A Novel Proteomic Approach Identifies New Interaction Partners for Proliferating Cell Nuclear Antigen

Abstract

During DNA replication and repair, many proteins bind to and dissociate in a highly specific and ordered manner from proliferating cell nuclear antigen (PCNA). We describe a combined approach of in silico searches at the genome level and combinatorial peptide synthesis to investigate the binding properties of hundreds of short PCNA-interacting peptides (PIP-peptides) to archaeal and eukaryal PCNAs. Biological relevance of our combined approach was demonstrated by identification an inactive complex of Pyrococcus abyssi ribonuclease HII with PCNA. Furthermore we show that PIP-peptides interact with PCNA largely in a sequence independent manner. Our experimental approach also identified many so far unidentified PCNA interacting peptides in a number of human proteins.

A fluorescent bimolecular complementation screen reveals MAF1, RNF7 and SETD3 as PCNA-associated proteins in human cells

“…Although PCNA was identified as critical for DNA replication and repair almost 20 years ago, 53 there still has not been a systematic investigation of the PCNA-interactome, although the need for such a study has been highlighted. 4,35 Instead there have been several independent studies using a variety of methodologies generating as many as 238 reported PCNA interaction partners to date (http://thebiogrid.org/111142). Here, we sought to design a strategy capable of detecting all potential PCNA interactions in human cells.…”

“…Given the importance of PCNA in regulating the processes that ensure genome and epigenome stability through replication, it has been previously noted that a full characterization of the PCNA-interactome would be desirable. 4,35 We developed an in-cell screening approach to identify PCNA interaction partners. The format of our screen will allow it to report on interactions that happen in the context of active replication sites, even those that are DNA-dependent or transient, interactions that could be missed in a purification-based strategy.…”

Archaeal DNA Repair Nucleases

“…To clarify the physiological role of type 2 RNases H in bacteria, archaea and eukaryotes, mutant strains containing one or two RNases H-encoding genes have been constructed. In single-celled species, deletions of all RNase H genes were not lethal, but showed modest sensitivity to DNA-damaging agents, indicating their requirement in DNA repair (Arudchandran et al, 2000;Fukushima et al, 2007;Itaya et al, 1999;Meslet-Cladiere et al, 2007). Conversely, in a multicellular organism, both type 1 and type 2 RNases H were shown to be essential.…”

“…Likewise, defect of the catalytic mutant MmuRNase H2 to hydrolyse single embedded ribonucleotides pointed toward a role for eukaryotic RNase H2 in DNA repair (Shaban et al, 2010). Furthermore, based on genetic and biochemical results, removal of single embedded ribonucleotides seems to involve at least type 2 RNases H, Fen1 (Flap Endonuclease 1) and PCNA (Bubeck et al, 2011;Meslet-Cladiere et al, 2007;Nick McElhinny et al, 2010a;Rumbaugh et al, 1997;Rydberg and Game, 2002), with PCNA:RNase HII/2 complex acting as a sensor of erroneous ribonucleotides. The association of such protein components likely suggests a role of type 2 RNases H in base excision repair (BER) to accomplish the removal of mutagenic ribonucleotides.…”

“…In the presence of Mg 2+ and 50 mM KCl, AfuRNase HII is shown to be active on DNA-RNA 1 -DNA/DNA, unless the ribonucleotide is positioned<4 bases from the 5' end or <2 bases from the 3' end, and cleavage occurs at the phosphodiester bond 5' of the junctional ribonucleotide (Bubeck et al, 2011). In addition, PCNA (Proliferating Cell Nuclear Antigen), described as a scaffold for DNA repair and replication enzymes (Maga and Hubscher, 2003;Meslet-Cladiere et al, 2007), enhances cleavage activity of AfuRNase HII on DNA-RNA 1 -DNA/DNA, with the exception of ribonucleotide located within the first ten 5'-bases of the strand containing it. Interestingly, this result is consistent with that observed previously for PabRNase HII .…”