Eukaryotic DNA Helicase Research Articles

P326 DNA replication initiator proteins stabilize the kinetochore in the human fungal pathogen Cryptococcus neoformans

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesDNA replication licensing ensures maintenance of the ploidy state by limiting DNA replication once per cell cycle. The mini-chromosome maintenance (MCM) complex is an evolutionarily conserved DNA helicase in eukaryotes that aids this process. Upon activation in the S phase by several protein kinases, the MCM complex unwinds the double-stranded DNA and moves away from DNA replication origins preventing re-initiation of replication in the remainder of the cell cycle. Deregulation of MCM function can lead to malignant transformation of proliferating cells as indicated by their upregulated expression in human cancer and pre-cancerous cells. MCMs, therefore, serve as important biomarkers and potential targets for anti-cancer drugs. This study aims to decipher the effect of deregulated expression of the MCM complex on the cell cycle events of the human fungal pathogen, Cryptococcus neoformans, which is more similar to metazoans than other budding yeast. Aneuploidy-mediated drug resistance is a common mechanism in many major human fungal pathogens including Cryptococcus and Candida.MethodsWe generated conditional mutants of individual subunits of the MCM complex in C. neoformans and assayed the impact of their altered expression on cell cycle events. We also tested the alternative role of MCM subunits in the assembly of the kinetochore, a vital component of the chromosome segregation machinery.ResultsOur screen with MCM conditional mutants identified two in vivo functional subcomplexes that comprise the MCM complex in C. neoformans. Although upregulated expression of either Mcm2 or Mcm3 does not affect cell cycle progression, overexpression of either Mcm6 or Mcm7 led to the accumulation of cells in the large bud stage with nuclear segregation defects. This work provides evidence for the first time for a mitotic role of pre-replication complex proteins, MCM 2-7 complex in Cryptococcus. Depletion of Mcm2 led to arrest of cells in the S and G2/M stages of the cell cycle with defects in nuclear segregation. Localization and expression of several kinetochore proteins upon depletion of Mcm2 established that Mcm2 is vital for kinetochore assembly/integrity. Although the centromeric histone H3 variant, CENP-A, remains largely clustered, the outer kinetochore did not mature indicating that Mcm2 alone or as part of the MCM complex plays a role in kinetochore assembly/integrity and thereby in chromosome segregation.ConclusionA conserved eukaryotic DNA helicase, MCM 2-7 complex, has an unexplored non-canonical role in kinetochore assembly/integrity and chromosome segregation.

G4-Interacting DNA Helicases and Polymerases: Potential Therapeutic Targets.

Guanine-rich DNA can fold into highly stable four-stranded DNA structures called G-quadruplexes (G4). In recent years, the G-quadruplex field has blossomed as new evidence strongly suggests that such alternately folded DNA structures are likely to exist in vivo. G4 DNA presents obstacles for the replication machinery, and both eukaryotic DNA helicases and polymerases have evolved to resolve and copy G4 DNA in vivo. In addition, G4-forming sequences are prevalent in gene promoters, suggesting that G4-resolving helicases act to modulate transcription. We have searched the PubMed database to compile an up-to-date and comprehensive assessment of the field's current knowledge to provide an overview of the molecular interactions of Gquadruplexes with DNA helicases and polymerases implicated in their resolution. Novel computational tools and alternative strategies have emerged to detect G4-forming sequences and assess their biological consequences. Specialized DNA helicases and polymerases catalytically act upon G4-forming sequences to maintain normal replication and genomic stability as well as appropriate gene regulation and cellular homeostasis. G4 helicases also resolve telomeric repeats to maintain chromosomal DNA ends. Bypass of many G4-forming sequences is achieved by the action of translesion DNS polymerases or the PrimPol DNA polymerase. While the collective work has supported a role of G4 in nuclear DNA metabolism, an emerging field centers on G4 abundance in the mitochondrial genome. Discovery of small molecules that specifically bind and modulate DNA helicases and polymerases or interact with the G4 DNA structure itself may be useful for the development of anticancer regimes.

Abstract 3684: Proteome-wide triple negative breast cancer mutant p53 association index identifies chromatin unwinding for precision therapeutics

Abstract Over 80% of TNBCs express mutant p53 (mtp53) proteins. We coupled cell fractionation with stable isotope labeling with amino acids in cell culture (SILAC) and inducible knockdown of endogenous mtp53 to determine the mtp53 driven proteome in the cytoplasm and chromatin of triple negative breast cancer (TNBC) cells. Using SILAC coupled to tandem mass spectrometry (LC-MS/MS) we identified that R273H mtp53 expression in MDA-MB-468 breast cancer cells both up and down-regulated multiple proteins and metabolic pathways. We sequenced 73,154 peptide pairs that corresponded to 3010 proteins detected under reciprocal labeling conditions in the cytoplasm and 48,825 peptide pairs that corresponded to 5195 proteins in the chromatin fraction. Pathway enrichment analysis ranked the DNA unwinding pathway as the highest chromatin associated pathway regulated by mtp53. Moreover, to summarize and quantify the degree of under- or over-expression of a protein from two reciprocal experiments, a mutant p53 association index (mPAI) was defined as the log (of base 2) ratio of two readings from the two reciprocal experiments. Values of mPAI were normally distributed with a mean close to zero, consistent with the fact that levels of the majority of proteins were not affected by mtp53 knockdown. Standard deviation of mPAI is close to one and mtp53 itself shows mPAI values of greater than 2.0 (z-score > 2.0) in both cytosol and chromatin fractions, consistent with the expectation that its levels are significantly reduced by knockdown experiments. We thereby identified proteins and pathways significantly affected by mtp53 knockdowns as those with mPAI > 1.0 (indicating positive association) or mPAI < -1.0 (indicating negative association). A pathway that was positively associated in both the cytoplasm and the chromatin was the DNA unwinding pathway, which was represented by associated changes for all the minichromosome maintenance protein complex proteins 2 through 7 (MCM2-7). This hexomeric protein ring structure complex is the eukaryotic DNA helicase complex required for DNA replication and elongation of the replication fork. We validated that the MCM4 protein level is positively associated with mtp53 R273H in the TNBC cell line MDA-MB-468 and in the colon adenocarcinoma cell line HT-29. This suggests that targeting inhibition of the MCM hexomeric complex may be a good precision medicine approach for TNBCs. Further studies are in progress to determine the efficacy of targeting inhibition of MCMs 2-7 as a mechanism to block TNBC. Citation Format: Wei-Gang Qiu, Alla Polotskaia, Gu Xiao, Lia Di, John Philip, Ronald C. Hendrikson, Jill Bargonetti. Proteome-wide triple negative breast cancer mutant p53 association index identifies chromatin unwinding for precision therapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3684.

Lack of DNA helicase Pif1 disrupts zinc and iron homoeostasis in yeast

The Saccharomyces cerevisiae gene PIF1 encodes a conserved eukaryotic DNA helicase required for both mitochondrial and nuclear DNA integrity. Our previous work revealed that a pif1Δ strain is tolerant to zinc overload. In the present study we demonstrate that this effect is independent of the Pif1 helicase activity and is only observed when the protein is absent from the mitochondria. pif1Δ cells accumulate abnormal amounts of mitochondrial zinc and iron. Transcriptional profiling reveals that pif1Δ cells under standard growth conditions overexpress aconitase-related genes. When exposed to zinc, pif1Δ cells show lower induction of genes encoding iron (siderophores) transporters and higher expression of genes related to oxidative stress responses than wild-type cells. Coincidently, pif1Δ mutants are less prone to zinc-induced oxidative stress and display a higher reduced/oxidized glutathione ratio. Strikingly, although pif1Δ cells contain normal amounts of the Aco1 (yeast aconitase) protein, they completely lack aconitase activity. Loss of Aco1 activity is also observed when the cell expresses a non-mitochondrially targeted form of Pif1. We postulate that lack of Pif1 forces aconitase to play its DNA protective role as a nucleoid protein and that this triggers a domino effect on iron homoeostasis resulting in increased zinc tolerance.

Does a helicase activity help mismatch repair in eukaryotes?

Mismatch repair (MMR) corrects innate DNA replication infidelities. Many components of eukaryotic MMR have been identified, the molecular mechanism of MMR has been largely demonstrated, and furthermore the nick-directed MMR reactions have been reconstituted with purified human proteins in vitro. However, some fundamental questions still remain to be answered. One such question is whether a DNA helicase activity is required for MMR in eukaryotes. This short review presents an overview of the interactions between eukaryotic DNA helicases and MMR factors, and suggests a possible mechanism for how DNA helicases may be involved in repair of DNA mismatches.

Hitting the bull's eye: Novel directed cancer therapy through helicase‐targeted synthetic lethality

Designing strategies for anti-cancer therapy have posed a significant challenge. One approach has been to inhibit specific DNA repair proteins and their respective pathways to enhance chemotherapy and radiation therapy used to treat cancer patients. Synthetic lethality represents an approach that exploits pre-existing DNA repair deficiencies in certain tumors to develop inhibitors of DNA repair pathways that compensate for the tumor-associated repair deficiency. Since helicases play critical roles in the DNA damage response and DNA repair, particularly in actively dividing and replicating cells, it is proposed that the identification and characterization of synthetic lethal relationships of DNA helicases will be of value in developing improved anti-cancer treatment strategies. In this review, we discuss this hypothesis and current evidence for synthetic lethal interactions of eukaryotic DNA helicases in model systems.

Prokaryotic and eukaryotic DNA helicases. Essential molecular motor proteins for cellular machinery.

DNA helicases are ubiquitous molecular motor proteins which harness the chemical free energy of ATP hydrolysis to catalyze the unwinding of energetically stable duplex DNA, and thus play important roles in nearly all aspects of nucleic acid metabolism, including replication, repair, recombination, and transcription. They break the hydrogen bonds between the duplex helix and move unidirectionally along the bound strand. All helicases are also translocases and DNA‐dependent ATPases. Most contain conserved helicase motifs that act as an engine to power DNA unwinding. All DNA helicases share some common properties, including nucleic acid binding, NTP binding and hydrolysis, and unwinding of duplex DNA in the 3′ to 5′ or 5′ to 3′ direction. The minichromosome maintenance (Mcm) protein complex (Mcm4/6/7) provides a DNA‐unwinding function at the origin of replication in all eukaryotes and may act as a licensing factor for DNA replication. The RecQ family of helicases is highly conserved from bacteria to humans and is required for the maintenance of genome integrity. They have also been implicated in a variety of human genetic disorders. Since the discovery of the first DNA helicase in Escherichia coli in 1976, and the first eukaryotic one in the lily in 1978, a large number of these enzymes have been isolated from both prokaryotic and eukaryotic systems, and the number is still growing. In this review we cover the historical background of DNA helicases, helicase assays, biochemical properties, prokaryotic and eukaryotic DNA helicases including Mcm proteins and the RecQ family of helicases. The properties of most of the known DNA helicases from prokaryotic and eukaryotic systems, including viruses and bacteriophages, are summarized in tables.

A Repressor with Similarities to Prokaryotic and Eukaryotic DNA Helicases Controls the Assembly of the CAAT Box Binding Complex at a Photosynthesis Gene Promoter

A single nucleotide exchange in a promoter region located immediately upstream of the CAAT box of the spinach photosynthesis gene AtpC (gene product is subunit gamma of the chloroplast ATP synthase) prevents the formation of a secondary structure and causes an unregulated, constitutive high level of expression (Kusnetsov, V., Landsberger, M., Oelmüller, R. (1999) J. Biol. Chem. 274, 36009-36014). We have isolated cDNAs for ATPC-2, a new polypeptide with homologies to pro- and eukaryotic helicases, which specifically binds to this promoter region. Binding of ATPC-2 competes strongly with that of a CAAT box binding factor (CBF), consistent with the idea that both complexes cannot be formed simultaneously because of sterical reasons. In gel mobility shift assays, high binding activities of ATPC-2 and low binding activities of CBF were observed with nuclear extracts from tissue with low AtpC expression levels, and the opposite was observed with extracts from tissues with high AtpC expression levels. Binding of ATPC-2 to the mutant sequence, which directs a constitutively high level expression in vivo and prevents the formation of a secondary structure in vitro, is significantly weaker than binding to the wild-type sequence. Again, the opposite results were obtained for the CBF. Thus, we conclude that the assembly of the CBF.DNA complex stimulates transcription of AtpC and that CBF binding is prevented if ATPC-2 is bound to the promoter region. The novel mechanism of gene regulation and the role of the helicase-like protein ATPC-2 as a potential transcriptional repressor is discussed in relation to its modular structure.

A Point Mutation in Escherichia coli DNA Helicase II Renders the Enzyme Nonfunctional in Two DNA Repair Pathways: EVIDENCE FOR INITIATION OF UNWINDING FROM A NICK IN VIVO

Biosynthetic errors and DNA damage introduce mismatches and lesions in DNA that can lead to mutations. These abnormalities are susceptible to correction by a number of DNA repair mechanisms, each of which requires a distinct set of proteins. Escherichia coli DNA helicase II has been demonstrated to function in two DNA repair pathways, methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair. To define further the role of UvrD in DNA repair a site-specific mutant was characterized. The mutation, uvrDQ251E, resides within helicase motif III, a conserved segment of amino acid homology found in a superfamily of prokaryotic and eukaryotic DNA helicases. The UvrD-Q251E protein failed to complement the mutator and ultraviolet light-sensitive phenotypes of a uvrD deletion strain indicating that the mutant protein is inactive in both mismatch repair and excision repair. Biochemical characterization revealed a significant defect in the ability of the mutant enzyme to initiate unwinding at a nick. The elongation phase of the unwinding reaction was nearly normal. Together, the biochemical and genetic data provide evidence that UvrD-Q251E is dysfunctional because the mutant protein fails to initiate unwinding at the nick(s) used to initiate excision and subsequent repair synthesis. These results provide direct evidence to support the notion that helicase II initiates unwinding from a nick in vivo in mismatch repair and excision repair.

DNA2 Encodes a DNA Helicase Essential for Replication of Eukaryotic Chromosomes

Although a number of eukaryotic DNA helicases have been identified biochemically and still more have been inferred from the amino acid sequences of the products of cloned genes, none of the cellular helicases or putative helicases has to date been implicated in eukaryotic chromosomal DNA replication. By the same token, numerous eukaryotic replication proteins have been identified, but none of these is a helicase. We have recently identified and characterized a temperature-sensitive yeast mutant, dna2ts, defective in DNA replication, and have cloned the corresponding gene (Kuo, C.-L., Huang, C,-H., and Campbell, J. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 30, 6465-6469; Budd, M. E., and Campbell, J. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7642-7646). The DNA2 gene is essential and encodes a 172-kDa protein with DNA helicase motifs in its C-terminal half and an N-terminal half with no similarity to any previously described protein (Budd, M. E., and Campbell, J. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7642-7646). Here we show that the helicase domain is required in vivo and that a 3' to 5' DNA helicase activity specific for forked substrates is intrinsic to the Dna2p. The N terminus is also essential for DNA replication. Thus, the structure of this new helicase is different from all previously characterized replicative helicases, which is consistent with the complex organization of eukaryotic replication forks, where the activities of not one but three essential DNA polymerases must be coordinated.

Formation of DNA triple helices inhibits DNA unwinding by the SV40 large T-antigen helicase.

Previous studies have indicated that d(TC)n.d(GA)n microsatellites may serve as arrest signals for mammalian DNA replication through the ability of such sequences to form DNA triple helices and thereby inhibit replication enzymes. To further test this hypothesis, we examined the ability of d(TC)i.d(GA)i.d(TC)i triplexes to inhibit DNA unwinding in vitro by a model eukaryotic DNA helicase, the SV40 large T-antigen. DNA substrates that were able to form triplexes, and non-triplex-forming control substrates, were tested. We found that the presence of DNA triplexes, as assayed by endonuclease S1 and osmium tetroxide footprinting, significantly inhibited DNA unwinding by T-antigen. Strong inhibition was observed not only at acidic pH values, in which the triplexes were most stable, but also at physiological pH values in the range 6.9-7.2. Little or no inhibition was detected at pH 8.7. Based on these results, and on previous studies of DNA polymerases, we suggest that DNA triplexes may form in vivo and cause replication arrest through a dual inhibition of duplex unwinding by DNA helicases and of nascent strand synthesis by DNA polymerases. DNA triplexes also have the potential to inhibit recombination and repair processes in which helicases and polymerases are involved.

Eukaryotic DNA helicases: essential enzymes for DNA transactions.

DNA in its double-stranded form is energetically favoured and therefore very stable. However, DNA is involved in metabolic events and thus has a continuous dynamic. Processes such as DNA replication, DNA repair, DNA recombination and transcription require that DNA occurs transiently in a single-stranded form. This status can be achieved by enzymes called DNA helicases. These enzymes have the power to melt the hydrogen bonds between the base pairs by using nucleoside 5'-triphosphate hydrolysis as an energy source. A variety of different DNA helicases have recently been identified from eukaryotic viruses and cells. We focus on the current knowledge of these DNA helicases and their possible function in DNA transactions.

Purification and characterization of delta helicase from fetal calf thymus.

A DNA helicase (delta helicase) which partially copurifies with DNA polymerase delta has been highly purified from fetal calf thymus. delta helicase differs in physical and enzymatic properties from other eukaryotic DNA helicases described thus far. The enzyme has an apparent mass of 57 kDa by gel filtration and is associated with polypeptides of 56 and 52 kDa by SDS-polyacrylamide gel electrophoresis. Photo-cross-linking of the purified enzyme with [alpha-32P]ATP resulted in labeling of a polypeptide of approximately 58 kDa, suggesting that the active site is present on the larger polypeptide. Unwinding of a partial duplex requires a nucleoside triphosphate which can be either ATP or dATP but not a nonhydrolyzable analogue of ATP. Other ribo- and deoxyribonucleoside triphosphates have little or no activity as cofactors. delta helicase also has DNA-dependent ATPase activity which has a relatively low Km for ATP (40 microM). delta helicase binds to single-stranded DNA but has little or no affinity for double-stranded DNA or single-stranded RNA. Similar to replicative DNA helicases from prokaryotes and the herpes simplex virus type 1 helicase-primase, delta helicase translocates in the 5'-3' direction along the strand to which it is bound and preferentially unwinds DNA substrates with a forklike structure.

A DNA helicase from Xenopus laevis ovaries.

A DNA helicase was extensively purified from Xenopus laevis ovaries. The most purified fraction was free of DNA topoisomerase, DNA polymerase, and nuclease activities. The enzyme had a Stokes radius of 54 A and a sedimentation coefficient of 6-7.3 S, from which a native molecular weight of 140,000-170,000 was calculated. DNA helicase activity required Mg2+ or Mn2+ and was dependent on hydrolysis of ATP or dATP. Monovalent cations, K+ and Na+, stimulated DNA unwinding with an optimum at 130 mM. DNA-dependent ATPase activity copurified with the X. laevis DNA helicase. Double-stranded and single-stranded DNA were both cofactors for the ATPase activity, but single-stranded DNA was more efficient. The molecular weight, monovalent cation dependence, cofactor requirements, and elution from single-stranded DNA-cellulose suggest that the X. laevis DNA helicase is different from previously described eukaryotic DNA helicases.

DNA helicase activity of SV40 large tumor antigen.

Large tumor antigen (T antigen) was extracted from SV40-infected African Green Monkey cells and purified to homogeneity by immunoaffinity chromatography. The purified T antigen preparations unwind DNA duplices of greater than 120 bp in a reaction which is dependent on magnesium ions and ATP hydrolysis. Based on these and other properties of the reaction we classify this newly discovered enzymatic activity as a eukaryotic DNA helicase. The helicase and the known ATPase function of T antigen cosediment with the mono- or dimeric 4-6 S form of T antigen, but not with higher T antigen aggregates. The helicase activity seems to be an intrinsic function of SV40 T antigen. First, several different T antigen-specific monoclonal antibodies interfere with the DNA unwinding activity; monoclonals which are known to reduce the T antigen-specific ATPase most strongly inhibited the helicase reaction. Second, mutant T antigens with impaired ATPase function also showed a reduced DNA unwinding activity.