Stuart A. MacNeill

ATP-dependent DNA ligases

SummaryBy 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.

The eukaryotic replicative DNA polymerases take shape

Three multi-subunit DNA polymerase enzymes lie at the heart of the chromosome replication machinery in the eukaryotic cell nucleus. Through a combination of genetic, molecular biological and biochemical analysis, significant advances have been made in understanding the essential roles played by each of these enzymes at the replication fork. Until very recently, however, little information was available on their three-dimensional structures. Lately, a series of crystallographic and electron microscopic studies has been published, allowing the structures of the complexes and their constituent subunits to be visualised in detail for the first time. Taken together, these studies provide significant insights into the molecular makeup of the replication machinery in eukaryotic cells and highlight a number of key areas for future investigation.

THE FISSION YEAST CDC1 PROTEIN, A HOMOLOGUE OF THE SMALL SUBUNIT OF DNA POLYMERASE DELTA , BINDS TO POL3 AND CDC27

cdc1+ is required for cell cycle progression in Schizosaccharomyces pombe. Cells carrying temperature‐sensitive cdc1 mutants undergo cell cycle arrest when shifted to the restrictive temperature, becoming highly elongated. Here we describe the cloning and sequencing of cdc1+, which is shown to encode a 462 residue protein that displays significant sequence similarity to the small subunit of mammalian DNA polymerase delta. cdc1+ interacts genetically with pol3+, which encodes the large subunit of DNA polymerase delta in fission yeast, and the Cdc1 protein binds to Pol3 in vitro, strongly suggesting that Cdc1 is likely to be the small subunit of Pol delta. In addition, we show that cdc1+ overexpression is sufficient to rescue cells carrying temperature‐sensitive cdc27 alleles and that the Cdc1 and Cdc27 proteins interact in vivo and in vitro. Deletion of either cdc1+ or cdc27+ results in cell cycle arrest with the arrested cells having a single nucleus with 2C DNA content. No evidence was obtained for a cut phenotype, indicating that neither cdc1+ nor cdc27+ is required for checkpoint function. cdc1 mutant cells are supersensitive to the DNA synthesis inhibitor hydroxyurea and to the DNA damaging agent MMS, display increased frequency of mini‐chromosome loss and have an extended S phase.

Structure and function of the GINS complex, a key component of the eukaryotic replisome.

High-fidelity chromosomal DNA replication is fundamental to all forms of cellular life and requires the complex interplay of a wide variety of essential and non-essential protein factors in a spatially and temporally co-ordinated manner. In eukaryotes, the GINS complex (from the Japanese go-ichi-ni-san meaning 5-1-2-3, after the four related subunits of the complex Sld5, Psf1, Psf2 and Psf3) was recently identified as a novel factor essential for both the initiation and elongation stages of the replication process. Biochemical analysis has placed GINS at the heart of the eukaryotic replication apparatus as a component of the CMG [Cdc45-MCM (minichromosome maintenance) helicase-GINS] complex that most likely serves as the replicative helicase, unwinding duplex DNA ahead of the moving replication fork. GINS homologues are found in the archaea and have been shown to interact directly with the MCM helicase and with primase, suggesting a central role for the complex in archaeal chromosome replication also. The present review summarizes current knowledge of the structure, function and evolution of the GINS complex in eukaryotes and archaea, discusses possible functions of the GINS complex and highlights recent results that point to possible regulation of GINS function in response to DNA damage.

Essential interaction between the fission yeast DNA polymerase δ subunit Cdc27 and Pcn1 (PCNA) mediated through a C-terminal p21Cip1-like PCNA binding motif

Direct interaction between DNA polymerase δ and its processivity factor proliferating cell nuclear antigen (PCNA) is essential for effective replication of the eukaryotic genome, yet the precise manner by which this occurs is unclear. We show that the 54 kDa subunit of DNA polymerase δ from Schizosaccharomyces pombe interacts directly with Pcn1 (PCNA) both in vivo and in vitro. Binding is effected via a short sequence at the C‐terminus of Cdc27 with significant similarity to the canonical PCNA binding motif first identified in the mammalian p21Cip1 protein. This motif is both necessary and sufficient for binding of Pcn1 by Cdc27 in vitro and is essential for Cdc27 function in vivo. We also show that the Pcn1 binding motif in Cdc27 is distinct from its binding site for Cdc1, the 55 kDa B‐subunit of polymerase δ, and present evidence that Cdc27 can bind to Pcn1 and Cdc1 simultaneously. Finally, we show that Cdc27 performs at least two distinct essential functions, one of which is independent of Pcn1 binding.

DNA replication: Partners in the Okazaki two-step

The correct processing of Okazaki fragments during lagging-strand DNA replication has a vital role in maintaining genome integrity. Recent findings suggest that, in eukaryotes, the processing of Okazaki fragments occurs by a two-step mechanism governed by the single-stranded DNA binding factor RPA.

Cdm1, the smallest subunit of DNA polymerase d in the fission yeast Schizosaccharomyces pombe, is non-essential for growth and division.

Abstract Highly purified DNA polymerase δ from the fission yeast Schizosaccharomyces pombe is a complex of at least four distinct subunits. Genes encoding three of these (pol3+/cdc6+, cdc1+ and cdc27+) have been characterised previously. Here we describe the isolation and characterisation of cdm1+, the gene encoding the smallest (22kDa) subunit of the Pol δ complex. Over-expression of cdm1+, which encodes a 160 amino-acid protein with no significant sequence similarity to proteins in current databases, is able to rescue cells carrying temperature-sensitive mutations in either pol3+/cdc6+, cdc1+ or cdc27+. Cells deleted for cdm1+ are viable, indicating that cdm1+ is non-essential for mitotic growth, and are no more sensitive to a variety of DNA replication inhibitors and DNA damaging agents than are wild-type cells. In addition, over-expression of cdm1+ suppresses the temperature-sensitive cdc24-M38 mutant suggesting that cdc24+ may also have a role in DNA polymerase δ function.

Genome Stability: A New Member of the RFC family

Three distinct forms of replication factor C are known to play vital roles in genome replication and integrity in eukaryotic cells. A fourth such complex has recently been identified; initial results suggest that this new family member plays an important role during S phase.

ATP‐ and NAD+‐dependent DNA ligases share an essential function in the halophilic archaeon Haloferax volcanii

DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP‐dependent ligases are found predominantly in the eukarya and archaea whereas NAD+‐dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile Haloferax volcanii as a model system, we describe the first genetic analysis of archaeal DNA ligase function. We show that the Hfx. volcanii ATP‐dependent DNA ligase family member, LigA, is non‐essential for cell viability, raising the question of how DNA strands are joined in its absence. We show that Hfx. volcanii also encodes an NAD+‐dependent DNA ligase family member, LigN, the first such enzyme to be identified in the archaea, and present phylogenetic analysis indicating that the gene encoding this protein has been acquired by lateral gene transfer (LGT) from eubacteria. As with LigA, we show that LigN is also non‐essential for cell viability. Simultaneous inactivation of both proteins is lethal, however, indicating that they now share an essential function. Thus the LigN protein acquired by LGT appears to have been co‐opted as a back‐up for LigA function, perhaps to provide additional ligase activity under conditions of high genotoxic stress.

Identification of essential and non-essential single-stranded DNA-binding proteins in a model archaeal organism

Single-stranded DNA-binding proteins (SSBs) play vital roles in all aspects of DNA metabolism in all three domains of life and are characterized by the presence of one or more OB fold ssDNA-binding domains. Here, using the genetically tractable euryarchaeon Haloferax volcanii as a model, we present the first genetic analysis of SSB function in the archaea. We show that genes encoding the OB fold and zinc finger-containing RpaA1 and RpaB1 proteins are individually non-essential for cell viability but share an essential function, whereas the gene encoding the triple OB fold RpaC protein is essential. Loss of RpaC function can however be rescued by elevated expression of RpaB, indicative of functional overlap between the two classes of haloarchaeal SSB. Deletion analysis is used to demonstrate important roles for individual OB folds in RpaC and to show that conserved N- and C-terminal domains are required for efficient repair of DNA damage. Consistent with a role for RpaC in DNA repair, elevated expression of this protein leads to enhanced resistance to DNA damage. Taken together, our results offer important insights into archaeal SSB function and establish the haloarchaea as a valuable model for further studies.