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SummaryHundreds of different protein complexes that perform important functions across all cellular processes, collectively comprising the “complexome” of an organism, have been identified1. However, less is known about the fraction of the interactome that exists outside the complexome, in the “outer-complexome”. To investigate features of “inner”- versus outer-complexome organisation in yeast, we generated a high-quality atlas of binary protein-protein interactions (PPIs), combining three previous maps2–4 and a new reference all-by-all binary interactome map. A greater proportion of interactions in our map are in the outer-complexome, in comparison to those found by affinity purification followed by mass spectrometry5–7 or in literature curated datasets8–11. In addition, recent advances in deep learning predictions of PPI structures12 mirror the existing experimentally resolved structures in being largely focused on the inner complexome and missing most interactions in the outer-complexome. Our new PPI network suggests that the outer-complexome contains considerably more PPIs than the inner-complexome, and integration with functional similarity networks13–15 reveals that interactions in the inner-complexome are highly detectable and correspond to pairs of proteins with high functional similarity, while proteins connected by more transient, harder-to-detect interactions in the outer-complexome, exhibit higher functional heterogeneity.

et al., Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype–phenotype relationships1,2. Here we present a human ‘all-by-all’ reference interactome map of human binary protein interactions, or ‘HuRI’. With approximately 53,000 protein–protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome3, transcriptome4 and proteome5 data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein–protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes., This work was primarily supported by the National Institutes of Health (NIH) National Human Genome Research Institute (NHGRI) grant U41HG001715 (M.V., F.P.R., D.E.H., M.A.C., G.D.B. and J.T.) with additional support from NIH grants P50HG004233 (M.V. and F.P.R.), U01HL098166 (M.V.), U01HG007690 (M.V.), R01GM109199 (M.A.C.), Canadian Institute for Health Research (CIHR) Foundation Grants (F.P.R. and J. Rak), the Canada Excellence Research Chairs Program (F.P.R.) and an American Heart Association grant 15CVGPS23430000 (M.V.). D.-K.K. was supported by a Banting Postdoctoral Fellowship through the Natural Sciences and Engineering Research Council (NSERC) of Canada and by the Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education (2017R1A6A3A03004385). C. Pons was supported by a Ramon Cajal fellowship (RYC-2017-22959). G.M.S. was supported by NIH Training Grant T32CA009361. M.V. is a Chercheurv Qualifié Honoraire from the Fonds de la Recherche Scientifique (FRS-FNRS, Wallonia-Brussels Federation, Belgium).

Global insights into cellular organization and function require comprehensive understanding of interactome networks. Similar to how a reference genome sequence revolutionized human genetics, a reference map of the human interactome network is critical to fully understand genotype-phenotype relationships. Here we present the first human “all-by-all” binary reference interactome map, or “HuRI”. With ~53,000 high-quality protein-protein interactions (PPIs), HuRI is approximately four times larger than the information curated from small-scale studies available in the literature. Integrating HuRI with genome, transcriptome and proteome data enables the study of cellular function within essentially any physiological or pathological cellular context. We demonstrate the use of HuRI in identifying specific subcellular roles of PPIs and protein function modulation via splicing during brain development. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms underlying tissue-specific phenotypes of Mendelian diseases. HuRI thus represents an unprecedented, systematic reference linking genomic variation to phenotypic outcomes.

In Escherichia coli the highly conserved DNA damage regulated dinB gene encodes DNA Polymerase IV (DinB), an error prone specialized DNA polymerase with a central role in stress-induced mutagenesis. Since DinB is the DNA polymerase with the highest intracellular concentrations upon induction of the SOS response, further regulation must exist to maintain genomic stability. Remarkably, we find that DinB DNA synthesis is inherently poor when using an RNA primer compared to a DNA primer, while high fidelity DNA polymerases are known to have no primer preference. Moreover, we show that the poor DNA synthesis from an RNA primer is conserved in DNA polymerase Kappa, the human DinB homolog. The activity of DinB is modulated by interactions with several other proteins, one of which is the equally evolutionarily conserved recombinase RecA. This interaction is known to positively affect DinB's fidelity on damaged templates. We find that upon interaction with RecA, DinB shows a significant reduction in DNA synthesis when using an RNA primer. Furthermore, with DinB or DinB:RecA a robust pause, sequence and lesion independent, occurs only when RNA is used as a primer. The robust pause is likely to result in abortive DNA synthesis when RNA is the primer. These data suggest a novel mechanism to prevent DinB synthesis when it is not needed despite its high concentrations, thus protecting genome stability. © 2017 Tashjian, Lin, Belt, Cafarelli and Godoy. National Institute of General Medical Sciences

Proteins effect a number of biological functions, from cellular signaling, organization, mobility, and transport to catalyzing biochemical reactions and coordinating an immune response. These varied functions are often dependent upon macromolecular interactions, particularly with other proteins. Small-scale studies in the scientific literature report protein–protein interactions (PPIs), but slowly and with bias towards well-studied proteins. In an era where genomic sequence is readily available, deducing genotype–phenotype relationships requires an understanding of protein connectivity at proteome-scale. A proteome-scale map of the protein–protein interaction network provides a global view of cellular organization and function. Here, we discuss a summary of methods for building proteome-scale interactome maps and the current status and implications of mapping achievements. Not only do interactome maps serve as a reference, detailing global cellular function and organization patterns, but they can also reveal the mechanisms altered by disease alleles, highlight the patterns of interaction rewiring across evolution, and help pinpoint biologically and therapeutically relevant proteins. Despite the considerable strides made in proteome-wide mapping, several technical challenges persist. Therefore, future considerations that impact current mapping efforts are also discussed.

Alkylation DNA lesions are ubiquitous, and result from normal cellular metabolism as well as from treatment with methylating agents and chemotherapeutics. DNA damage tolerance by translesion synthesis DNA polymerases has an important role in cellular resistance to alkylating agents. However, it is not yet known whether Escherichia coli (E. coli) DNA Pol IV (DinB) alkylation lesion bypass efficiency and fidelity in vitro are similar to those inferred by genetic analyses. We hypothesized that DinB-mediated bypass of 3-deaza-3-methyladenine, a stable analog of 3-methyladenine, the primary replication fork-stalling alkylation lesion, would be of high fidelity. We performed here the first kinetic analyses of E. coli DinB•RecA binary complexes. Whether alone or in a binary complex, DinB inserted the correct deoxyribonucleoside triphosphate (dNTP) opposite either lesion-containing or undamaged template; the incorporation of other dNTPs was largely inefficient. DinB prefers undamaged DNA, but the DinB•RecA binary complex increases its catalytic efficiency on lesion-containing template, perhaps as part of a regulatory mechanism to better respond to alkylation damage. Notably, we find that a DinB derivative with enhanced affinity for RecA, either alone or in a binary complex, is less efficient and has a lower fidelity than DinB or DinB•RecA. This finding contrasts our previous genetic analyses. Therefore, mutagenesis resulting from alkylation lesions is likely limited in cells by the activity of DinB•RecA. These two highly conserved proteins play an important role in maintaining genomic stability when cells are faced with ubiquitous DNA damage. Kinetic analyses are important to gain insights into the mechanism(s) regulating TLS DNA polymerases.

Escherichia coli strains overproducing DinB undergo survival loss; however, the mechanisms regulating this phenotype are poorly understood. Here we report a genetic selection revealing DinB residues essential to effect this loss-of-survival phenotype. The selection uses strains carrying both an antimutator allele of DNA polymerase III (Pol III) α-subunit ( dnaE915 ) and either chromosomal or plasmid-borne dinB alleles. We hypothesized that dnaE915 cells would respond to DinB overproduction differently from dnaE + cells because the dnaE915 allele is known to have an altered genetic interaction with dinB + compared to its interaction with dnaE + . Notably, we observe a loss-of-survival phenotype in dnaE915 strains with either a chromosomal catalytically inactive dinB ( D103N ) allele or a low-copy-number plasmid-borne dinB + upon DNA damage treatment. Furthermore, we find that the loss-of-survival phenotype occurs independently of DNA damage treatment in a dnaE915 strain expressing the catalytically inactive dinB ( D103N ) allele from a low-copy-number plasmid. The selective pressure imposed resulted in suppressor mutations that eliminated growth defects. The dinB intragenic mutations examined were either base pair substitutions or those that we inferred to be loss of function (i.e., deletions and insertions). Further analyses of selected novel dinB alleles, generated by single-base-pair substitutions in the dnaE915 strain, indicated that these no longer effect loss of survival upon overproduction in dnaE + strains. These mutations are mapped to specific areas of DinB; this permits us to gain insights into the mechanisms underlying the DinB-mediated overproduction loss-of-survival phenotype.

Alkylation DNA lesions are ubiquitous, and result from normal cellular metabolism as well as from treatment with methylating agents and chemotherapeutics. DNA damage tolerance by translesion synthesis DNA polymerases has an important role in cellular resistance to alkylating agents. However, it is not yet known whether Escherichia coli (E. coli) DNA Pol IV (DinB) alkylation lesion bypass efficiency and fidelity in vitro are similar to those inferred by genetic analyses. We hypothesized that DinB-mediated bypass of 3-deaza-3-methyladenine, a stable analog of 3-methyladenine, the primary replication fork-stalling alkylation lesion, would be of high fidelity. We performed here the first kinetic analyses of E. coli DinB•RecA binary complexes. Whether alone or in a binary complex, DinB inserted the correct deoxyribonucleoside triphosphate (dNTP) opposite either lesion-containing or undamaged template; the incorporation of other dNTPs was largely inefficient. DinB prefers undamaged DNA, but the DinB•RecA binary complex increases its catalytic efficiency on lesion-containing template, perhaps as part of a regulatory mechanism to better respond to alkylation damage. Notably, we find that a DinB derivative with enhanced affinity for RecA, either alone or in a binary complex, is less efficient and has a lower fidelity than DinB or DinB•RecA. This finding contrasts our previous genetic analyses. Therefore, mutagenesis resulting from alkylation lesions is likely limited in cells by the activity of DinB•RecA. These two highly conserved proteins play an important role in maintaining genomic stability when cells are faced with ubiquitous DNA damage. Kinetic analyses are important to gain insights into the mechanism(s) regulating TLS DNA polymerases.

The activity of DinB is governed by the formation of a multiprotein complex (MPC) with RecA and UmuD. We identified two highly conserved surface residues in DinB, cysteine 66 (C66) and proline 67 (P67). Mapping on the DinB tertiary structure suggests these are noncatalytic, and multiple-sequence alignments indicate that they are unique among DinB-like proteins. To investigate the role of the C66-containing surface in MPC formation, we constructed the dinB(C66A) derivative. We found that DinB(C66A) copurifies with its interacting partners, RecA and UmuD, to a greater extent than DinB. Notably, copurification of RecA with DinB is somewhat enhanced in the absence of UmuD and is further increased for DinB(C66A). In vitro pulldown assays also indicate that DinB(C66A) binds RecA and UmuD better than DinB. We note that the increased affinity of DinB(C66A) for UmuD is RecA dependent. Thus, the C66-containing binding surface appears to be critical to modulate interaction with UmuD, and particularly with RecA. Expression of dinB(C66A) from the chromosome resulted in detectable differences in dinB-dependent lesion bypass fidelity and homologous recombination. Study of this DinB derivative has revealed a key surface on DinB, which appears to modulate the strength of MPC binding, and has suggested a binding order of RecA and UmuD to DinB. These findings will ultimately permit the manipulation of these enzymes to deter bacterial antibiotic resistance acquisition and to gain insights into cancer development in humans.

We report a powerful method to replace wild type genes on the chromosome of Escherichia coli. Employing a unique form of PCR, we generate easily constructible gene fusions bearing single point mutations. Used in conjunction with homologous recombination, this method eliminates cloning procedures previously used for this purpose.