DNA Helicase

In this special Methods collection on DNA helicases, I have solicited articles from leading experts in the field with a priority to gather a defined series of papers on highly relevant topics that encompass biological, biochemical, and biophysical aspects of helicase function. The experimental approaches described provide an opportunity for both new and more experienced scientists to use the information for the design of their own investigations. The reader will find detailed methods for single-molecule studies, novel biochemical experiments, genetic analyses, and cell biological assays in a variety of systems with an emphasis placed on state-of-the-art techniques to measure helicase function. Contributing authors were strongly encouraged to provide a carefully constructed description of the methods employed so that others might use this information in a manner that will be useful for their own particular application and helicase of interest. This special issue of Methods dedicated to DNA helicases offers readers a treasure chest of unique experimental approaches and protocols focused on rapidly developing techniques that are useful for studying both in vivo and in vitro aspects of helicase function.

Bloom syndrome (BS) is a rare autosomal recessive disorder characterized by growth deficiency, immunodeficiency, genomic instability, and the early development of cancers of many types. BLM, the protein encoded by BLM, the gene mutated in BS, is localized in nuclear foci and absent from BS cells. BLMencodes a DNA helicase, and proteins from three missense alleles lack displacement activity. BLM transfected into BS cells reduces the frequency of sister chromatid exchanges and restores BLM in the nucleus. Missense alleles fail to reduce the sister chromatid exchanges in transfected BS cells or restore the normal nuclear pattern. BLM complements a phenotype of aSaccharomyces cerevisiae sgs1 top3 strain, and the missense alleles do not. This work demonstrates the importance of the enzymatic activity of BLM for its function and nuclear localization pattern.

The R2TP complex which comprises of RUVBL1, RUVBL2, PIH1D1 and RPAP3 in humans is known to be a specialized Co-chaperone of Hsp-90 protein. This multimeric-protein complex is involved in the assembly and maturation of several multi-subunit complexes including RNA polymerase II, small nucleolar ribonucleoproteins, and complexes containing phosphatidylinositol-3-kinase-like kinases. Since their discovery as a co-chaperone of Hsp90, the R2TP complex is involved in multitude of cellular processes including, chromatin remodelling, transcription regulation, ribonucleoprotein complex biogenesis, mitotic assembly, telomerase complex assembly, and apoptosis. Lymphoma arises from the abnormal proliferation of B-cells and the R2TP complex have been reported to play an important role in the activation of p53 and RB. Therefore, the inactivation in any of the tumor suppressor pathways can drive cells to malignancy.However, there are multiple factors which may contribute towards malignancy but the...

The post-entry events of HIV-1 infection occur within reverse transcription complexes derived from the viral cores entering the target cell. HIV-1 cores contain host proteins incorporated from virus-producing cells. In this report, we show that MCM5, a subunit of the hexameric minichromosome maintenance (MCM) DNA helicase complex, associates with Gag polyprotein and is incorporated into HIV-1 virions. The progeny virions depleted of MCM5 demonstrated reduced reverse transcription in newly infected cells, but integration and subsequent replication steps were not affected. Interestingly, increased packaging of MCM5 into the virions also led to reduced reverse transcription, but here viral replication was impaired. Our data suggest that incorporation of physiological amounts of MCM5 promotes aberrant reverse transcription, leading to partial incapacitation of cDNA, whereas increased MCM5 abundance leads to reduced reverse transcription and infection. Therefore, MCM5 has the properties of an inhibitory factor that interferes with production of an integration-competent cDNA product.

Susceptibility to breast cancer is significantly increased in individuals with germ line mutations in RECQ1 (also known as RECQL or RECQL1 ), a gene encoding a DNA helicase essential for genome maintenance. We previously reported that RECQ1 expression predicts clinical outcomes for sporadic breast cancer patients stratified by estrogen receptor (ER) status.

Chromosomal behaviour during megasporogenesis and microsporogenesis has been studied in ornamental Delphinium ajacis L. Meiosis in female sex cell initiates later than male. The floral buds which carry egg mother cell (EMC) at diplotene stage has pollen mother cells (PMCs) at tetrad stage of meiosis suggesting protandry. Although the 16 chromosomes formed regular eight bivalents in both the sex cells, they differed in overall chiasma frequency which was 32.95% higher in EMCs and found to be 18.52 ± 2.12 per cell. In PMCs, the average chiasma frequency recorded was 13.93 ± 1.40 per cell. Interestingly, this variation in chiasma frequency was largely confined to the two large bivalents which shared 42.61% chiasma per EMC. The use of Q-Q plot, Box plot and Whisker plot showed departure in the chiasma frequency distributions in EMCs and PMCs from the normal distribution pattern. The difference in chiasma frequency in the two sex cells was significant at all levels as indicated by the low P values of 3.094 9 10-11 obtained from nonparametric test, i.e. Wilcoxon rank-sum test. It is suggested that the two different mechanisms of recombination are operational in the two sex cells, and the sex differences of chiasma frequency could have arisen due to differential epigenetic modifications of the chromatin which pattern the double-strand breaks, and the position and frequency of crossing over visible as chiasmata.

RuvB protein belongs to AAA+ family of enzymes involved in diverse cellular activities. In addition to the annotated two RuvB proteins in Plasmodium falciparum database, we report that a third RuvB protein is also present. The amino acid sequence analysis has revealed that P. falciparum RuvB3 (PfRuvB3) possesses Walker motif A, Walker motif B, sensor I and sensor II conserved motifs similar to yeast and human RuvB like proteins. The phylogenetic analysis revealed that PfRuvB3 is closely related to yeast RuvB like proteins which are essential for the survival of yeast. The biochemical characterization of recombinant PfRuvB3 confirms its ssDNA dependent ATPase activity. Using the truncated derivatives we show that Walker motif A is essential for the enzymatic activity of PfRuvB3. Using the immunodepletion assays we further show that the ATPase activity is attributable to PfRuvB3 protein. The endogenous P. falciparum RuvB3 contains the characteristic ATPase and some DNA helicase activities. The confocal microscopy analysis showed that this protein is mainly expressed during intraerythrocytic schizont stages of the parasite and is localized to the nuclear region. Once merozoite comes out from schizont, PfRuvB3 protein distinctly relocalized to the subnuclear region. The co-localization studies with a nucleolar marker PfNop1 further suggest that in P. falciparum RuvB3 localizes into a discrete nuclear compartment. On the basis of these studies it can be speculated that P. falciparum RuvB3 is most likely required for intraerythrocytic schizogony.

Bloom syndrome protein (BLM) is one of five human RecQ helicases that participate in DNA metabolism. RecQ C-terminal (RQC) domain is the main DNA binding module of BLM and specifically recognizes G-quadruplex (G4) DNA structures. Because G4 processing by BLM is essential for regulating replication and transcription, both G4 and BLM are considered as potential targets for anticancer therapy. Although several studies have revealed the detailed mechanism of G4 unwinding by BLM, the initial recognition of the G4 structure by the RQC domain is unclear. Here, we investigated the interaction between BLM RQC and the G4 DNA from the c-Myc promoter by NMR spectroscopy. While the signals broadened upon reciprocal titrations, the β-wing of RQC had significant chemical shift perturbations and experienced millisecond timescale dynamics upon G4 binding. A point mutation in the β-wing (N1164A) reduced G4 binding affinity. Our hydrogen–deuterium exchange data indicate that imino protons of G4 were e...

DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of po...

The eukaryotic MCM2-7 replicative helicase is a hetero-complex composed by 6 homologues belonging to the AAA+ ATPase superfamily. These proteins form a ring-like structure which is essential for the initiation and the progression of the replication fork. In order to better understand the cellular role and mechanism of action of these proteins, I carried out structural and functional studies on two different systems: the archaeal MCM proteins, that forms a homomeric ring representing a simplified model of the eukaryotic complex, and the less well studied human MCM8, whose exact role in eukaryotic cells has not been elucidated. MCM interacts in two ways with the DNA: the canonical "loaded" mode where the MCM protein is encircling the DNA, and a "associated" mode where the DNA is wrapped around the enzyme. Using the protein from the archaeon Methanothermobacter thermautotrophicum, I biochemically shown that binding of MCM proteins affect the degree of supercoiling o...

have determined the structure of the Rep protein from cryptic plasmid pSTK1 of Geobacillus stearothermophilus (Gst), and several variants of RepD from Staphylococcus aureus (Sau), representing the first structural information on this class of initiators. Cloning and expression of a construct derived from the from pSTK1 Rep yielded a product that relaxed plasmid substrates encoding an inverted repeat sequence from pSTK1, which resembles the replication origin of the pT181 family, and activated the cognate Gst PcrA helicase. The crystal structure for the 31 kDa fragment of Gst Rep has been solved at 2.3 Å , showing a novel, ring-shaped dimer with a 20Å diameter pore. The inner surface is formed by an 18-stranded b-sheet, while the outer surface is decorated with 18 a-helices. The protein has a novel fold, but the extended sheet exhibits similarities to that in TATA-binding protein (TBP). The active site Tyr179 residues, one from each subunit, lie 26 Å apart across the pore, with a nearby catalytic magnesium ion coordinated by three carboxylate side-chains. Crystal structures for the Sau Rep variants RepDE, RepDN and RepDC have been solved by molecular repacement using the Gst Rep as a model, and show similar structural features. The implications for the mechanism of rolling circle replication will be discussed in the light of extensive functional data available for Sau RepD.

The obligate human pathogen Neisseria gonorrhoeae alters its cell surface antigens to evade the immune system in a process known as antigenic variation (AV). During pilin AV, portions of the expressed pilin gene (pilE) are replaced with segments of silent pilin genes (pilS) through homologous recombination. The pilE-pilS exchange is initiated by formation of a parallel guanine quadruplex (G4) structure near the pilE gene, which recruits the homologous recombination machinery. The RecQ helicase, which has been proposed to aid AV by unwinding the pilE G4 structure, is an important component of this machinery. However, RecQ also promotes homologous recombination through G4-independent duplex DNA unwinding, leaving the relative importance of its G4 unwinding activity unclear. Previous investigations revealed a guanine-specific pocket (GSP) on the surface of RecQ that is required for G4, but not duplex, DNA unwinding. To determine whether RecQmediated G4 resolution is required for AV, N. gonorrhoeae strains that encode a RecQ GSP variant that cannot unwind G4 DNA were created. In contrast to the hypothesis that G4 unwinding by RecQ is important for AV, the RecQ GSP variant N. gonorrhoeae strains had normal AV levels. Analysis of a purified RecQ GSP variant confirmed that it retained duplex DNA unwinding activity but had lost its ability to unwind antiparallel G4 DNA. Interestingly, neither the GSP-deficient RecQ variant nor the wild-type RecQ could unwind the parallel pilE G4 nor the prototypical c-myc G4. Based on these results, we conclude that N. gonorrhoeae AV occurs independently of RecQ-mediated pilE G4 resolution. IMPORTANCE The pathogenic bacteria Neisseria gonorrhoeae avoids clearance by the immune system through antigenic variation (AV), the process by which immunogenic surface features of the bacteria are exchanged for novel variants. RecQ helicase is critical in AV and its role has been proposed to stem from its ability to unwind a DNA secondary structure known as a guanine quadruplex (G4) that is central to AV. In this work, we demonstrate that the role of RecQ in AV is independent of its ability to resolve G4s and that RecQ is incapable of unwinding the G4 in question. We propose a new model of RecQ's role in AV where the G4 might recruit or orient RecQ to facilitate homologous recombination. KEYWORDS antigen variation, DNA helicase, G quadruplex, Neisseria gonorrhoeae M ore than 550,000 Americans are infected annually with Neisseria gonorrhoeae, the causative agent of gonorrhea (1). Untreated gonorrhea infections can lead to serious complications, including septic arthritis, pelvic inflammatory disease, and infertility (2). Although gonorrhea can be treated using antibiotics, increasing levels of