Remote homology detection identifies a eukaryotic RPA DBD-C-like DNA binding domain as a conserved feature of archaeal Rpa1-like proteins

Open Access publication fees were provided by the University of St Andrews.The eukaryotic single-stranded DNA binding factor replication protein A (RPA) is essential for DNA replication, repair and recombination. RPA is a heterotrimer containing six related OB folds and a winged helix-turn-helix (wH) domain. The OB folds are designated DBD-A through DBD-F, with DBD-A through DBD-D being directly involved in ssDNA binding. DBD-C is located at the C-terminus of the RPA1 protein and has a distinctive structure that includes an integral C4 zinc finger, while the wH domain is found at the C-terminus of the RPA2 protein. Previously characterised archaeal RPA proteins fall into a number of classes with varying numbers of OB folds, but one widespread class includes proteins that contain a C4 or C3H zinc finger followed by a 100-120 amino acid C-terminal region reported to lack detectable sequence or structural similarity. Here, the sequences spanning this zinc finger and including the C-terminal region are shown to comprise a previously unrecognised DBD-C-like OB fold, confirming the evolutionary relatedness of this group of archaeal RPA proteins to eukaryotic RPA1. The evolutionary relationship between eukaryotic and archaeal RPA is further underscored by the presence of RPA2-like proteins comprising an OB fold and C-terminal winged helix (wH) domain in multiple species and crucially, suggests that several biochemically characterised archaeal RPA proteins previously thought to exist as monomers are likely to be RPA1-RPA2 heterodimers.Publisher PDFPeer reviewe

Genome Stability: A Self-Sufficient DNA Repair Machine

AbstractThe repair of DNA double-strand breaks often requires the broken ends to be processed prior to religation. New results describe a bacterial enzyme with processing and rejoining activities encoded in a single polypeptide chain

Genome Sequencing: And Then There Were Six

AbstractThe genome of the fission yeast Schizosaccharomyces pombe has been sequenced, bringing the number of sequenced eukaryotic genomes to six. Analysis of the sequence predicts only 4824 protein coding genes, the smallest number yet recorded for a free-living eukaryote

PCNA-binding proteins in the archaea : novel functionality beyond the conserved core

Sliding clamps play an essential role in coordinating protein activity in DNA metabolism in all three domains of life. In eukaryotes and archaea the sliding clamp is PCNA (proliferating cell nuclear antigen). Across the diversity of the archaea PCNA interacts with a highly conserved set of proteins with key roles in DNA replication and repair, including DNA polymerases B and D, replication factor C, the Fen1 nuclease and RNAseH2, but this core set of factors is likely to represent a fraction of the PCNA interactome only. Here I review three recently characterised non-core archaeal PCNA binding proteins NusS, NreA/NreB and TIP, highlighting what is known of their interactions with PCNA and their functions in vivo and in vitro. Gaining a detailed understanding of the non-core PCNA interactome will provide significant insights into key aspects of chromosome biology in divergent archaeal lineages.Publisher PDFPeer reviewe

Inhibiting NAD+-dependent DNA ligase activity with 2-(cyclopentyloxy)-5'-deoxyadenosine (CPOdA) offers a new tool for DNA replication and repair studies in the model archaeon Haloferax volcanii

DNA ligases play an essential role in many aspects of DNA metabolism in all three domains of life. The haloarchaeal organism Haloferax volcanii encodes both ATP- and NAD+-dependent DNA ligase enzymes designated LigA and LigN, respectively. Neither LigA nor LigN alone is required for cell viability but they share an essential function, most likely the ligation of Okazaki fragments during chromosome replication. Here we show that 2-(cyclopentyloxy)-5’-deoxyadenosine (referred to as CPOdA), originally developed as a inhibitor of bacterial NAD+-dependent DNA ligases, is a potent inhibitor of the growth of Hfx. volcanii cells expressing LigN alone, causing chromosome fragmentation and cell death, while cells expressing LigA are unaffected. Growth inhibition occurs at significantly lower CPOdA concentrations (MIC ≤ 50 ng/ml) than those required for inhibition of bacterial growth (≥ 2 µg/ml). CPOdA has the potential to become a vital tool in DNA replication and repair studies in this important model organism.PostprintPeer reviewe

Biochemical characterisation of LigN, an NAD(+)-dependent DNA ligase from the halophilic euryarchaeon Haloferax volcanii that displays maximal in vitro activity at high salt concentrations

BACKGROUND: DNA ligases are required for DNA strand joining in all forms of cellular life. NAD(+)-dependent DNA ligases are found primarily in eubacteria but also in some eukaryotic viruses, bacteriophage and archaea. Among the archaeal NAD(+)-dependent DNA ligases is the LigN enzyme of the halophilic euryarchaeon Haloferax volcanii, the gene for which was apparently acquired by Hfx.volcanii through lateral gene transfer (LGT) from a halophilic eubacterium. Genetic studies show that the LGT-acquired LigN enzyme shares an essential function with the native Hfx.volcanii ATP-dependent DNA ligase protein LigA. RESULTS: To characterise the enzymatic properties of the LigN protein, wild-type and three mutant forms of the LigN protein were separately expressed in recombinant form in E.coli and purified to apparent homogeneity by immobilised metal ion affinity chromatography (IMAC). Non-isotopic DNA ligase activity assays using λ DNA restriction fragments with 12 bp cos cohesive ends were used to show that LigN activity was dependent on addition of divalent cations and salt. No activity was detected in the absence of KCl, whereas maximum activity could be detected at 3.2 M KCl, close to the intracellular KCl concentration of Hfx.volcanii cells. CONCLUSION: LigN is unique amongst characterised DNA ligase enzymes in displaying maximal DNA strand joining activity at high (> 3 M) salt levels. As such the LigN enzyme has potential both as a novel tool for biotechnology and as a model enzyme for studying the adaptation of proteins to high intracellular salt levels

ATP-dependent DNA ligases

By catalyzing the joining of breaks in the phosphodiester backbone of duplex DNA, DNA ligases play a vital role in the diverse processes of DNA replication, recombination and repair. Three related classes of ATP-dependent DNA ligase are readily apparent in eukaryotic cells. Enzymes of each class comprise catalytic and non-catalytic domains together with additional domains of varying function. DNA ligase I is required for the ligation of Okazaki fragments during lagging-strand DNA synthesis, as well as for several DNA-repair pathways; these functions are mediated, at least in part, by interactions between DNA ligase I and the sliding-clamp protein PCNA. DNA ligase III, which is unique to vertebrates, functions both in the nucleus and in mitochondria. Two distinct isoforms of this enzyme, differing in their carboxy-terminal sequences, are produced by alternative splicing: DNA ligase IIIα has a carboxy-terminal BRCT domain that interacts with the mammalian DNA-repair factor XrccI, but both α and β isoforms have an amino-terminal zinc-finger motif that appears to play a role in the recognition of DNA secondary structures that resemble intermediates in DNA metabolism. DNA ligase IV is required for DNA non-homologous end joining pathways, including recombination of the V(D)J immunoglobulin gene segments in cells of the mammalian immune system. DNA ligase IV forms a tight complex with Xrcc4 through an interaction motif located between a pair of carboxy-terminal BRCT domains in the ligase. Recent structural studies have shed light on the catalytic function of DNA ligases, as well as illuminating protein-protein interactions involving DNA ligases IIIα and IV

Non‐canonical binding of the Chaetomium thermophilum PolD4 N‐terminal PIP motif to PCNA involves Q‐pocket and compact 2‐fork plug interactions but no 310 helix

Funding: Carnegie Trust for the Universities of Scotland (Grant Number(s): 70668). Chinese Scholarship Council.DNA polymerase δ (Pol δ) is a key enzyme for the maintenance of genome integrity in eukaryotic cells, acting in concert with the sliding clamp processivity factor PCNA (proliferating cell nuclear antigen). Three of the four subunits of human Pol δ interact directly with the PCNA homotrimer via a short, conserved protein sequence known as a PCNA interacting protein (PIP) motif. Here, we describe the identification of a PIP motif located towards the N-terminus of the PolD4 subunit of Pol δ (equivalent to human p12) from the thermophilic filamentous fungus Chaetomium thermophilum and present the X-ray crystal structure of the corresponding peptide bound to PCNA at 2.45 Å. Like human p12, the fungal PolD4 PIP motif displays non-canonical binding to PCNA. However, the structures of the human p12 and fungal PolD4 PIP motif peptides are quite distinct, with the fungal PolD4 PIP motif lacking the 310 helical segment that characterises most previously identified PIP motifs. Instead, the fungal PolD4 PIP motif binds PCNA via a conserved glutamine that inserts into the Q-pocket on the surface of PCNA and with conserved leucine and phenylalanine sidechains forming a compact 2-fork plug that inserts into the hydrophobic pocket on PCNA. Despite the unusual binding mode of the fungal PolD4, isothermal calorimetry (ITC) measurements show that its affinity for PCNA is similar to that of its human orthologue. These observations add to a growing body of information on how diverse proteins interact with PCNA and highlight how binding modes can vary significantly between orthologous PCNA partner proteins.Publisher PDFPeer reviewe

Canonical binding of Chaetomium thermophilum DNA polymerase δ/ζ subunit PolD3 and flap endonuclease Fen1 to PCNA

Funding: This work was funded by the Carnegie Trust for the Universities of Scotland through a Research Incentive Grant (grant reference 70668) and by the School of Biology, University of St Andrews. Article processing charges (APCs) and Open Access charges were covered by the University of St Andrews.The sliding clamp PCNA is a key player in eukaryotic genome replication and stability, acting as a platform onto which components of the DNA replication and repair machinery are assembled. Interactions with PCNA are frequently mediated via a short protein sequence motif known as the PCNA-interacting protein (PIP) motif. Here we describe the binding mode of a PIP motif peptide derived from C-terminus of the PolD3 protein from the thermophilic ascomycete fungus C. thermophilum, a subunit of both DNA polymerase δ (Pol δ) and the translesion DNA synthesis polymerase Pol ζ, characterised by isothermal titration calorimetry (ITC) and protein X-ray crystallography. In sharp contrast to the previously determined structure of a Chaetomium thermophilum PolD4 peptide bound to PCNA, binding of the PolD3 peptide is strictly canonical, with the peptide adopting the anticipated 310 helix structure, conserved Gln441 inserting into the so-called Q-pocket on PCNA, and Ile444 and Phe448 forming a two-fork plug that inserts into the hydrophobic surface pocket on PCNA. The binding affinity for the canonical PolD3 PIP-PCNA interaction determined by ITC is broadly similar to that previously determined for the non-canonical PolD4 PIP-PCNA interaction. In addition, we report the structure of a PIP peptide derived from the C. thermophilum Fen1 nuclease bound to PCNA. Like PolD3, Fen1 PIP peptide binding to PCNA is achieved by strictly canonical means. Taken together, these results add to an increasing body of information on how different proteins bind to PCNA, both within and across species.Publisher PDFPeer reviewe

The archaeal RecJ-like proteins : nucleases and ex-nucleases with diverse roles in replication and repair

RecJ proteins belong to the DHH superfamily of phosphoesterases that has members in all three domains of life. In bacteria, the archetypal RecJ is a 5’→3’ ssDNA exonuclease that functions in homologous recombination, base excision repair and mismatch repair, while in eukaryotes, the RecJ-like protein Cdc45 (which has lost its nuclease activity) is a key component of the CMG (Cdc45-MCM-GINS) complex, the replicative DNA helicase that unwinds double-stranded DNA at the replication fork. In archaea, database searching identifies genes encoding one or more RecJ family proteins in almost all sequenced genomes. Biochemical analysis has confirmed that some but not all of these proteins are components of archaeal CMG complexes and has revealed a surprising diversity in mode of action and substrate preference. In addition to this, some archaea encode catalytically inactive RecJ-like proteins, and others a mix of active and inactive proteins, with the inactive proteins being confined to structural roles only. Here, I summarise current knowledge of the structure and function of the archaeal RecJ-like proteins, focusing on similarities and differences between proteins from different archaeal species, between proteins within species, and between the archaeal proteins and their bacterial and eukaryotic relatives. Models for RecJ-like function are described and key areas for further study highlighted.PostprintPeer reviewe