Molecular insight into the PCNA-binding mode of FBH1

Highlights

• Divergent PIP and APIM motifs of FBH1 bind PCNA

• The PIP and APIM binding sites of PCNA overlap

• PCNA shows greater affinity to the PIP motif

Summary

F-box DNA helicase 1 (FBH1) is involved in the regulation of cell responses to replicative stress. FBH1 is recruited to stalled DNA replication fork by PCNA where it inhibits homologous recombination and catalyzes fork regression. Here, we report the structural basis for the molecular recognition of two distinctly different motifs of FBH1, FBH1_PIP and FBH1_APIM, by PCNA. The crystal structure of PCNA in complex with FBH1_PIP and analysis of NMR perturbations reveal overlapped FBH1_PIP and FBH1_APIM binding sites of PCNA and the dominant contribution of FBH1_PIP in this interaction.

Introduction

F-box DNA helicase 1 (FBH1) plays a major role in maintenance of the genome integrity and stability.^1,2 It mediates the response to stalled or damaged DNA replication forks by impeding homologous recombination (HR). The anti-recombinogenic helicase activity of FBH1 was shown to be essential for repairing and restarting the replication fork.^3,4 FBH1 is recruited to the DNA damaged sites by proliferating cell nuclear antigen (PCNA) (Figure 1A) and afterwards is polyubiquitinated and degraded through the Cullin-ring ligase 4-Cdt2 (CRL4Cdt2)–PCNA pathway.⁵ It has been proposed that FBH1 polyubiquitination occurs simultaneously with PCNA monoubiquitination, which facilitates the recruitment of translesion DNA synthesis polymerase η to the DNA damaged site. In response to the DNA replication stalling arising from oncogenic stress, FBH1 promotes DNA double strand breaks and induces cellular apoptosis.^6,7 FBH1 deletion is frequently observed in melanoma, and defects in FBH1 are associated with the resistance of cancer cells to pharmacological treatment.⁸ FBH1 also catalyzes the regression of stalled replication forks, triggering the response mediated by stress-activated protein kinases.¹ FBH1 is predicted to have two folded domains: a small F-box and a large catalytic ATP-binding UvrD-like helicase domain,⁹ and additionally two short motifs important for binding to PCNA were identified near the N- and C-terminal regions of the protein.⁵

PCNA is a key component of the chromatin remodeling and DNA replication and repair machineries in eukaryotic cells.¹⁰ This large homotrimeric protein encircles DNA and tethers DNA-editing complexes required for DNA synthesis, flap cleavage, and ligation to the replication fork.^11,12,13 PCNA is also a critical DNA damage response (DDR) factor that recruits DDR proteins to the DNA damaged sites to complete repair.^14,15 PCNA has a large number of binding partners, the majority of which contains a conserved PCNA-interacting protein (PIP) motif, characterized by the sequence Qxxhxxaa, where h is a hydrophobic aliphatic residue, a is an aromatic residue, and x is any residue.^16,17 Along with the canonical PIP motif (FBH1_PIP) near its N-terminus, FBH1 contains a second short motif within the C-terminal helicase domain. The AlkB homolog 2 PCNA-interacting motif (FBH1_APIM) is defined by the +ahx+ sequence, where + is a positively charged residue, a is an aromatic residue, h is a hydrophobic aliphatic residue, and x is any residue. It has been shown that both FBH1_PIP and FBH1_APIM motifs associate with PCNA, contributing to the accumulation of FBH1 at the stalled replication fork after DNA damage,⁵ however the mechanistic basis underlying this association remains unclear.

Here, we describe the molecular mechanism by which FBH1 binds to PCNA via its FBH1_PIP and FBH1_APIM motifs. The crystal structure of the PCNA-FBH1_PIP complex and analysis of NMR resonance perturbations in PCNA suggest overlapped binding sites for FBH1_PIP and FBH1_APIM and tighter binding of FBH1_PIP.