, Linda Z Shi, Hailong Wang, Dong Wang, Xiaohua Wu.

  The MRE11/RAD50/NBS1 (MRN) complex plays multiple roles in the maintenance of genome stability. MRN is associated with replication forks to preserve fork integrity and is also required for end resection at double-strand breaks (DSBs) to facilitate homologous recombination (HR). The critical need for proper control of the MRE11 nuclease activity is highlighted by the extensive nascent strand DNA degradation driven by MRE11 in BRCA-deficient cells, leading to genome instability and increased sensitivity to chemotherapeutics. In this study, we identified a tightly controlled mechanism, elicited by sequential phosphorylation of MRE11 by ATM and ATR to regulate MRE11 nuclease activities through its DNA binding. Specifically, at DSBs, MRE11 phosphorylation by ATM at the C-terminal S676/S678 primes it for subsequent phosphorylation by ATR, whose activation is triggered by end resection which requires the MRE11 nuclease activity. This ATR-mediated phosphorylation in turn induces MRE11 dissociation from DNA, providing a feedback mechanism to regulate the extent of end resection. At stalled replication forks, however, without ATM priming, MRN is stably associated with forks despite ATR activation. Furthermore, the ATR phosphorylation-defective MRE11 mutants are retained at single-ended DSBs formed by fork reversal upon replication stress, leading to extensive degradation of nascent DNA strands. Importantly, this end resection-coupled MRE11 phosphorylation elicits another critical layer of fork protection of nascent DNA in addition to BRCA2, ensuring proper end resection that is sufficient for replication restart at reversed forks while maintaining fork stability.

Keywords:  ATM; ATR; MRE11; end resection; fork protection

DOI:  https://doi.org/10.1073/pnas.2422720122
bioRxiv. 2025 Apr 01. pii: 2025.04.01.643995. [Epub ahead of print]

Mechanistic insights into direct DNA and RNA strand transfer and dynamic protein exchange of SSB and RPA.

Tapas Paul, I-Ren Lee, Sushil Pangeni, Fahad Rashid, Olivia Yang, Edwin Antony, James M Berger, Sua Myong, Taekjip Ha.

Single-stranded DNA-binding proteins (SSBs) are essential for genome stability, facilitating replication, repair, and recombination by binding ssDNA, recruiting other proteins, and dynamically relocating in response to cellular demands. Using single-molecule fluorescence resonance energy transfer (smFRET) assays, we elucidated the mechanisms underlying direct strand transfer from one locale to another, protein exchange, and RNA interactions at high resolution. Both bacterial SSB and eukaryotic replication protein A (RPA) exhibited direct strand transfer to competing ssDNA, with rates strongly influenced by ssDNA length. Strand transfer proceeded through multiple failed attempts before a successful transfer, forming a ternary intermediate complex with transient interactions, supporting a direct transfer mechanism. Both proteins efficiently exchanged DNA-bound counterparts with freely diffusing molecules, while hetero-protein exchange revealed that SSB and RPA could replace each other on ssDNA in a length-dependent manner, indicating that protein exchange does not require specific protein-protein interactions. Additionally, both proteins bound RNA and underwent strand transfer to competing RNA, with RPA demonstrating faster RNA transfer kinetics. Competitive binding assays confirmed a strong preference for DNA over RNA. These findings provide critical insights into the dynamic behavior of SSB and RPA in nucleic acid interactions, advancing our understanding of their essential roles in genome stability, regulating RNA metabolism, and orchestrating nucleic acid processes.

DOI: https://doi.org/10.1101/2025.04.01.643995
Sci Adv. 2025 Apr 18. 11(16): eadu0437

Mechanism of DNA replication fork breakage and PARP1 hyperactivation during replication catastrophe.

Pedro Ortega, Elodie Bournique, Junyi Li, Ambrocio Sanchez, Gisselle Santiago, Brooke R Harris, Josefine Striepen, John Maciejowski, Abby M Green, Rémi Buisson.

Ataxia telangiectasia and Rad3-related (ATR) inhibition triggers a surge in origin firing, resulting in increased levels of single-stranded DNA (ssDNA) that rapidly deplete all available RPA. This leaves ssDNA unprotected and susceptible to breakage, a phenomenon known as replication catastrophe. However, the mechanism by which unprotected ssDNA breaks remains unclear. Here, we reveal that APOBEC3B is the key enzyme targeting unprotected ssDNA at replication forks, initiating a reaction cascade that induces fork collapse and poly(ADP-ribose) polymerase 1 (PARP1) hyperactivation. Mechanistically, we demonstrate that uracils generated by APOBEC3B at replication forks are removed by UNG2, resulting in abasic sites that are subsequently cleaved by APE1 endonuclease. Moreover, we show that APE1-mediated DNA cleavage is the critical enzymatic step for PARP1 hyperactivation in cells, regardless of how abasic sites are generated on DNA. Last, we demonstrate that APOBEC3B-induced PARP1 trapping and DNA double-strand breaks drive cell sensitivity to ATR inhibition, creating a context of synthetic lethality when coupled with PARP inhibitors.

DOI: https://doi.org/10.1126/sciadv.adu0437