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BACKGROUND The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring-shaped protein. This protein ("sliding clamp') encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity factors are ring-shaped,(More)
Although three human genes encoding DNA ligases have been isolated, the molecular mechanisms by which these gene products specifically participate in different DNA transactions are not well understood. In this study, fractionation of a HeLa nuclear extract by DNA ligase I affinity chromatography resulted in the specific retention of a replication protein,(More)
Human replication factor C (hRFC) is a multi-subunit protein complex capable of supporting proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis by DNA polymerases delta and epsilon. The hRFC complex consists of five different subunits with apparent molecular masses of 140, 40, 38, 37, and 36 kDa. We have previously reported the expression of a(More)
The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ring-shaped protein, the beta clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The beta clamp is assembled onto DNA by the multiprotein gamma complex clamp loader(More)
Escherichia coli expression vectors, based on the pET system, were constructed to allow fusion of a protein kinase (PK) recognition motif, a hemagglutinin (HG) epitope-tag and a His6-tag at the N-terminal portion of a protein of interest. The fusion proteins, that result from expression using these vectors, can be phosphorylated in vitro using(More)
Chromosomal replicases of cellular organisms utilize a ring shaped protein that encircles DNA as a mobile tether for high processivity in DNA synthesis. These "sliding clamps" have sufficiently large linear diameters to encircle duplex DNA and are perhaps even large enough to slide over certain DNA secondary structural elements. This report examines the(More)
Human replication factor C (RFC, also called activator 1) is a five-subunit protein complex (p140, p40, p38, p37, and p36) required for proliferating cell nuclear antigen (PCNA)-dependent processive DNA synthesis catalyzed by DNA polymerase delta or epsilon. Here we report the reconstitution of the RFC complex from its five subunits simultaneously(More)
Bacterial substrate-binding proteins exist in an equilibrium among four forms: open/substrate-free, open/substrate-bound, closed/substrate-free, and closed/substrate-bound. Ligands stabilize the closed conformation, whereas the open conformation predominates in the substrate-free species. In its closed form, the NH2-terminal and COOH-terminal domains of(More)
Human replication factor C (hRFC) is a five-subunit protein complex (p140, p40, p38, p37, and p36) that acts to catalytically load proliferating cell nuclear antigen onto DNA, where it recruits DNA polymerase delta or epsilon to the primer terminus at the expense of ATP, leading to processive DNA synthesis. We have previously shown that a subcomplex of hRFC(More)
Class III myosins (Myo3) and actin-bundling protein Espin play critical roles in regulating the development and maintenance of stereocilia in vertebrate hair cells, and their defects cause hereditary hearing impairments. Myo3 interacts with Espin1 through its tail homology I motif (THDI), however it is not clear how Myo3 specifically acts through Espin1 to(More)
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