FC0089
PCNA-associated factor, p15^PAF  -  DNA

Biological function
p15^PAF regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp.

Domain organization/sequence features
The N-terminal region contains an ²¹APRK²⁴ sequence similar to the known DNA-binding motif SP(R/K)(R/K) that is present in p15 homologues as well as in the N-terminal histone H3 tail. Owing to the enrichment in basic residues the N-tail has been proposed to unspecifically bind DNA. For detailed description see FC00088.

Structural evidence
In the complex of p15 with 24 bp DNA, several resonances were perturbed as compared to the free protein, with largest CPS around the basic residues in the N-terminal region and smallest CPS for the acidic residues in the C-terminal region. The poor H^N signal dispersion and only minor changes in C^a chemical shifts indicate that p15 does not acquire a defined structure on binding to the DNA; rather, its backbone remains flexible and disordered. In the presence of PCNA, p15 shows similar NMR pattern attenuation with and without DNA, which is also consistent with weak binding without gaining a definite structure. Transmission electron microscopy shows increased electron density within the PCNA ring.

Biochemical evidence
The affinity of p15 for DNA is 0.2-0.4 μM, that is decreased by an order of magnitude upon increasing the salt concentration. The affinity of p15^ΔN (missing 2-31 AA) is reduced by two orders of magnitude, indicating that the basic N- terminal region of p15 contributes most to the DNA affinity. The central fragment p15^50–77 binds DNA with even lower affinity, and no DNA binding was observed with the core PIP-box fragment p15^59–70. The slightly reduced DNA affinity shows that PCNA interferes only weakly with p15 binding to DNA. Similarly, when DNA- bound p15 was titrated with PCNA, the PCNA affinity was comparable to that of isolated p15 (or p15^50–77). Thus, a ternary p15–PCNA– DNA complex forms in solution in which the direct p15–PCNA and p15–DNA interactions are largely independent of each other.

Structure/Mechanism
p15 binds DNA, mainly through its histone-like N-terminal tail. While PCNA alone does not interact with DNA, p15 binds to PCNA and DNA simultaneously via independent sites. Several clusters of positively charged residues in the N- and C-terminal region of p15 indicate DNA binding via electrostatic interactions. The binding is achieved via the disordered tails of p15, which in the ternary complex is transiently located within the PCNA ring.

Mechanism category
Flanking

Significance
The fuzzy N-terminal tail of p15 may act as an extended arm that captures and binds the DNA fibre at the back-face of PCNA.

Submitted by
Francisco Blanco   fblanco@cicbiogune.es