Abstract
Proliferating Cell Nuclear Antigen (PCNA) is an essential factor for DNA replication and repair. PCNA forms a toroidal, ring shaped structure of 90 kDa by the symmetric association of three identical monomers. The ring encircles the DNA and acts as a platform where polymerases and other proteins dock to carry out different DNA metabolic processes. The amino acid sequence of human PCNA is 35% identical to the yeast homolog, and the two proteins have the same 3D crystal structure. In this report, we give evidence that the budding yeast (sc) and human (h) PCNAs have highly similar structures in solution but differ substantially in their stability and dynamics. hPCNA is less resistant to chemical and thermal denaturation and displays lower cooperativity of unfolding as compared to scPCNA. Solvent exchange rates measurements show that the slowest exchanging backbone amides are at the β-sheet, in the structure core, and not at the helices, which line the central channel. However, all the backbone amides of hPCNA exchange fast, becoming undetectable within hours, while the signals from the core amides of scPCNA persist for longer times. The high dynamics of the α-helices, which face the DNA in the PCNA-loaded form, is likely to have functional implications for the sliding of the PCNA ring on the DNA since a large hole with a flexible wall facilitates the establishment of protein-DNA interactions that are transient and easily broken. The increased dynamics of hPCNA relative to scPCNA may allow it to acquire multiple induced conformations upon binding to its substrates enlarging its binding diversity.
Highlights
DNA replication, a fundamental event for the maintenance and transfer of the hereditary information through generations, requires a complex molecular apparatus called replisome [1]
We present a detailed comparative analysis showing that the yeast homolog of Proliferating Cell Nuclear Antigen (PCNA) is more resistant to chemical and thermal denaturation and that it is characterized by higher cooperativity of unfolding
Results scPCNA and hPCNA sequence and structure comparison The human and yeast PCNA homologs share 35% of sequence identity and their crystal structures are highly superimposable over the elements of secondary structure
Summary
DNA replication, a fundamental event for the maintenance and transfer of the hereditary information through generations, requires a complex molecular apparatus called replisome [1]. A central component of the replisome is represented by the DNA sliding clamps. These consist of multimeric, toroidal-shaped structures with pseudo-six fold symmetry that encircle the DNA duplex and act as processivity factors during replication by tethering the replicative polymerases to the genomic template. The bacterial clamp (DNA polymerase III b subunit) is formed by homo-dimeric association of two protomers, each one with three topologically similar domains [3,4]. The archaeal and eukaryotic clamps (PCNAs) assemble into trimeric rings in which each 29 kDa protomer contains two similar domains with 2 b-sheets, 2 a-helices and a long interdomainconnecting loop (IDCL) (Figure 1) [4,5]. The clamp loader (replication factor C) mediates the assembly of PCNA onto DNA in an ATP dependent process [8]
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