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Results for “DNA REPLICATION”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

TORC1 signaling regulates DNA replication via DNA replication protein levels

Highlights: • TORC1 protein kinase reduced the levels of proteins critical for DNA replication initiation and retarded DNA replication. • TORC1 inactivation promoted Mcm3 degradation in a manner dependent on Skp1–Cullin–F-box (SCF)-Grr1 and proteasome. • TORC1-downstream factors PP2A-Cdc55 protein phosphatase and protein kinase A regulated Mcm3 degradation. Accurate DNA replication is at the heart of faithful genome transmission in dividing cells. DNA replication is strictly controlled by various factors. However, how environmental stresses such as nutrient starvation impact on these factors and DNA replication is largely unknown. Here we show that DNA replication is regulated by target of rapamycin complex 1 (TORC1) protein kinase, which is a central regulator of cell growth and proliferation in response to nutrients. TORC1 inactivation reduced the levels of various proteins critical for DNA replication initiation, such as Mcm3, Orc3, Cdt1, and Sld2, and retarded DNA replication. TORC1 inactivation promoted proteasome-mediated Mcm3 degradation. Skp1–Cullin–F-box (SCF)-Grr1 and PEST motif mediated Mcm3 degradation. TORC1-downstream factors PP2A-Cdc55 protein phosphatase and protein kinase A regulated Mcm3 degradation. This study showed that TORC1 signaling modulates DNA replication to coordinate cell growth and genome replication in response to nutrient availability.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

High-fidelity DNA ligation enforces accurate Okazaki fragment maturation during DNA replication

DNA ligase 1 (LIG1, Cdc9 in yeast) finalizes eukaryotic nuclear DNA replication by sealing Okazaki fragments using DNA end-joining reactions that strongly discriminate against incorrectly paired DNA substrates. Whether intrinsic ligation fidelity contributes to the accuracy of replication of the nuclear genome is unknown. Here, we show that an engineered low-fidelity LIG1 Cdc9 variant confers a novel mutator phenotype in yeast typified by the accumulation of single base insertion mutations in homonucleotide runs. The rate at which these additions are generated increases upon concomitant inactivation of DNA mismatch repair, or by inactivation of the Fen1 Rad27 Okazaki fragment maturation (OFM) nuclease. Biochemical and structural data establish that LIG1 Cdc9 normally avoids erroneous ligation of DNA polymerase slippage products, and this protection is compromised by mutation of a LIG1 Cdc9 high-fidelity metal binding site. Collectively, our data indicate that high-fidelity DNA ligation is required to prevent insertion mutations, and that this may be particularly critical following strand displacement synthesis during the completion of OFM.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Asymmetric loading of TnsE regulates Tn7 targeting of DNA replication structures

Abstract Tn7 transposable elements are known for their sophisticated target-site selection mechanisms. For the prototypical Tn7 element, dedicated transposon-encoded proteins direct insertions to either a conserved site in the chromosome or replicating DNA structures in conjugal plasmids, ensuring the vertical and horizontal spread of the element. While the pathway targeting the attTn7 site in the bacterial chromosome has been extensively studied, the pathway targeting DNA replication structures remains poorly understood. We have used an integrative structural biology approach to elucidate how the Tn7-encoded protein TnsE recognizes replication sites. Using native mass spectrometry, we found that TnsE forms 1:1 and 2:1 (TnsE:DNA) complexes on 3′-recessed DNA, with gain-of-function TnsE variants favoring the formation of 2:1 complexes. Structural characterization confirms that two TnsE molecules bind to DNA with the C-terminal domain of the protein recognizing duplex DNA, leaving the N-terminal domain to impose DNA substrate specificity and recruit the core transposition machinery. Collectively, our work is consistent with a model where TnsE-mediated target-site selection relies on the formation of an asymmetric TnsE:DNA complex to recruit the Tn7 transposase to DNA replication structures.

Biochemistry & Molecular Biology↗
DOE OSTI2015

Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication

To initiate DNA replication, cells first load an MCM helicase double hexamer at origins in a reaction requiring ORC, Cdc6, and Cdt1, also called pre-replicative complex (pre-RC) assembly. The essential mechanistic role of Cdc6 ATP hydrolysis in this reaction is still incompletely understood. Here, we show that although Cdc6 ATP hydrolysis is essential to initiate DNA replication, it is not essential for MCM loading. Using purified proteins, an ATPase-defective Cdc6 mutant ‘Cdc6-E224Q’ promoted MCM loading on DNA. Cdc6-E224Q also promoted MCM binding at origins in vivo but cells remained blocked in G1-phase. If after loading MCM, Cdc6-E224Q was degraded, cells entered an apparently normal S-phase and replicated DNA, a phenotype seen with two additional Cdc6 ATPase-defective mutants. Cdc6 ATP hydrolysis is therefore required for Cdc6 disengagement from the pre-RC after helicase loading to advance subsequent steps in helicase activation in vivo.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Analysis of JC virus DNA replication using a quantitative and high-throughput assay

Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Identification of genetic interactions with priB links the PriA/PriB DNA replication restart pathway to double-strand DNA break repair in Escherichia coli

Collisions between DNA replication complexes (replisomes) and impediments such as damaged DNA or proteins tightly bound to the chromosome lead to premature dissociation of replisomes at least once per cell cycle in Escherichia coli. Left unrepaired, these events produce incompletely replicated chromosomes that cannot be properly partitioned into daughter cells. DNA replication restart, the process that reloads replisomes at prematurely terminated sites, is therefore essential in E. coli and other bacteria. Three replication restart pathways have been identified in E. coli: PriA/PriB, PriA/PriC, and PriC/Rep. A limited number of genetic interactions between replication restart and other genome maintenance pathways have been defined, but a systematic study placing replication restart reactions in a broader cellular context has not been performed. We have utilized transposon-insertion sequencing to identify new genetic interactions between DNA replication restart pathways and other cellular systems. Known genetic interactors with the priB replication restart gene (uniquely involved in the PriA/PriB pathway) were confirmed and several novel priB interactions were discovered. Targeted genetic and imaging-based experiments with priB and its genetic partners revealed significant double-strand DNA break accumulation in strains with mutations in dam, rep, rdgC, lexA, or polA. Modulating the activity of the RecA recombinase partially suppressed the detrimental effects of rdgC or lexA mutations in ΔpriB cells. Taken together, our results highlight roles for several genes in double-strand DNA break homeostasis and define a genetic network that facilitates DNA repair/processing upstream of PriA/PriB-mediated DNA replication restart in E. coli.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

5-Formylcytosine mediated DNA–protein cross-links block DNA replication and induce mutations in human cells

5-Formylcytosine (5fC) is an epigenetic DNA modification introduced via TET protein-mediated oxidation of 5-methyl-dC. We recently reported that 5fC form reversible DNA–protein conjugates (DPCs) with histone proteins in living cells (Ji et al. (2017) Angew. Chem. Int. Ed., 56:14130–14134). We now examined the effects of 5fC mediated DPCs on DNA replication. Synthetic DNA duplexes containing site-specific DPCs between 5fC and lysine-containing proteins and peptides were subjected to primer extension experiments in the presence of human translesion synthesis DNA polymerases η and κ. We found that DPCs containing histones H2A or H4 completely inhibited DNA replication, but the replication block was removed when the proteins were subjected to proteolytic digestion. Cross-links to 11-mer or 31-mer peptides were bypassed by both polymerases in an error-prone manner, inducing targeted C→T transitions and –1 deletions. Similar types of mutations were observed when plasmids containing 5fC-peptide cross-links were replicated in human embryonic kidney (HEK) 293T cells. Molecular simulations of the 11-mer peptide-dC cross-links bound to human polymerases η and κ revealed that the peptide fits well on the DNA major groove side, and the modified dC forms a stable mismatch with incoming dATP via wobble base pairing in the polymerase active site.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Pot1 promotes telomere DNA replication via the Stn1-Ten1 complex in fission yeast

Abstract Telomeres are nucleoprotein complexes that protect the chromosome-ends from eliciting DNA repair while ensuring their complete duplication. Pot1 is a subunit of telomere capping complex that binds to the G-rich overhang and inhibits the activation of DNA damage checkpoints. In this study, we explore new functions of fission yeast Pot1 by using a pot1-1 temperature sensitive mutant. We show that pot1 inactivation impairs telomere DNA replication resulting in the accumulation of ssDNA leading to the complete loss of telomeric DNA. Recruitment of Stn1 to telomeres, an auxiliary factor of DNA lagging strand synthesis, is reduced in pot1-1 mutants and overexpression of Stn1 rescues loss of telomeres and cell viability at restrictive temperature. We propose that Pot1 plays a crucial function in telomere DNA replication by recruiting Stn1-Ten1 and Polα-primase complex to telomeres via Tpz1, thus promoting lagging-strand DNA synthesis at stalled replication forks.

Carvalho Borges, Pâmela C. (ORCID:0000000244919874↗
DOE OSTI2019

Protection of abasic sites during DNA replication by a stable thiazolidine protein-DNA cross-link

Abasic (AP) sites are one of the most common DNA lesions that block replicative polymerases. 5-hydroxymethylcytosine binding, embryonic stem cell-specific protein (HMCES) recognizes and processes these lesions in the context of single-stranded DNA (ssDNA). A HMCES DNA-protein cross-link (DPC) intermediate is thought to shield the AP site from endonucleases and error-prone polymerases. The highly evolutionarily conserved SOS-response associated peptidase (SRAP) domain of HMCES and its Escherichia coli ortholog YedK mediate lesion recognition. Here we uncover the basis of AP site protection by SRAP domains from a crystal structure of the YedK DPC. We report YedK forms a stable thiazolidine linkage between a ring-opened AP site and the α-amino and sulfhydryl substituents of its amino-terminal cysteine residue. The thiazolidine linkage explains the remarkable stability of the HMCES DPC, its resistance to strand cleavage and the proteolysis requirement for resolution. Furthermore, its structure reveals that HMCES has specificity for AP sites in ssDNA at junctions found when replicative polymerases encounter the AP lesion.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

A chimeric protein composed of NuMA fused to the DNA binding domain of LANA is sufficient for the ori-P-dependent DNA replication

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome is stably maintained in KSHV-infected PEL cell lines during cell division. We previously showed that accumulation of LANA in the nuclear matrix fraction could be important for the latent DNA replication, and that the functional significance of LANA should be its recruitment of ori-P to the nuclear matrix. Here, we investigated whether the forced localization of the LANA-DNA binding domain (DBD) to the nuclear matrix facilitated ori-P-containing plasmid replication. We demonstrated that chimeric proteins constructed by fusion of LANA DBD with the nuclear mitotic apparatus protein (NuMA), which is one of the components of the nuclear matrix, could bind with ori-P and enhance replication of an ori-P-containing plasmid, compared with that in the presence of DBD alone. These results further suggested that the ori-P recruitment to the nuclear matrix through the binding with DBD is important for latent viral DNA replication. - Highlights: •KSHV replication in latency depends on LANA localization to the nuclear matrix. •LANA DBD was fused with NuMA, a nuclear matrix protein, at the N- and C-terminus. •NuMA-DBD was in the nuclear matrix and supported the ori-P dependent replication. •LANA in the nuclear matrix should be important for the KSHV replication in latency.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Coupling between DNA replication, segregation, and the onset of constriction in Escherichia coli

Escherichia coli cell cycle features two critical cell-cycle checkpoints: initiation of replication and the onset of constriction. While the initiation of DNA replication has been extensively studied, it is less clear what triggers the onset of constriction and when exactly it occurs during the cell cycle. Here, using high-throughput fluorescence microscopy in microfluidic devices, we determine the timing for the onset of constriction relative to the replication cycle in different growth rates. Our single-cell data and modeling indicate that the initiation of constriction is coupled to replication-related processes in slow growth conditions. Furthermore, our data suggest that this coupling involves the mid-cell chromosome blocking the onset of constriction via some form of nucleoid occlusion occurring independently of SlmA and the Ter linkage proteins. This work highlights the coupling between replication and division cycles and brings up a new nucleoid mediated control mechanism in E. coli.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Role of Chromatin assembly factor 1 in DNA replication of Plasmodium falciparum

Highlights: • PfCAF1 interacts with several proteins including PfCAF2, Histones and PfHP1. • PfCAF1 and PfHP1 share similar binding sites in the genome of P. falciparum during ring and schizont stages. • PfCAF1 co-localizes with newly synthesized histone mark H3K56Ac. • PfCAF1 expression upregulates after DNA damage along with PCNA. Nucleosome assembly in P. falciparum could be the key process in maintaining its genomic integrity as DNA replicates more than once per cell cycle during several stages of its life cycle. Here, we report the functional characterization of P. falciparum chromatin assembly factor 1 (CAF1), which interacts with several proteins namely PfCAF2, Histones, PfHP1 and others. Consistent with the above findings, we demonstrate the presence of PfCAF1 at the telomeric repeat regions, central and subtelomeric var genes of multiple var gene family along with PfHP1. Further, we report the upregulation of PfCAF1 after treatment with genotoxic agents like MMS and HU. Together, these findings establish role of PfCAF1 in heterochromatin maintenance and as histone chaperone in nucleosome assembly and DNA damage repair.

60 APPLIED LIFE SCIENCES↗