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Molecular data storage is an attractive alternative for dense and durable information storage, which is sorely needed to deal with the growing gap between information production and the ability to store data. DNA is a clear example of effective archival data storage in molecular form. In this Review, we provide an overview of the process, the state of the art in this area and challenges for mainstream adoption. We also survey the field of in vivo molecular memory systems that record and store information within the DNA of living cells, which, together with in vitro DNA data storage, lie at the growing intersection of computer systems and biotechnology.Throughout evolution, DNA has been the primary medium of biological information storage. In this article, Ceze, Nivala and Strauss discuss how DNA can be adopted as a storage medium for custom data, as a potential future complement to current data storage media such as computer hard disks, optical disks and tape. They discuss strategies for coding, decoding and error correction and give examples of implementation both in vitro and in vivo.

Poly(ADP-ribose) polymerase 1 (PARP1) is implicated in the detection and processing of unligated Okazaki fragments and other DNA replication intermediates, highlighting such structures as potential sources of genome breakage induced by PARP inhibition. Here, we show that PARP1 activity is greatly elevated in chicken and human S phase cells in which FEN1 nuclease is genetically deleted and is highest behind DNA replication forks. PARP inhibitor reduces the integrity of nascent DNA strands in both wild-type chicken and human cells during DNA replication, and does so in FEN1 − / − cells to an even greater extent that can be detected as postreplicative single-strand nicks or gaps. Collectively, these data show that PARP inhibitors impede the maturation of nascent DNA strands during DNA replication, and implicate unligated Okazaki fragments and other nascent strand discontinuities in the cytotoxicity of these compounds. Biochemical and cell-based assays reveal that PARP inhibitors impede the maturation of nascent DNA strands during DNA replication, and implicate unligated Okazaki fragments and other nascent strand discontinuities in the cytotoxicity of these anti-cancer compounds.

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.

Loop-mediated isothermal amplification (LAMP) is increasingly used in molecular diagnostics as an alternative to PCR based methods. There are numerous reported techniques to detect the LAMP amplification including turbidity, bioluminescence and intercalating fluorescent dyes. In this report we show that quenched fluorescent labels on various LAMP primers can be used to quantify and detect target DNA molecules down to single copy numbers. By selecting different fluorophores, this method can be simply multiplexed. Moreover this highly specific LAMP detection technique can reduce the incidence of false positives originating from mispriming events. Attribution of these events to particular primers will help inform and improve LAMP primer design.

The intricate orchestration of enzymatic activities involving nicotinamide adenine dinucleotide (NAD + ) is essential for maintaining metabolic homeostasis and preserving genomic integrity. As a co-enzyme, NAD + plays a key role in regulating metabolic pathways, such as glycolysis and Kreb’s cycle. ADP-ribosyltransferases (PARPs) and sirtuins rely on NAD + to mediate post-translational modifications of target proteins. The activation of PARP1 in response to DNA breaks leads to rapid depletion of cellular NAD + compromising cell viability. Therefore, the levels of NAD + must be tightly regulated. Here we show that exogenous NAD + , but not its precursors, has a direct effect on mitochondrial activity. Short-term incubation with NAD + boosts Kreb’s cycle and the electron transport chain and enhances pyrimidine biosynthesis. Extended incubation with NAD + results in depletion of pyrimidines, accumulation of purines, activation of the replication stress response and cell cycle arrest. Moreover, a combination of NAD + and 5-fluorouridine selectively kills cancer cells that rely on de novo pyrimidine synthesis. We propose an integrated model of how NAD + regulates nucleotide metabolism, with relevance to healthspan, ageing and cancer therapy. Munk et al. show that exogenous NAD + , but not its precursors, induces metabolic changes in mitochondria affecting nucleotide metabolism with impacts on genomic DNA synthesis and genome integrity.

DNA double-strand breaks (DSBs) are deleterious lesions that challenge genome integrity. To mitigate this threat, human cells rely on the activity of multiple DNA repair machineries that are tightly regulated throughout the cell cycle 1 . In interphase, DSBs are mainly repaired by non-homologous end joining and homologous recombination 2 . However, these pathways are completely inhibited in mitosis 3 – 5 , leaving the fate of mitotic DSBs unknown. Here we show that DNA polymerase theta 6 (Polθ) repairs mitotic DSBs and thereby maintains genome integrity. In contrast to other DSB repair factors, Polθ function is activated in mitosis upon phosphorylation by Polo-like kinase 1 (PLK1). Phosphorylated Polθ is recruited by a direct interaction with the BRCA1 C-terminal domains of TOPBP1 to mitotic DSBs, where it mediates joining of broken DNA ends. Loss of Polθ leads to defective repair of mitotic DSBs, resulting in a loss of genome integrity. This is further exacerbated in cells that are deficient in homologous recombination, where loss of mitotic DSB repair by Polθ results in cell death. Our results identify mitotic DSB repair as the underlying cause of synthetic lethality between Polθ and homologous recombination. Together, our findings reveal the critical importance of mitotic DSB repair in the maintenance of genome integrity. In mitosis, genome integrity is maintained by DNA polymerase theta-dependent repair of DNA double-strand breaks, which is regulated by Polo-like kinase 1 activity.

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45–MCM–GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo . Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication. It has long been a goal to reconstitute eukaryotic DNA replication; here a purified in vitro system from budding yeast containing 16 factors, themselves composed of 42 polypeptides, fulfils the staged process of origin-dependent initiation, including its regulation by kinases. An origin-al in vitro system for DNA replication It has been a long-desired goal to be able to reconstitute a eukaryotic system of DNA replication from its earliest stages of origin firing using purified proteins. However, the greater complexity of eukaryotes compared to bacterial and phage systems has hampered this development. But now John Diffley and colleagues have successfully reconstituted the initial events of budding yeast DNA replication in vitro . The purified system contains 42 proteins, comprising 16 complexes, and fulfills the staged process of origin-dependent initiation, including its regulation by kinases.

The mammalian DNA polymerase-α–primase (Polα–primase) complex is essential for DNA metabolism, providing the de novo RNA–DNA primer for several DNA replication pathways 1 – 4 such as lagging-strand synthesis and telomere C-strand fill-in. The physical mechanism underlying how Polα–primase, alone or in partnership with accessory proteins, performs its complicated multistep primer synthesis function is unknown. Here we show that CST, a single-stranded DNA-binding accessory protein complex for Polα–primase, physically organizes the enzyme for efficient primer synthesis. Cryogenic electron microscopy structures of the CST-Polα–primase preinitiation complex (PIC) bound to various types of telomere overhang reveal that template-bound CST partitions the DNA and RNA catalytic centres of Polα–primase into two separate domains and effectively arranges them in RNA–DNA synthesis order. The architecture of the PIC provides a single solution for the multiple structural requirements for the synthesis of RNA–DNA primers by Polα–primase. Several insights into the template-binding specificity of CST, template requirement for assembly of the CST-Polα–primase PIC and activation are also revealed in this study. A structural analysis demonstrates how the single-stranded DNA-binding accessory protein complex CST physically organizes the human DNA polymerase-α–primase complex for efficient primer synthesis during telomere replication.