DNA helicase E and DNA polymerase .epsilon. functionally interact for displacement synthesis

Turchi, John J.; Siegal, Gregg; Bambara, Robert A.

doi:10.1021/bi00152a043

Cited by 12 publications

(11 citation statements)

References 44 publications

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“…Unlike HDH III, HDH II works somewhat more efficiently if the substrate has a 5' end tail as compared with a 3' end tail, suggesting that HDH II translocates in the 3' to 5' direction. By using the direction-specific substrate we confirmed that it moves in a 3' to 5' direction along the bound strand, like the previously described HDH I , HDH III (Tuteja et al, 1992), HDH V , DNA helicase a and DNA helicase £ from HeLa cells (Seo et al, 1991;Seo and Hurwitz, 1993), 47 kDa calf thymus DNA helicase (Thommes and Huibscher, 1990), calf thymus DNA helicase E (Turchi et al, 1992) and I (Zhang and Grosse, 1991), SV40 large-T antigen (Stahl and Knippers, 1987) and polyoma T-antigen (Seki et al, 1987).…”

Section: Discussionsupporting

confidence: 83%

Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen.

et al. 1994

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Human DNA helicase II (HDH II) is a novel ATP‐dependent DNA unwinding enzyme, purified to apparent homogeneity from HeLa cells, which (i) unwinds exclusively DNA duplexes, (ii) prefers partially unwound substrates and (iii) proceeds in the 3′ to 5′ direction on the bound strand. HDH II is a heterodimer of 72 and 87 kDa polypeptides. It shows single‐stranded DNA‐dependent ATPase activity, as well as double‐stranded DNA binding capacity. All these activities comigrate in gel filtration and glycerol gradients, giving a sedimentation coefficient of 7.4S and a Stokes radius of approximately 46 A, corresponding to a native molecular weight of 158 kDa. The antibodies raised in rabbit against either polypeptide can remove from the solution all the activities of HDH II. Photoaffinity labelling with [alpha‐32P]ATP labelled both polypeptides. Microsequencing of the separate polypeptides of HDH II and cross‐reaction with specific antibodies showed that this enzyme is identical to Ku, an autoantigen recognized by the sera of scleroderma and lupus erythematosus patients, which binds specifically to duplex DNA ends and is regulator of a DNA‐dependent protein kinase. Recombinant HDH II/Ku protein expressed in and purified from Escherichia coli cells showed DNA binding and helicase activities indistinguishable from those of the isolated protein. The exclusively nuclear location of HDH II/Ku antigen, its highly specific affinity for double‐stranded DNA, its abundance and its newly demonstrated ability to unwind exclusively DNA duplexes, point to an additional, if still unclear, role for this molecule in DNA metabolism.

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Section: Discussionsupporting

confidence: 83%

Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen.

et al. 1994

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“…No pausing is observed at ϩ2 for 3.5B and 4.5B, but the ϩ2 pause reappears in 5.5B, which suggests that the predicted helix melting may not occur until incorporation of the 3rd nucleotide in 5.5B rather than the predicted 2nd nucleotide. Regardless, more processive synthesis is observed through the ϩ5, ϩ6, and ϩ7 pause sites for substrates 5.5B, 6.5B, and 7.5B.…”

Section: Hiv-1 Rt Performs Limited Rna Displacement Synthesis On a Longmentioning

confidence: 92%

“…Not all DNA polymerases can perform displacement synthesis and often require accessory factors in the form of helicases and single-stranded DNA-binding proteins (5)(6)(7)(8)(9). Although retroviral nucleocapsid can enhance displacement synthesis, retroviral RTs have an intrinsic ability to synthesize DNA through duplex structures in the absence of other factors (10 -16).…”

mentioning

confidence: 99%

Single Unpaired Nucleotides Facilitate HIV-1 Reverse Transcriptase Displacement Synthesis through Duplex RNA

Lanciault

Champoux

2004

Journal of Biological Chemistry

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During reverse transcription of viral RNA, HIV-1 reverse transcriptase (RT) encounters RNA stem-loop structures that require displacement synthesis activity in which RT disrupts the RNA helix to access the template strand. A primer extension assay was developed to assess HIV-1 RT RNA displacement synthesis activity in vitro. Initial results revealed that HIV-1 RT performs only limited amounts of RNA displacement through long stretches of RNA duplex, with the majority of synthesis stalling at sequence-dependent pause positions. DNA displacement synthesis through the same sequence, however, proceeded rapidly to the end of the template. The RNA folding algorithm mfold indicated that the presence of an unpaired nucleotide, or "bulge," along the RNA duplex would promote helix melting ahead of the DNA primer terminus to create a small gap of nondisplacement synthesis. Primer extension assays using substrates possessing single-nucleotide bulges in the nontemplate strand near pause sites resulted in diminished pausing at positions within the predicted melted region. Surprisingly, the bulges also reduced pausing distal to the bulge at positions that are expected to remain base-paired. Further analysis revealed that stalling during RNA displacement synthesis results from the displaced RNA re-annealing to the template strand thus forcing the primer terminus to become unpaired and, therefore, not extendable. Introduction of a bulge facilitates displacement synthesis through distal regions by increasing RT processivity in the vicinity of a bulge and reducing the impact of branch migration on primer extension. HIV-11 reverse transcriptase (RT) is a heterodimer consisting of 66-and 51-kDa subunits and is the viral polymerase necessary to convert the positive-sense, single-stranded RNA genome to double-stranded DNA for subsequent integration into the host cell genome. HIV-1 RT is capable of synthesizing DNA using DNA and RNA in all possible primer-template combinations (1). Reverse transcriptases in general possess two other activities in addition to the polymerase function. The first is a ribonuclease H (RNase H) activity, which hydrolyzes RNA in RNA-DNA hybrid duplexes (1). The second is a displacement synthesis activity, which allows the enzyme to synthesize DNA through regions of duplex nucleic acid (2-4).Not all DNA polymerases can perform displacement synthesis and often require accessory factors in the form of helicases and single-stranded DNA-binding proteins (5-9). Although retroviral nucleocapsid can enhance displacement synthesis, retroviral RTs have an intrinsic ability to synthesize DNA through duplex structures in the absence of other factors (10 -16). In vitro primer extension assays show that the RTs from Moloney murine leukemia virus (15, 16), feline immunodeficiency virus (17, 18), avian myeloblastic virus (19,20), and HIV-1 (2-4) all possess strand displacement activity. Importantly, mutating the Phe-61 residue in HIV-1 RT revealed that displacement activity is functionally separable from primertemplate bindi...

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“…Our initial report on hel E demonstrated partial co-elution of pol c with hel E over the chromatographic matrices used during purification (4). These data suggest that hel E may form a loose physical complex with DNA pol e. Recent work from this laboratory has demonstrated a functional coordination between hel E and pol E resulting in DNA synthesis through a downstream primer dependent on helicase activity (10). The functional interaction with hel E was demonstrated to be specific for pol c. The results suggested that hel E may participate with pol E at the eukaryotic replication fork or in DNA repair.…”

Section: Introductionmentioning

confidence: 81%

“…at 37°C unless stated otherwise. Products were analyzed by 10% native polyacrylamide gel electrophoresis, and visualized by autoradiography as previously described (10).…”

Section: Enzymesmentioning

confidence: 99%

DNA substrate specificity of DNA helicase E from calf thymus

1992

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DNA helicase E from calf thymus has been characterized with respect to DNA substrate specificity. The helicase was capable of displacing DNA fragments up to 140 nucleotides in length, but was unable to displace a DNA fragment 322 nucleotides in length. DNA competition experiments revealed that helicase E was moderately processive for translocation on single strand M13mp18 DNA, and that the helicase would dissociate and rebind during a 15 minute reaction. Comparison of the rate of ATPase activity catalyzed by helicase E on single strand DNA substrates of different lengths, suggested a processivity consistent with the competition experiments. The helicase displayed a preference for displacing primers whose 5' terminus was fully annealed as opposed to primers with a 12 nucleotide 5' unannealed tail. The presence of a 12 nucleotide 3' tail had no effect on the rate of displacement. DNA helicase E was capable of displacing a primer downstream of either a four nucleotide gap, a one nucleotide gap or a nick in the DNA substrate. Helicase E was inactive on a fully duplex DNA 30 base pairs in length. Calf thymus RP-A stimulated the DNA displacement activity of helicase E. These properties are consistent with a role for DNA helicase E in chromosomal DNA repair.

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DNA helicase E and DNA polymerase .epsilon. functionally interact for displacement synthesis

Cited by 12 publications

References 44 publications

Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen.

Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen.

Single Unpaired Nucleotides Facilitate HIV-1 Reverse Transcriptase Displacement Synthesis through Duplex RNA

DNA substrate specificity of DNA helicase E from calf thymus

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