Abstract
BackgroundSliding clamps, such as Proliferating Cell Nuclear Antigen (PCNA) in eukaryotes, are ring-shaped protein complexes that encircle DNA and enable highly processive DNA replication by serving as docking sites for DNA polymerases. In an ATP-dependent reaction, clamp loader complexes, such as the Replication Factor-C (RFC) complex in eukaryotes, open the clamp and load it around primer-template DNA.ResultsWe built a model of RFC bound to PCNA and DNA based on existing crystal structures of clamp loaders. This model suggests that DNA would enter the clamp at an angle during clamp loading, thereby interacting with positively charged residues in the center of PCNA. We show that simultaneous mutation of Lys 20, Lys 77, Arg 80, and Arg 149, which interact with DNA in the RFC-PCNA-DNA model, compromises the ability of yeast PCNA to stimulate the DNA-dependent ATPase activity of RFC when the DNA is long enough to extend through the clamp. Fluorescence anisotropy binding experiments show that the inability of the mutant clamp proteins to stimulate RFC ATPase activity is likely caused by reduction in the affinity of the RFC-PCNA complex for DNA. We obtained several crystal forms of yeast PCNA-DNA complexes, measuring X-ray diffraction data to 3.0 Å resolution for one such complex. The resulting electron density maps show that DNA is bound in a tilted orientation relative to PCNA, but makes different contacts than those implicated in clamp loading. Because of apparent partial disorder in the DNA, we restricted refinement of the DNA to a rigid body model. This result contrasts with previous analysis of a bacterial clamp bound to DNA, where the DNA was well resolved.ConclusionMutational analysis of PCNA suggests that positively charged residues in the center of the clamp create a binding surface that makes contact with DNA. Disruption of this positive surface, which had not previously been implicated in clamp loading function, reduces RFC ATPase activity in the presence of DNA, most likely by reducing the affinity of RFC and PCNA for DNA. The interaction of DNA is not, however, restricted to one orientation, as indicated by analysis of the PCNA-DNA co-crystals.
Highlights
Sliding clamps, such as Proliferating Cell Nuclear Antigen (PCNA) in eukaryotes, are ring-shaped protein complexes that encircle DNA and enable highly processive DNA replication by serving as docking sites for DNA polymerases
A Model for PCNA-DNA Interaction During Clamp Loading To determine if PCNA-DNA interactions have relevance for clamp loading, a Replication Factor-C (RFC)-PCNA-DNA model was generated to visualize the path of the DNA through PCNA during the clamp loading process
X-ray crystallography has provided snapshots of several steps of the clamp loader cycle, including the apo-gcomplex [38], nucleotide-bound g-complex [39], RFC bound to closed PCNA [31], and DNA-bound g-complex [30]. These structures, along with other biochemical studies, have established that loops facing the interior of the clamp loader recognize DNA by presenting conserved positively charged and polar residues that are positioned to track the negatively charged phosphate backbone of a bound DNA molecule [31,40,41], and that DNA binding helps create a geometry of clamp loader subunits that is activated for ATPase catalysis [30]
Summary
Sliding clamps, such as Proliferating Cell Nuclear Antigen (PCNA) in eukaryotes, are ring-shaped protein complexes that encircle DNA and enable highly processive DNA replication by serving as docking sites for DNA polymerases. The faithful and efficient copying of chromosomal DNA is performed by chromosomal replicases, which generate double-stranded DNA from primed, single-stranded DNA [1,2]. These replicases achieve great speed and processivity by tethering to ring-shaped protein complexes called sliding clamps, which encircle DNA and have the ability to slide freely along the DNA strand [3,4]. Similar conclusions have been reached for the replicase of T4 bacteriophage [7,8]
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