PCNA tightens its hold on the nucleus

The sliding clamp protein PCNA is a key controller of multiple processes in DNA and chromatin metabolism, regulating replication, repair and chromatin assembly through interaction with a huge number of partner proteins (Fig 1). In many of these transactions, the partners bind via a conserved PCNA-interacting peptide or PIP, first identified in CDKN1/p21. Sequential and competitive binding of these PIPs within the interdomain connector loop (IDCL) of PCNA provides a means to ensure ordered reactions. Adding to the roll call of PCNA partners, Cooper et al. have now employed bimolecular fluorescence complementation (BiFC) screening to look for proteins that bind to PCNA ‘bait’ in normally proliferating human cells; unlike in vitro interaction studies and heterologous yeast 2 hybrid screens, BiFC screening in cycling human cells provides a platform for physiologically relevant protein interaction discovery. Combining FACS sorting with the speed and depth of reads possible with next generation sequencing also allows the scaleup of this type of screen for identification of drug targets. Notably, this screen identified novel PCNA partners that do not necessarily interact through the canonical PIP (though a number of the expected partners were also detected). Interactions were not universal across all cells – perhaps because the initial screen did not enrich for S phase cells – nor particularly robust (the signal was lost on detergent treatment), while use of a skeletal muscle cDNA library probably influenced the range of partner proteins identified – SetD3, for instance, is important in muscle differentiation. Nevertheless, this screen is powerful and interactions with RNF7, Maf1 and SetD3 were validated by a range of direct assays. So what might these novel partners tell us about how PCNA acts in cells? SetD3 is a histone H3 lysine methyltransferase that adjusts the histone code to promote a transcription-competent chromatin conformation. Perhaps interaction here simply adds to PCNA’s repertoire in assisting copying of the histone code immediately after replication fork passage, as postulated from its association with Caf1 and Asf1. While SetD3 does not have every residue of a classical PIP, the motif QKGLSVTF may be adequate for association with PCNA’s IDCL. Notably the aromatic residues play a critical role in binding affinity with tyrosine (e.g. p21 PIP D QTSMTDFY) conferring much tighter PCNA binding than phenylalanine (e.g., Fen1 PIPD QGRLDDFF). Maf1 and RNF7 are small proteins without identifiable PIPs; but lack of a PIP does not prevent other partners such as RF-C from binding PCNA with high affinity, nor proteins that bind through the alternative APIMmotif. Regulation of the cell cycle through PIPdependent degradation of key protein such as p27, Cdt1 and – most recently Cdc6 provides yet another critical role for PCNA. Its