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Chromosome replication is performed by a complex and intricate ensemble of proteins termed the replisome, where the DNA polymerases Polδ and Polε, DNA polymerase α-primase (Polα) and accessory proteins including AND-1, CLASPIN and TIMELESS–TIPIN (respectively known as Ctf4, Mrc1 and Tof1–Csm3 in Saccharomyces cerevisiae ) are organized around the CDC45–MCM–GINS (CMG) replicative helicase 1 – 7 . Because a functional human replisome has not been reconstituted from purified proteins, how these factors contribute to human DNA replication and whether additional proteins are required for optimal DNA synthesis are poorly understood. Here we report the biochemical reconstitution of human replisomes that perform fast and efficient DNA replication using 11 purified human replication factors made from 43 polypeptides. Polε, but not Polδ, is crucial for optimal leading-strand synthesis. Unexpectedly, Polε-mediated leading-strand replication is highly dependent on the sliding-clamp processivity factor PCNA and the alternative clamp loader complex CTF18–RFC. We show how CLASPIN and TIMELESS–TIPIN contribute to replisome progression and demonstrate that, in contrast to the budding yeast replisome 8 , AND-1 directly augments leading-strand replication. Moreover, although AND-1 binds to Polα 9 , 10 , the interaction is dispensable for lagging-strand replication, indicating that Polα is functionally recruited via an AND-1-independent mechanism for priming in the human replisome. Collectively, our work reveals how the human replisome achieves fast and efficient leading-strand and lagging-strand DNA replication, and provides a powerful system for future studies of the human replisome and its interactions with other DNA metabolic processes. A biochemical reconstitution of human replisomes that provides a system for future studies of DNA metabolic processes.

Although a rich record of Pleistocene human-associated archaeological assemblages exists, the scarcity of hominin fossils often impedes the understanding of which hominins occupied a site. Using targeted enrichment of mitochondrial DNA, we show that cave sediments represent a rich source of ancient mammalian DNA that often includes traces of hominin DNA, even at sites and in layers where no hominin remains have been discovered. By automation-assisted screening of numerous sediment samples, we detected Neandertal DNA in eight archaeological layers from four caves in Eurasia. In Denisova Cave, we retrieved Denisovan DNA in a Middle Pleistocene layer near the bottom of the stratigraphy. Our work opens the possibility of detecting the presence of hominin groups at sites and in areas where no skeletal remains are found.

Shifts in soil microbial communities over altitudinal gradients and the driving factors are poorly studied. Their elucidation is indispensable to gain a comprehensive understanding of the response of ecosystems to global climate change. Here, we investigated soil archaeal, bacterial, and fungal communities at four Alpine forest sites representing a climosequence, over an altitudinal gradient from 545 to 2000 m above sea level (asl), regarding abundance and diversity by using qPCR and Illumina sequencing, respectively. Archaeal community was dominated by Thaumarchaeota, and no significant shifts were detected in abundance or community composition with altitude. The relative bacterial abundance increased at higher altitudes, which was related to increasing levels of soil organic matter and nutrients with altitude. Shifts in bacterial richness and diversity as well as community structure (comprised basically of Proteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes) significantly correlated with several environmental and soil chemical factors, especially soil pH. The site at the lowest altitude harbored the highest bacterial richness and diversity, although richness/diversity community properties did not show a monotonic decrease along the gradient. The relative size of fungal community also increased with altitude and its composition comprised Ascomycota, Basidiomycota, and Zygomycota. Changes in fungal richness/diversity and community structure were mainly governed by pH and C/N, respectively. The variation of the predominant bacterial and fungal classes over the altitudinal gradient was the result of the environmental and soil chemical factors prevailing at each site.

Rapid advancements in long-read sequencing technologies have transformed read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly challenging in plants and fungi. To overcome this, we present a protocol collection; high-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. We optimised a gentle magnetic bead based high-molecular weight DNA extraction, which is presented here in detail. The protocol circumvents spin columns and high-centrifugation, to limit DNA fragmentation. The protocol is scalable based on tissue input, which can be used on many species of plants, fungi, reptiles and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale in low resource settings. An optional sorbitol wash is listed and is highly recommended for plant and fungal tissues. To further remove any remaining contaminants such as phenols and polysaccharides, optional DNA clean-up and size selection strategies are given. This protocol collection is suitable for all common long-read sequencing platforms, such as technologies offered by PacBio and Oxford Nanopore. Using these protocols, sequencing on the Oxford Nanopore MinION can achieve read length N50 values of 30–50 kb, with reads exceeding 200 kb and outputs ranging from 15–30 Gbp. This has been routinely achieved with various plant, fungi, animal and bacteria samples.

DNA samples derived from vertebrate skin, bodily cavities and body fluids contain both host and microbial DNA; the latter often present as a minor component. Consequently, DNA sequencing of a microbiome sample frequently yields reads originating from the microbe(s) of interest, but with a vast excess of host genome-derived reads. In this study, we used a methyl-CpG binding domain (MBD) to separate methylated host DNA from microbial DNA based on differences in CpG methylation density. MBD fused to the Fc region of a human antibody (MBD-Fc) binds strongly to protein A paramagnetic beads, forming an effective one-step enrichment complex that was used to remove human or fish host DNA from bacterial and protistan DNA for subsequent sequencing and analysis. We report enrichment of DNA samples from human saliva, human blood, a mock malaria-infected blood sample and a black molly fish. When reads were mapped to reference genomes, sequence reads aligning to host genomes decreased 50-fold, while bacterial and Plasmodium DNA sequences reads increased 8-11.5-fold. The Shannon-Wiener diversity index was calculated for 149 bacterial species in saliva before and after enrichment. Unenriched saliva had an index of 4.72, while the enriched sample had an index of 4.80. The similarity of these indices demonstrates that bacterial species diversity and relative phylotype abundance remain conserved in enriched samples. Enrichment using the MBD-Fc method holds promise for targeted microbiome sequence analysis across a broad range of sample types.

Effective DNA recovery from bone material is essential for applications in biomedical research, clinical diagnostics, and forensic and archaeogenetic investigations. In this study, DNA isolation performance was evaluated in an exploratory manner in human bone samples representing three preservation states: fresh, cryopreserved, and ancient. All samples were processed using a unified bead mill homogenization and magnetic bead–based extraction workflow in order to maintain procedural consistency. DNA quantity and purity were evaluated by spectrophotometry, and amplifiability was assessed using nuclear and mitochondrial PCR assays as well as representative STR profiling. Fresh and cryopreserved samples yielded higher DNA concentrations and more consistent amplification than ancient specimens, in which recovery was primarily constrained by postmortem degradation. PCR success demonstrated a clear dependence on amplicon length, with shorter mitochondrial and nuclear targets amplifying more reliably across all sample types. Due to the limited sample size and the use of a single individual per preservation group, the results are presented as qualitative observations rather than as statistically generalizable conclusions. Within these constraints, the study demonstrates the feasibility of using a standardized mechanical disruption and extraction workflow across bone samples of differing preservation status and provides a methodological reference for future larger-scale studies involving both modern and degraded skeletal material. •Bead-mill homogenization evaluated across fresh, frozen and ancient bone.•DNA recovery varied primarily with preservation status.•Short amplicons amplified more reliably in degraded samples.•STR profiles illustrated fragment length–dependent allele dropout.•Workflow provides a qualitative reference for future bone DNA studies.

PAXX is a recently identified component of the nonhomologous end joining (NHEJ) DNA repair pathway. The molecular mechanisms of PAXX action remain largely unclear. Here we characterise the interactomes of PAXX and its paralogs, XLF and XRCC4, to show that these factors share the ability to interact with DNA polymerase λ (Pol λ), stimulate its activity and are required for recruitment of Pol λ to laser-induced DNA damage sites. Stimulation of Pol λ activity by XRCC4 paralogs requires a direct interaction between the SP/8 kDa domain of Pol λ and their N-terminal head domains to facilitate recognition of the 5′ end of substrate gaps. Furthermore, PAXX and XLF collaborate with Pol λ to promote joining of incompatible DNA ends and are redundant in supporting Pol λ function in vivo. Our findings identify Pol λ as a novel downstream effector of PAXX function and show XRCC4 paralogs act in synergy to regulate polymerase activity in NHEJ. PAXX functions as part of the nonhomologous end-joining pathway to repair double-strand DNA breaks. Here the authors show PAXX and its paralogs interact with polymerase lambda to promote joining of incompatible ends.

Environmental DNA (eDNA) is increasingly popular as a useful non-invasive method to monitor and study biodiversity and community structure in freshwater and marine environments. To effectively extract eDNA from the filter surface is a fundamental factor determining the representativeness of the samples. We improved the eDNA extraction efficiency of an established Sterivex method by 12- to 16-fold using a larger volume of lysis buffer mix coupled with backflushing the cartridges. The DNeasy extraction column could be overloaded when the environmental sample input is high, possibly due to a higher nonspecific binding present in environmental samples, thus resulting in a relatively lower quantity measured. Therefore, we included an internal control DNA in the extraction to monitor the extraction and purification efficiencies in field samples, which is crucial for quantification of original eDNA concentration. The use of Takara Probe qPCR Mix supplemented with protein-based additives improved the robustness of the real time PCR assay on inhibitor-rich environmental samples, but prior purification by Qiagen PowerClean Pro Cleanup kit could be essential for inhibitor-rich water samples, even though the recovery rate was unexpectedly low (average 33.0%). The improved extraction and quantification complement the qualitative analyses including metabarcoding and metagenomics in field application.

We describe continuing work to develop restriction endonucleases as tools to enrich targeted genomes of interest from diverse populations. Two approaches were developed in parallel to segregate genomic DNA based on cytosine methylation. First, the methyl-sensitive endonuclease HpaII was used to bind non-CG methylated DNA. Second, a truncated fragment of McrB was used to bind CpG methylated DNA. Enrichment levels of microbial genomes can exceed 100-fold with HpaII allowing improved genomic detection and coverage of otherwise trace microbial genomes from sputum. Additionally, we observe interesting enrichment results that correlate with the methylation states not only of bacteria, but of fungi, viruses, a protist and plants. The methods presented here offer promise for testing biological samples for pathogens and global analysis of population methylomes.

Circulating cell-free nucleic acids (NAs), in particular plasma-derived cell-free DNA, have evolved into promising clinical analytes for prenatal diagnostics, cancer analysis, and cancer surveillance and therapy monitoring. Nevertheless, salivary extracellular and extracellular vesicle (EV)-derived DNA and microRNA have recently gained attention as potential non-invasive biomarkers for a variety of diseases, including cancer, cardiovascular, autoimmune, and infectious diseases. Our goal in this study was therefore to evaluate and optimize commercially available approaches for cell-free nucleic acid isolation, focusing specifically on DNA and miRNA present in cell-free saliva or saliva-derived EVs. Along these lines, we investigated various commercially available kits, which enable parallel isolation of cell-free DNA and RNA in separate fractions from cell-free saliva and salivary EVs, respectively, and compared them to single analyte extraction kits. The efficiency of all tested nucleic acid extraction methods was determined by comparing DNA and RNA fluorescence spectroscopy measurements and quantitative PCR values obtained from a selection of different DNA- and microRNA targets. We found the Norgen Plasma/Serum RNA/DNA Purification Mini kit in combination with the miRCURY exosome isolation kit to work best in our hands and to provide the highest yields of EV-derived nucleic acids. Having tested and identified effective protocols for isolating salivary extracellular nucleic acids, we present with this comparison study, among others, a sound basis for future circulating small nucleic acid and epigenetic biomarker research aiming for early disease diagnosis, prognosis, and prediction from cell-free saliva, representing an easy-to-collect and readily available diagnostic fluid.