P15PAF binding to PCNA modulates the DNA sliding surface.

Matteo De March,Miguel Romano-Moreno,Giovanni Maga,Ramon Crehuet,Silvia Onesti,Francisco J Blanco,Elisa Mentegari,Alfredo De Biasio,Susana Barrera-Vilarmau,Amaia Gonzalez-Magaña,Emmanuele Crespan,Nekane Merino

doi:10.1093/nar/gky723

Abstract

p15PAF is an oncogenic intrinsically disordered protein that regulates DNA replication and lesion bypass by interacting with the human sliding clamp PCNA. In the absence of DNA, p15PAF traverses the PCNA ring via an extended PIP-box that contacts the sliding surface. Here, we probed the atomic-scale structure of p15PAF–PCNA–DNA ternary complexes. Crystallography and MD simulations show that, when p15PAF occupies two subunits of the PCNA homotrimer, DNA within the ring channel binds the unoccupied subunit. The structure of PCNA-bound p15PAF in the absence and presence of DNA is invariant, and solution NMR confirms that DNA does not displace p15PAF from the ring wall. Thus, p15PAF reduces the available sliding surfaces of PCNA, and may function as a belt that fastens the DNA to the clamp during synthesis by the replicative polymerase (pol δ). This constraint, however, may need to be released for efficient DNA lesion bypass by the translesion synthesis polymerase (pol η). Accordingly, our biochemical data show that p15PAF impairs primer synthesis by pol η–PCNA holoenzyme against both damaged and normal DNA templates. In light of our findings, we discuss the possible mechanistic roles of p15PAF in DNA replication and suppression of DNA lesion bypass.

Highlights

Sliding clamps are ring-shaped proteins that tether polymerases and other factors of the replisome to the genomic template, enabling DNA replication and repair
Crystals including the p1550–77 peptide diffracted to 3.2 Aresolution, and the Fourier difference map calculated after placing and refining the Proliferating Cell Nuclear Antigen (PCNA) ring alone in the asymmetric unit showed two PIP-box sites occupied by the p1550–77 peptide and electron density features in the channel that may be attributed to one strand of the DNA duplex
Because of the partial disorder of DNA and the presence of a symmetry related loop that plugs the top of the PCNA channel, we resorted to MD simulations to corroborate the crystallographic results and gain further structural insights on the ternary assembly

Summary

Introduction

Sliding clamps are ring-shaped proteins that tether polymerases and other factors of the replisome to the genomic template, enabling DNA replication and repair. Experimental and computational analyses of the human PCNA–DNA complex showed that the DNA in the channel is tilted and its phosphates transiently interact with a set of basic residues forming a right-hand spiral that matches the DNA pitch [5,7]. This dynamic interaction may allow the clamp to slide by rotationally tracking the DNA helix, or by a linear motion uncoupled from the helical pitch, or by a combination of the two modes [8]. Acetylation of K20 negatively affects the processivity of the replicative polymerase ␦ (pol ␦), but not that of the translesion synthesis (TLS) polymerase ␩ (pol ␩), spe-

Methods

Results

Conclusion