bims-replis 2025-07-13 papers

bioRxiv. 2025 Jul 02. pii: 2025.07.02.662785. [Epub ahead of print]

DNA polymerase α-primase can function as a translesion DNA polymerase.

Ryan Mayle, Roxana Georgescu, Michael E O'Donnell.

Replication of cellular chromosomes requires a primase to generate short RNA primers to initiate genomic replication. While bacterial and archaeal primase generate short RNA primers, the eukaryotic primase, Polα-primase, contains both RNA primase and DNA polymerase (Pol) subunits that function together to form a >20 base hybrid RNA-DNA primer. Interestingly, the DNA Pol1 subunit of Polα lacks a 3'-5' proofreading exonuclease, contrary to the high fidelity normally associated with DNA replication. However, Polο and Polδ synthesize the majority of the eukaryotic genome and both contain 3'-5' exonuclease activity for high fidelity. None the less, even the small amount of DNA produced by Pol1 in each of the many RNA/DNA primers during chromosome replication adds up to tens of millions of nucleotides in a human genome. Thus it has been a longstanding question why Pol1 lacks a proofreading exonuclease. We show here that Polα is uniquely capable of traversing common oxidized or hydrolyzed template nucleotides and propose that Polα evolved to bypass these common template lesions when they are encountered during chromosome replication.
Significance statement: Eukaryotic Polα-primase contains DNA polymerase (Pol1) and RNA primase subunits that together synthesize a >20 nucleotide hybrid RNA-DNA primer. Bacteria and archaea only require a dozen or less RNA residues to prime DNA synthesis. Therefore, why do eukaryotes require the additional DNA? We propose, and demonstrate here that Pol1, which lacks a proofreading 3'-5' exonuclease, is capable of traversing some common template lesions produced in the normal hydrolytic and metabolic oxidative environment of cells. Thus, we hypothesize that Pol1 activity within the eukaryotic primase evolved to help replisomes bypass template damage. Bypassed damaged sites can be dealt with by repair processes after replication has occurred.

DOI: https://doi.org/10.1101/2025.07.02.662785

Nucleic Acids Res. 2025 Jul 08. pii: gkaf530. [Epub ahead of print]53(13):

CDK-driven phosphorylation of TRAIP is essential for mitotic replisome disassembly and MiDAS.

Divyasree Poovathumkadavil, Alicja Reynolds-Winczura, Aggeliki Skagia, Paolo Passaretti, Martina Muste Sadurni, Georgia Kingsley, Alexander Leitner, Marco Saponaro, Agnieszka Gambus.

Disassembly of the replication machinery (replisome) from chromatin is an active process driven by two ubiquitin ligases Cul2LRR1 and TRAIP, which both target the Mcm7 subunit of the replicative helicase for ubiquitylation. Uncontrolled unloading of replisomes during S-phase would be disastrous for genome stability and cell viability. On the other hand, replisomes retained on under-replicated DNA in mitosis require removal to allow access and processing of the DNA before cell division. TRAIP ubiquitylates replisomes in mitosis but can also act in specific situations during S-phase. However, we do not know how TRAIP's activity is regulated to stop uncontrolled replisome unloading. Here we show that TRAIP activity towards replisomes is not regulated at the level of interaction with the substrate: it interacts with terminated replisomes in S-phase without ubiquitylation. However, in mitosis, TRAIP is phosphorylated by cyclin-dependent kinases (CDKs) and this phosphorylation is essential for mitotic replisome unloading. CDK phosphorylation of TRAIP stimulates its autoubiquitylation activity and ubiquitylation of replisomes isolated from mitotic chromatin. The phosphorylation of TRAIP is also important in human cells for TRAIP functions during MiDAS. Although essential during mitosis, the CDK-driven phosphorylation of TRAIP is not sufficient to activate uncontrolled unloading of replisomes in S-phase.

DOI: https://doi.org/10.1093/nar/gkaf530

bioRxiv. 2025 Jul 03. pii: 2025.07.02.662830. [Epub ahead of print]

PCNA is a Nucleotide Exchange Factor for the Clamp Loader ATPase Complex.

Joshua Pajak, Jacob T Landeck, Xingchen Liu, Krishna Anand, Sasha Litvak, Brian A Kelch.

All life requires loading ring-shaped sliding clamp protein complexes onto DNA. The sliding clamp loader is a conserved AAA+ ATPase that binds the sliding clamp, opens the ring, and places it onto DNA. While recent structural work on both the canonical and 'alternative' clamp loaders has shed light into how these machines perform their task once, it remains unclear how clamp loaders are recycled to load multiple sliding clamps. Here, we present structures of the Saccharomyces cerevisiae clamp loader Replication Factor C (RFC) in absence of sliding clamp or supplemented nucleotide. Our structures indicate that RFC holds onto ADP tightly in at least two of its four ATPase active sites, suggesting that nucleotide exchange is regulated. Our molecular dynamics simulations and biochemical data indicate that binding of the sliding clamp PCNA causes rapid exchange of tightly bound ADP. Our data suggests that PCNA acts as a nucleotide exchange factor by prying apart adjacent subunits, providing a pathway for ADP release. We propose that, by using its own substrate as a nucleotide exchange factor, RFC excludes off-pathway states that would arise from binding DNA prior to PCNA.

DOI: https://doi.org/10.1101/2025.07.02.662830

Exp Ther Med. 2025 Aug;30(2): 163

Role and mechanism of RPA1 in the development and progression of glioma.

Zhiming Zhao, Yu Sun, Ping Yin, Quan Zhang, Jianfu Zhang.

Glioma is a highly malignant primary tumor of the central nervous system, characterized by highly invasive behavior and a high recurrence rate. The current treatment options for gliomaoften have unsatisfactory outcomes. Replication protein A1 (RPA1), a component of the RPA heterotrimer, has been identified as a potential oncogene implicated in the development and clinical prognosis of various types of solid tumors, including liver cancer, nasopharyngeal carcinoma and gastrointestinal cancer. However, studies on the associations between RPA1 expression and glioma cell proliferation or patient prognosis are limited, and the underlying mechanisms remain unclear. The aim of the present study was to explore the expression of RPA1 in glioma and its clinical relevance in the clinicopathology and prognosis of glioma. In addition, the biological functions and signal transduction pathways mediated by RPA1 were predicted using bioinformatics analysis to provide basic theoretical support for further mechanistic research and in vivo experiments. The Cancer Genome Atlas (TCGA) data analysis, and reverse transcription-quantitative PCR, western blot analysis and immunohistochemical (IHC) staining were performed to analyze RPA1 expression in glioma cells or tissues of different grades. The associations between RPA1 expression and various pathological parameters related to tumor cell proliferation and patient prognosis were also investigated. Furthermore, the biological processes and signaling pathways influenced by RPA1-related target genes were predicted. The results revealed that RPA1 expression varies among different grades of gliomas, with higher World Health Organization (WHO) grades exhibiting higher RPA expression levels. Furthermore, both bioinformatics analysis and IHC staining results revealed that high RPA1 expression was associated with a shorter overall survival (OS). In addition, RPA1 expression exhibited a significant association with WHO glioma grade and key markers of malignancy, namely Ki-67 expression and p53 mutation status. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses suggested that RPA1 may promote the development of glioma through pathways including 'DNA replication', 'Fanconi anemia pathway', 'mismatch repair', 'homologous recombination', 'nucleotide excision repair' and 'cell cycle'. In conclusion, RPA1 expression in glioma tissues and cells is positively associated with the degree of glioma malignancy, and high RPA1 expression is associated with a shorter OS in patients with glioma.

Keywords: RPA1; clinical pathology; glioma; prognosis

DOI: https://doi.org/10.3892/etm.2025.12913