Steps Of DNA Replication Research Articles

Structures of LIG1 provide a mechanistic basis for understanding a lack of sugar discrimination against a ribonucleotide at the 3'-end of nick DNA

Human DNA ligase 1 (LIG1) is the main replicative ligase that seals Okazaki fragments during nuclear replication and finalizes DNA repair pathways by joining DNA ends of the broken strand breaks in the three-steps of ligation reaction. LIG1 can tolerate the RNA strand at the upstream of the nick, yet an atomic insight into the sugar discrimination mechanism by LIG1 against a ribonucleotide at the 3'-terminus of nick DNA is unknown. Here, we determined X-ray structures of LIG1/3'-RNA-DNA hybrids and captured the ligase during pre- and post-step 3 the ligation reaction. Furthermore, the overlays of 3'-rA:T and 3'-rG:C step 3 structures with step 2 structures of canonical 3'-dA:T and 3'-dG:C uncover a network of LIG1/DNA interactions through Asp570 and Arg871 side chains with 2'-OH of the ribose at nick showing a final phosphodiester bond formation and the other ligase active site residues surrounding the AMP site. Finally, we demonstrated that LIG1 can ligate the nick DNA substrates with preinserted 3'-ribonucleotides as efficient as Watson-Crick base-paired ends in vitro. Together, our findings uncover a novel atomic insight into a lack of sugar discrimination by LIG1 and the impact of improper sugar on the nick sealing of ribonucleotides at the last step of DNA replication and repair.

TICRR serves as a prognostic biomarker in lung adenocarcinoma with implications in RNA epigenetic modification, DDR pathway, and RNA metabolism.

TOPBP1 interacting checkpoint and replication regulator (TICRR), a hub gene of the Cdk2-mediated initiation step of DNA replication, has been shown an essential role in tumorigenesis by accelerating the DNA replication of tumor cells. RT-qPCR was used to detect the mRNA expression of TICRR in LUAD tumors and adjacent normal tissues. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database of LUAD were acquired to analyze the critical role of TICRR expression in survival prognosis and clinicopathology characters in LUAD. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) were performed using the R package. The correlation of TICRR expression with immune cellinfiltration, RNA epigenetic modification, DNA damage repair (DDR) pathway, and cell metabolism of LUAD was further explored to verify significant conclusions. TICRR was significantly upregulated in most cancer types, including LUAD, lung squamous cell carcinoma (LUSC), and others. Cox regression analysis indicated the overexpression of TICRR was associated with poor survival in several cancers. In LUAD, TICRR expression was positively correlated with tumor stage and was increased in smoking, male, and high tumor mutational burden (TMB) patients. Enrichment analysis revealed that TICRR could influence tumor proliferation and prognosis via activating pathways involving cell cycle, DNA repair, DNA replication, cysteine metabolism, oxidative phosphorylation, and ubiquitin-mediated proteolysis pathways. Interestingly, high TICRR expression correlated with DDR pathway signature (34 genes), 37 m6A/m5C regulated genes, and some metabolism-regulated genes. Silencing the TICRR gene affects cysteine metabolism and modifies cancer-related pathways, with decreased cell cycle and increased B/T cell receptor signaling. Our TICRR risk model accurately predicts LUAD patient prognosis, validated across GEO datasets, and is integrated with clinical characteristics via a nomogram, facilitating personalized treatment strategies and enhancing patient management. Taken together, TICRR has emerged as a promising prognostic biomarker in lung adenocarcinoma (LUAD), with implications in immune activation, cell cycle regulation, RNA modification, and tumor energy metabolism. These findings suggest that TICRR could serve as a viable therapeutic target and a reliable prognostic indicator for LUAD.

G1 Dynamics at the Crossroads of Pluripotency and Cancer.

G1 cell cycle phase dynamics are regulated by intricate networks involving cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors, which control G1 progression and ensure proper cell cycle transitions. Moreover, adequate origin licensing in G1 phase, the first committed step of DNA replication in the subsequent S phase, is essential to maintain genome integrity. In this review, we highlight the intriguing parallels and disparities in G1 dynamics between stem cells and cancer cells, focusing on their regulatory mechanisms and functional outcomes. Notably, SOX2, OCT4, KLF4, and the pluripotency reprogramming facilitator c-MYC, known for their role in establishing and maintaining stem cell pluripotency, are also aberrantly expressed in certain cancer cells. In this review, we discuss recent advances in understanding the regulatory role of these pluripotency factors in G1 dynamics in the context of stem cells and cancer cells, which may offer new insights into the interconnections between pluripotency and tumorigenesis.

ATP-Dependent Mismatch Recognition in DNA Replication Mismatch Repair

Mismatch repair is a critical step in DNA replication that occurs after base selection and proofreading, significantly increasing fidelity. However, the mechanism of mismatch recognition has not been established for any repair enzyme. Speculations in this area mainly focus on exploiting thermodynamic equilibrium and free energy. Nevertheless, non-equilibrium processes may play a more significant role in enhancing mismatch recognition accuracy by utilizing adenosine triphosphate (ATP). This study aimed to investigate this possibility. Considering our limited knowledge of actual mismatch repair enzymes, we proposed a hypothetical enzyme that operates as a quantum system with three discrete energy levels. When the enzyme is raised to its highest energy level, a quantum transition occurs, leading to one of two low-energy levels representing potential recognition outcomes: a correct match or a mismatch. The probabilities of the two outcomes are exponentially different, determined by the energy gap between the two low energy levels. By flipping the energy gap, discrimination between mismatches and correct matches can be achieved. Within a framework that combines quantum mechanics with thermodynamics, we established a relationship between energy cost and the recognition error.

RNF4 and USP7 cooperate in ubiquitin-regulated steps of DNA replication.

DNA replication requires precise regulation achieved through post-translational modifications, including ubiquitination and SUMOylation. These modifications are linked by the SUMO-targeted E3 ubiquitin ligases (STUbLs). Ring finger protein 4 (RNF4), one of only two mammalian STUbLs, participates in double-strand break repair and resolving DNA-protein cross-links. However, its role in DNA replication has been poorly understood. Using CRISPR/Cas9 genetic screens, we discovered an unexpected dependency of RNF4 mutants on ubiquitin specific peptidase 7 (USP7) for survival in TP53-null retinal pigment epithelial cells. TP53-/-/RNF4-/-/USP7-/- triple knockout (TKO) cells displayed defects in DNA replication that cause genomic instability. These defects were exacerbated by the proteasome inhibitor bortezomib, which limited the nuclear ubiquitin pool. A shortage of free ubiquitin suppressed the ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint response, leading to increased cell death. In conclusion, RNF4 and USP7 work cooperatively to sustain a functional level of nuclear ubiquitin to maintain the integrity of the genome.

Abstract 1477: TICRR is a prognosis biomarker associated with RNA epigenetic modification, DDR pathway, and RNA metabolism of lung adenocarcinoma

Abstract Background: TOPBP1 interacting checkpoint and replication regulator (TICRR), a hub gene of the Cdk2-mediated initiation step of DNA replication, has been identified as a prognostic biomarker in papillary renal cell carcinoma (PRCC). Nevertheless, there are few studies on the comprehensive analysis between mRNA expression of TICRR and lung adenocarcinoma (LUAD). Methods: The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database of LUAD were acquired to analyze the mRNA expression between tumor and normal tissues, survival prognosis, and clinicopathology characters. The GO, KEGG, and gene set enrichment analysis (GSEA) were performed using the R package. The correlation of TICRR with immune cell infiltration, RNA epigenetic modification, DDR pathway, and RNA metabolism of LUAD were further explored using TCGA and GEO database. Results: TICRR was confirmed significantly upregulated in several cancers, like LUAD, Lung squamous cell carcinoma (LUSC), Uterine Corpus Endometrial Carcinoma (UCEC), except for Prostate adenocarcinoma (PRAD), Thyroid carcinoma (THCA), which was downregulated in tumor tissues. Cox analysis indicated the overexpression of TICRR was related to poor survival in several cancers. The KEGG enrichment showed TICRR expression positively related to cell cycle, homologous recombination, mismatch repair, DNA replication, and cysteine metabolism in LUAD. Pathway enrichment showed a close relationship with spliceosome, oxidative phosphorylation, and ubiquitin-mediated proteolysis. Immune infiltration analysis revealed TICRR expression was negatively correlated with B cell expression, immune score, stromal score, and immunostimulators like IL2 (P<0.001), and positively correlated with neutrophil cell expression, immune inhibitors like PD-L1 and CTLA4. Interestingly, high mRNA expression of TICRR correlates with DDR pathway signature (37 genes), 37 m6A/m5C regulated genes, and some metabolism-regulated genes. Single-cell analysis of six LUAD samples showed that TICRR was significantly enriched in macrophage and T cells. Conclusions: TICRR has been identified to be a prognosis biomarker in LUAD, which is involved in immune activation, pathway transportation, RNA modification, and tumor energy metabolism. TICRR can be used as a promising therapeutic target and prognosis indicator for LUAD. Citation Format: Xunbo Zheng, Jun Guan, Chenteng Chen, Li Han, Hanxing Huang. TICRR is a prognosis biomarker associated with RNA epigenetic modification, DDR pathway, and RNA metabolism of lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1477.

Human DNA polymerase α has a strong mutagenic potential at the initial steps of DNA synthesis.

DNA polymerase α (Polα) is essential for DNA replication initiation and makes a notable contribution to genome mutagenesis. The activity and fidelity of Polα during the early steps of DNA replication have not been well studied. Here we show that at the beginning of DNA synthesis, when extending the RNA primer received from primase, Polα is more mutagenic than during the later DNA elongation steps. Kinetic and binding studies revealed substantially higher activity and affinity to the template:primer when Polα interacts with ribonucleotides of a chimeric RNA-DNA primer. Polα activity greatly varies during first six steps of DNA synthesis, and the bias in the rates of correct and incorrect dNTP incorporation leads to impaired fidelity, especially upon the second step of RNA primer extension. Furthermore, increased activity and stability of Polα/template:primer complexes containing RNA-DNA primers result in higher efficiency of mismatch extension.

Coordinating transcription and replication to mitigate their conflicts in early Drosophila embryos.

Collisions between transcribing RNA polymerases and DNA replication forks are disruptive. The threat of collisions is particularly acute during the rapid early embryonic cell cycles of Drosophila when S phase occupies the entirety of interphase. We hypothesize that collision-avoidance mechanisms safeguard this early transcription. Real-time imaging of endogenously tagged RNA polymerase II (RNAPII) and a reporter for nascent transcripts in unperturbed embryos shows clustering of RNAPII at around 2min after mitotic exit, followed by progressive dispersal as associated nascent transcripts accumulate later in interphase. Abrupt inhibition of various steps in DNA replication, including origin licensing, origin firing, and polymerization, suppresses post-mitotic RNAPII clustering and transcription in nuclear cycles. We propose that replication dependency defers the onset of transcription so that RNAPII transcribes behind advancing replication forks. The resulting orderly progression can explain how early embryos circumvent transcription-replication conflicts to express essential developmental genes.

(Invited) DNA Sequencing with Carbon Nanotube Electronics

Nanoscale materials and devices provide new opportunities to monitor and understand biomolecules. This presentation will review novel devices in which single molecules of DNA polymerase have been joined to single-walled carbon nanotube (SWNT) transistors.DNA polymerases are the enzymes that convert single-stranded DNA into double-stranded helices. This biochemical process is the primary step in DNA replication, amplification, and most sequencing technologies. When one of these polymerases is attached to a SWNT transistor, the enzyme’s native activity is simultaneously transduced into a high-resolution electrical signal [1]. Each time the enzyme works its way down a DNA strand, it generates a nucleotide-by-nucleotide signal that encodes the DNA sequence.This hybrid, biofunctional device is the key element for developing a new, electronic DNA sequencing technology. As a solid-state, manufacturable element, the SWNT transistors provide exciting possibilities for high-density, high-throughput, and low cost sequencing solutions. In addition, the technique is easily generalized to DNA polymerases with specialized properties. Proof-of-principle measurements have been accomplished with DNA polymerases from five different organisms, including the highly processive polymerase of φ29 phage and the high-temperature polymerase Thermus aquaticus (Taq), which is the foundation of the polymerase chain reaction (PCR) and commercial DNA amplification. The SWNT transistor technique operates equally well at room temperature and the elevated PCR temperature of 72 °C, which opens opportunities for novel, high-temperature sequencing strategies with Taq or similar thermostable polymerases.[1] T. J. Olsen et. al., JACS 135, 7855 (2013); O. T. Gul et. al., Biosensors 6, 29 (2016)

Dimerization of Firing Factors for Replication Origin Activation in Eukaryotes: A Crucial Process for Simultaneous Assembly of Bidirectional Replication Forks?

Simple SummaryChromosomal DNA must be faithfully duplicated and segregated into two daughter cells when cells divide. DNA synthesis initiates from specific regions known as the origins of replication. When it starts, a pair of the replication fork is established, and each replication fork moves away from replication origins. In each replication fork, replicative helicase unwinds DNA from a double to a single strand. This implies that two sets of active helicase are generated from each replication origin. To make this possible, two sets of replicative helicases are loaded onto replication origins as inactive dimers first. When S-phase specific cyclin-dependent kinases, S-CDKs, are activated, the inactive helicase is converted into the active helicase with the aid of other factors called firing factors. Although two sets of firing factors seem to be required to activate two sets of helicase, it is largely unknown whether two sets of firing factors function simultaneously to establish bidirectional replication forks in a coordinated way. We introduce our current understanding of firing factor dimerization and discuss its potential contribution to bidirectional replication fork formation in this review.Controlling the activity of the heterohexameric Mcm2–7 replicative helicase is crucial for regulation of replication origin activity in eukaryotes. Because bidirectional replication forks are generated from every replication origin, when origins are licensed for replication in the first step of DNA replication, two inactive Mcm2–7 heterohexiameric complexes are loaded around double stranded DNA as a head-to-head double hexamer. The helicases are subsequently activated via a ‘firing’ reaction, in which the Mcm2–7 double hexamer is converted into two active helicase units, the CMG complex, by firing factors. Dimerization of firing factors may contribute to this process by allowing simultaneous activation of two sets of helicases and thus efficient assembly of bidirectional replication forks. An example of this is dimerization of the firing factor Sld3/Treslin/Ticrr via its binding partner, Sld7/MTBP. In organisms in which no Sld7 ortholog has been identified, such as the fission yeast Schizosaccharomyces pombe, Sld3 itself has a dimerization domain, and it has been suggested that this self-interaction is crucial for the firing reaction in this organism. Dimerization induces a conformational change in Sdl3 that appears to be critical for the firing reaction. Moreover, Mcm10 also seems to be regulated by self-interaction in yeasts. Although it is not yet clear to what extent dimerization of firing factors contributes to the firing reaction in eukaryotes, we discuss the possible roles of firing factor dimerization in simultaneous helicase activation.

High abundance of CDC45 inhibits cell proliferation through elevation of HSPA6.

ObjectivesCDC45 is the core component of CMG (CDC45‐MCMs‐GINS) complex that plays important role in the initial step of DNA replication in eukaryotic cells. The expression level of cdc45 is under the critical control for the accurate cell cycle progression. Loss‐of‐function of cdc45 has been demonstrated to inhibit cell proliferation and leads to cell death due to the inhibition of DNA replication and G1‐phase arrest. An increasing of CDC45 inhibits cell proliferation as well. Nevertheless, a systematic analysis of the effect of high dose of CDC45 on cell physiology and behaviors is unclear. In the present study, we aimed to investigate the effects and mechanisms of high dose of CDC45 on cell behaviors.Materials and MethodsWe overexpressed cdc45 in cultured cell lines, Ciona and Drosophila embryos, respectively. The cell cycle progression was examined by the BrdU incorporation experiment, flow cytometry and PH3 (phospho‐Histone 3) staining. RNA‐sequencing analysis and qRT‐PCR were carried out to screen the affected genes in HeLa cells overexpressing cdc45. siRNA‐mediated knockdown was performed to investigate gene functions in HeLa cells overexpressing cdc45.ResultsWe found that high level of cdc45 from different species (human, mammal, ascidian, and Drosophila) inhibited cell cycle in vitro and in vivo. High dose of CDC45 blocks cells entering into S phase. However, we failed to detect DNA damage and cell apoptosis. We identified hspa6 was the most upregulated gene in HeLa cells overexpressing cdc45 via RNA‐seq analysis and qRT‐PCR validation. Overexpression of Hs‐hspa6 inhibited proliferation rate and DNA replication in HeLa cells, mimicking the phenotype of cdc45 overexpression. RNAi against hspa6 partially rescued the cell proliferation defect caused by high dose of CDC45.ConclusionsOur study suggests that high abundance of CDC45 stops cell cycle. Instead of inducing apoptosis, excessive CDC45 prevents cell entering S phase probably due to promoting hspa6 expression.

Deletion of Orc4 during oogenesis severely reduces polar body extrusion and blocks zygotic DNA replication†.

Origin recognition complex subunit 4 (ORC4) is a DNA-binding protein required for DNA replication. During oocyte maturation, after the last oocyte DNA replication step and before zygotic DNA replication, the oocyte undergoes two meiotic cell divisions in which half the DNA is ejected in much smaller polar bodies. We previously demonstrated that ORC4 forms a cytoplasmic cage around the DNA that is ejected in both polar body extrusion (PBE) events. Here, we used ZP3 activated Cre to delete exon 7 of Orc4 during oogenesis to test how it affected both predicted functions of ORC4: its recently discovered role in PBE and its well-known role in DNA synthesis. Orc4 deletion severely reduced PBE. Almost half of Orc4-depleted germinal vesicle (GV) oocytes cultured in vitro were arrested before anaphase I (48%), and only 25% produced normal first polar bodies. This supports the role of ORC4 in PBE and suggests that transcription of the full-length Orc4 during oogenesis is required for efficient PBE. Orc4 deletion also abolished zygotic DNA synthesis. Fewer Orc4-depleted oocytes developed to the metaphase II (MII) stage, and after activation these oocytes were arrested at the two-cell stage without undergoing DNA synthesis. This confirms that transcription of full-length Orc4 after the primary follicle stage is required for zygotic DNA replication. The data also suggest that MII oocytes do not have a replication licensing checkpoint as cytokinesis progressed without DNA synthesis. Together, the data confirm that oocyte ORC4 is important for both PBE and zygotic DNA synthesis.

Characterization of the Chimeric PriB-SSBc Protein.

PriB is a primosomal protein required for the replication fork restart in bacteria. Although PriB shares structural similarity with SSB, they bind ssDNA differently. SSB consists of an N-terminal ssDNA-binding/oligomerization domain (SSBn) and a flexible C-terminal protein–protein interaction domain (SSBc). Apparently, the largest difference in structure between PriB and SSB is the lack of SSBc in PriB. In this study, we produced the chimeric PriB-SSBc protein in which Klebsiella pneumoniae PriB (KpPriB) was fused with SSBc of K. pneumoniae SSB (KpSSB) to characterize the possible SSBc effects on PriB function. The crystal structure of KpSSB was solved at a resolution of 2.3 Å (PDB entry 7F2N) and revealed a novel 114-GGRQ-117 motif in SSBc that pre-occupies and interacts with the ssDNA-binding sites (Asn14, Lys74, and Gln77) in SSBn. As compared with the ssDNA-binding properties of KpPriB, KpSSB, and PriB-SSBc, we observed that SSBc could significantly enhance the ssDNA-binding affinity of PriB, change the binding behavior, and further stimulate the PriA activity (an initiator protein in the pre-primosomal step of DNA replication), but not the oligomerization state, of PriB. Based on these experimental results, we discuss reasons why the properties of PriB can be retrofitted when fusing with SSBc.

Single-molecule studies of helicases and translocases in prokaryotic genome-maintenance pathways

Helicases involved in genomic maintenance are a class of nucleic-acid dependent ATPases that convert the energy of ATP hydrolysis into physical work to execute irreversible steps in DNA replication, repair, and recombination. Prokaryotic helicases provide simple models to understand broadly conserved molecular mechanisms involved in manipulating nucleic acids during genome maintenance. Our understanding of the catalytic properties, mechanisms of regulation, and roles of prokaryotic helicases in DNA metabolism has been assembled through a combination of genetic, biochemical, and structural methods, further refined by single-molecule approaches. Together, these investigations have constructed a framework for understanding the mechanisms that maintain genomic integrity in cells. This review discusses recent single-molecule insights into molecular mechanisms of prokaryotic helicases and translocases.

Downregulation of MCM8 expression restrains the malignant progression of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly aggressive malignant tumor with an extremely poor prognosis. Minichromosome maintenance 8 homologous recombination repair factor (MCM8) is a helicase involved in the elongation step of DNA replication and tumorigenesis. In the present study, the clinical significance and biological function of MCM8 in CCA were investigated. The expression levels of MCM8 in CCA and paracancerous tissues were analyzed using immunohistochemical staining. The potential mechanisms underlying MCM8 and the biological effects of MCM8 in CCA cells were explored using in vitro assays and in vivo mouse xenograft models. The high expression levels of MCM8 in CCA has important clinical significance in predicting disease progression. Knockdown of MCM8 decreased proliferation, promoted apoptosis and suppressed migration of CCA cells. MCM8 knockdown also suppressed tumor growth in vivo. Mechanistically, MCM8 knockdown led to the abnormal downregulation of survivin, XIAP, HSP27, IGF-1sR, sTNF-R1, sTNF-R2, TNF-α and TNF-β. Furthermore, downregulation of MCM8 expression inhibited the PI3K/Akt signaling pathway and induced the MAPK9 signaling pathway. MCM8 promoted the malignant progression of CCA, indicating that inhibition of MCM8 may have the potential to serve as a novel molecular targeted therapy.

DNA polymerase Gp90 activities and regulations on strand displacement DNA synthesis revealed at single-molecule level.

Strand displacement DNA synthesis (SDDS) is an essential step in DNA replication. With magnetic tweezers, we investigated SDDS kinetics of wild-type gp90 and its exonuclease-deficient polymerase gp90 exo- at single-molecule level. A novel binding state of gp90 to the fork flap was confirmed prior to SDDS, suggesting an intermediate in the initiation of SDDS. The rate and processivity of SDDS by gp90 exo- or wt-gp90 are increased with force and dNTP concentration. The rate and processivity of exonuclease by wt-gp90 are decreased with force. High GC content decreases SDDS and exonuclease processivity but increases exonuclease rate for wt-gp90. The high force and dNTP concentration and low GC content facilitate the successive SDDS but retard the successive exonuclease for wt-gp90. Furthermore, increasing GC content accelerates the transition from SDDS or exonuclease to exonuclease. This work reveals the kinetics of SDDS in detail and offers a broader cognition on the regulation of various factors on SDDS at single-polymerase level.

Identification of the Unwinding Region in the Clostridioides difficile Chromosomal Origin of Replication.

Faithful DNA replication is crucial for viability of cells across all kingdoms. Targeting DNA replication is a viable strategy for inhibition of bacterial pathogens. Clostridioides difficile is an important enteropathogen that causes potentially fatal intestinal inflammation. Knowledge about DNA replication in this organism is limited and no data is available on the very first steps of DNA replication. Here, we use a combination of in silico predictions and in vitro experiments to demonstrate that C. difficile employs a bipartite origin of replication that shows DnaA-dependent melting at oriC2, located in the dnaA-dnaN intergenic region. Analysis of putative origins of replication in different clostridia suggests that the main features of the origin architecture are conserved. This study is the first to characterize aspects of the origin region of C. difficile and contributes to our understanding of the initiation of DNA replication in clostridia.

Distinct and sequential re-replication barriers ensure precise genome duplication.

Achieving complete and precise genome duplication requires that each genomic segment be replicated only once per cell division cycle. Protecting large eukaryotic genomes from re-replication requires an overlapping set of molecular mechanisms that prevent the first DNA replication step, the DNA loading of MCM helicase complexes to license replication origins, after S phase begins. Previous reports have defined many such origin licensing inhibition mechanisms, but the temporal relationships among them are not clear, particularly with respect to preventing re-replication in G2 and M phases. Using a combination of mutagenesis, biochemistry, and single cell analyses in human cells, we define a new mechanism that prevents re-replication through hyperphosphorylation of the essential MCM loading protein, Cdt1. We demonstrate that Cyclin A/CDK1 can hyperphosphorylate Cdt1 to inhibit MCM re-loading in G2 phase. The mechanism of inhibition is to block Cdt1 binding to MCM independently of other known Cdt1 inactivation mechanisms such as Cdt1 degradation during S phase or Geminin binding. Moreover, our findings suggest that Cdt1 dephosphorylation at the mitosis-to-G1 phase transition re-activates Cdt1. We propose that multiple distinct, non-redundant licensing inhibition mechanisms act in a series of sequential relays through each cell cycle phase to ensure precise genome duplication.

(Invited) Single-Molecule Electronics for DNA Sequencing

Nanoscale materials provide new opportunities to interface solid-state electronics with biomolecules and biochemical activity. For example, single-walled carbon nanotubes (SWNTs) have the special property of electronic resistance that is sensitive to single electrons. We have exploited this sensitivity to build nanoelectronic biosensors that monitor the biochemical activity of individual proteins [1]. As an attached protein moves, binds, or performs catalysis, its charged amino acid sidechains induce resistance fluctuations in the SWNT device that may be monitored with microsecond resolution [2].Recently, we have used this measurement platform for single-molecule measurements of DNA polymerases [3]. Polymerases are the key enzymes for converting single-stranded DNA to double-stranded helices, the primary step in DNA replication, amplification, and most sequencing technologies. When a polymerase processing DNA is also attached to a SWNT device, the electrical signal provides a high-resolution readout of single-nucleotide incorporations and exciting possibilities for high-density, high-throughput electronic DNA sequencing.To investigate the feasibility of electronic DNA sequencing, we have compared single-molecule transduction by DNA polymerases from three different organisms. By working with multiple families of DNA polymerases, we have tested the applicability of the electronic technique while also generating detailed records of differences among the enzymes. For example, we observe an anomalous rate variability when measuring the polymerase from the bacillus phage φ29. Base incorporation rates average 20 s-1 for most the enzymes processing single-stranded DNA templates, but rates up to 200 and 400 s-1 occurred when φ29 encountered homopolymeric sequences of poly(dT) or poly(dC), respectively. When processing poly(dA) and poly(dG) sequences, on the other hand, φ29 had bursts of activity interrupted by pauses lasting 50 to 300 s. This sequence-dependent activity illustrates one way that single-molecule methods reveal information hidden in ensemble-based techniques.Another workhorse protein in DNA sequencing technologies is the DNA polymerase derived from the thermophilic bacteria Thermus aquaticus (Taq). Anomalous stability at high temperatures makes Taq a unique enzyme for the polymerase chain reaction (PCR) and commercial amplification of DNA. In a first for single-molecule biophysics, SWNT devices have recorded Taq activity over a wide temperature range from 22 to 94 °C, including the typical PCR operating temperature of 72 °C. Even operating at this high temperature, the technique resolved Taq testing incoming nucleotides for complementarity and incorporating correct matches in the base-by-base construction of Watson-Crick pairs. The detailed recordings reveal the similarities of Taq’s operation to other, room temperature polymerases.[1] Y. Choi, et. al., Science 335, 319 (2012). [2] M. V. Akhterov, et. al., ACS Chem. Biol. 10, 1495 (2015). [3] T. J. Olsen et. al., JACS 135, 7855 (2013); O. T. Gul et. al., Biosensors 6, 29 (2016)

Human RPA activates BLM's bidirectional DNA unwinding from a nick.

BLM is a multifunctional helicase that plays critical roles in maintaining genome stability. It processes distinct DNA substrates, but not nicked DNA, during many steps in DNA replication and repair. However, how BLM prepares itself for diverse functions remains elusive. Here, using a combined single-molecule approach, we find that a high abundance of BLMs can indeed unidirectionally unwind dsDNA from a nick when an external destabilizing force is applied. Strikingly, human replication protein A (hRPA) not only ensures that limited quantities of BLMs processively unwind nicked dsDNA under a reduced force but also permits the translocation of BLMs on both intact and nicked ssDNAs, resulting in a bidirectional unwinding mode. This activation necessitates BLM targeting on the nick and the presence of free hRPAs in solution whereas direct interactions between them are dispensable. Our findings present novel DNA unwinding activities of BLM that potentially facilitate its function switching in DNA repair.