238 research outputs found
Composition and Dynamics of the Eukaryotic Replisome: A Brief Overview
No full textHigh-fidelity chromosomal DNA replication is vital for maintaining the integrity of the genetic material in all forms of cellular life. In eukaryotic cells, around 40-50 distinct conserved polypeptides are essential for chromosome replication, the majority of which are themselves component parts of a series of elaborate molecular machines that comprise the replication apparatus or replisome. How these complexes are assembled, what structures they adopt, how they perform their functions, and how those functions are regulated, are key questions for understanding how genome duplication occurs. Here I present a brief overview of current knowledge of the dynamic molecular events underlying chromosomal DNA replication in eukaryotic cells and how these events are regulated
Purification and functional inactivation of the fission yeast MCMMCM–BP complex
No full textAbstractThe MCM (mini-chromosome maintenance) complex is the core of the eukaryotic replicative helicase and comprises six proteins, Mcm2–Mcm7. In humans, a variant form of the complex has Mcm2 replaced by the MCM–BP protein. Recent results suggest that a similar complex exists in fission yeast with an essential role in DNA replication and cell cycle progression. Here, we describe the purification and subunit composition of the fission yeast MCMMcb1 complex. Using newly generated temperature-sensitive alleles, we show that loss of MCMMcb1 function leads to accumulation of DNA damage, checkpoint activation and cell cycle arrest, and provide evidence for a role for MCMMcb1 in meiosis.Structured summary of protein interactionsMcb1 physically interacts with Mcm4 by pull down (View interaction)Mcb1 physically interacts with Mcm5 by pull down (View interaction)Mcb1 physically interacts with Mcm6 by pull down (View interaction)Mcm4 and Mcb1 physically interact by bimolecular fluorescence complementation (View interaction)Mcb1 physically interacts with Mcm3, Mcm4, Mcm5, Mcm6 and Mcm7 by tandem affinity purification (View interaction)Mcb1 physically interacts with Mcm7 by pull down (View interaction
Genetics of lagging strand DNA synthesis and maturation in fission yeast:suppression analysis links the Dna2-Cdc24 complex to DNA polymerase delta
No full textThe Cdc24 protein is essential for the completion of chromosomal DNA replication in fission yeast. Although its precise role in this process is unclear, Cdc24 forms a complex with Dna2, a conserved endonuclease-helicase implicated in the removal of the RNA-DNA primer during Okazaki fragment processing. To gain further insights into Cdc24-Dna2 function, we screened for chromosomal suppressors of the temperature-sensitive cdc24-M38 allele and mapped the suppressing mutations into six complementation groups. Two of these mutations defined genes encoding the Pol3 and Cdc27 subunits of DNA polymerase delta. Sequence analysis revealed that all the suppressing mutations in Cdc27 resulted in truncation of the protein and loss of sequences that included the conserved C-terminal PCNA binding motif, previously shown to play an important role in maximizing enzyme processivity in vitro. Deletion of this motif is shown to be sufficient for suppression of both cdc24-M38 and dna2-C2, a temperature-sensitive allele of dna2(+), suggesting that disruption of the interaction between Cdc27 and PCNA renders the activity of the Cdc24-Dna2 complex dispensable.</p
The archaeo-eukaryotic GINS proteins and the archaeal primase catalytic subunit PriS share a common domain
No full textThis work was funded by the Scottish Universities Life Sciences Alliance (SULSA).Primase and GINS are essential factors for chromosomal DNA replication in eukaryotic and archaeal cells. Here we describe a previously undetected relationship between the C-terminal domain of the catalytic subunit (PriS) of archaeal primase and the B-domains of the archaeo-eukaryotic GINS proteins in the form of a conserved structural domain comprising a three-stranded antiparallel beta-sheet adjacent to an alpha-helix and a two-stranded beta-sheet or hairpin. The presence of a shared domain in archaeal PriS and GINS proteins, the genes for which are often found adjacent on the chromosome, suggests simple mechanisms for the evolution of these proteins.Peer reviewe
The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines
No full textThe hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies.Peer reviewe
The fission yeast <em>pfh1</em> gene encodes an essential 5' to 3' DNA helicase required for the completion of S-phase
No full textThe Cdc24 protein plays an essential role in chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe, most likely via its direct interaction with Dna2, a conserved endonuclease-helicase protein required for Okazaki fragment processing. To gain insights into Cdc24 function, we isolated cold-sensitive chromosomal suppressors of the temperature-sensitive cdc24-M38 allele. One of the complementation groups of such suppressors defined a novel gene, pfh1(+), encoding an 805 amino acid nuclear protein highly homologous to the Saccharomyces cerevisiae Pif1p and Rrm3p DNA helicase family proteins. The purified Pfh1 protein displayed single-stranded DNA-dependent ATPase activity as well as 5' to 3' DNA helicase activity in vitro. Reverse genetic analysis in S.pombe showed that helicase activity was essential for the function of the Pfh1 protein in vivo. Schizosaccharomyces pombe cells carrying the cold-sensitive pfh1-R20 allele underwent cell cycle arrest in late S/G2-phase of the cell cycle when shifted to the restrictive temperature. This arrest was dependent upon the presence of a functional late S/G2 DNA damage checkpoint, suggesting that Pfh1 is required for the comple tion of DNA replication. Furthermore, at their permissive temperature pfh1-R20 cells were highly sensitive to the DNA-alkylating agent methyl methanesulphonate, implying a further role for Pfh1 in the repair of DNA damage.</p
Molecular biology of giant viruses' DNA replication machinery
No full textViruses are the most widespread and abundant entity on this planet, further constituting the largest part of the genosphere. The majority of these infectious agents are miniature, having been described as being smaller than the smallest bacteria. Even though they encode a limited number of viral proteins, they still obtain the bulk of the material they require for their replication and propagation from the infected host cell. Recently, this traditional concept of viruses has been shaken up by the breakthrough finding of a new group of viruses, the Giant Viruses. They have been assigned this definition due to their amazingly and surprisingly large genomic size. The vast majority have their own replication machinery. They have been discovered in the sea, where they prefer to infect amoebas and other marine microorganisms. For the purpose of this study, we focused on three of these giant viruses; Mimivirus, Marseillevirus, and Cafeteria roenbergensis virus (CroV). The aim of the study was to comprehend how these giant viruses replicate and propagate their genetic material through the generations, to have reached a point where their genome size is comparable to normal-sized bacteria. For this reason, an extensive biochemical analysis on the molecular biology of giant viruses' DNA replication machinery was performed, hoping to obtain new insights into the evolution and lifestyle of these unique viruses. We specifically focused on what we considered to be two of the most important DNA replication proteins; the Proliferating Cell Nuclear Antigen (PCNA) and Flap structure-specific Endonuclease 1 (FEN1). Our goal was to determine their properties. The protocols performed were a series of protein expression procedures, during which the particular synthetic genes were cloned in a selection of expression vectors and were then expressed in bacteria (i.e. E.coli host expression strains). Depending on the protein expression efficiencies, some trial protein purification procedures followed. For the first few months of the project, however, it was impossible to obtain any conclusive results concerning the expression of the proteins. The synthetic genes were proving to be extremely difficult to express in vectors containing an expression tag. Only when we switched to un-tagged expression vectors, much later on in the project, did we start getting better and more promising results. This was a particularly useful outcome in itself, as it revealed that enhanced expression of the PCNA and FEN1 proteins preferentially occurs when no expression tags are present. Towards the end of the project, some protein purification trials were performed, but unfortunately they only resulted in an incredibly low protein purity level. The discovery of these distinctive viruses has not only incited scientists to maybe rethink and change their view about the general nature of viruses, but it has also begun to alter and question the outlook regarding the history of life as a whole. As the investigation is still in its very early stages, there are many aspects concerning the giant viruses still to be discovered. This in the end could essentially teach us a great deal more than we ever hoped to expect, and therefore it is of great significance and importance to continue with this research
The eukaryotic replisome: a guide to protein structure and function
No full textSuccessful chromosome replication is vital for maintaining the integrity of the genetic material in all forms of cellular life. In humans, there are clear links between chromosome replication defects and genome instability, genetic disease and cancer, making a detailed understanding of the molecular mechanisms of genome duplication vital for future advances in diagnosis and treatment. Inspired by recent exciting breakthroughs in protein structure determination and written by leading experts in the field, The Eukaryotic Replisome: a guide to protein structure and function takes the reader on a guided journey through the intricate molecular machinery of eukaryotic chromosomal DNA replication, from replication origin recognition and the assembly of the pre-replicative complexes in G1 through to the final processing of Okazaki fragments at the end of S-phase. This extensively illustrated book is an invaluable source of information, ideas and inspiration for all those with an interest in chromosome replication, whether from a basic science, translational biology and medical research perspective
The origin recognition complex: a biochemical and structural view
No full textThe origin recognition complex (ORC) was first discovered in the baker's yeast in 1992. Identification of ORC opened up a path for subsequent molecular level investigations on how eukaryotic cells initiate and control genome duplication each cell cycle. Twenty years after the first biochemical isolation, ORC is now taking on a three-dimensional shape, although a very blurry shape at the moment, thanks to the recent electron microscopy and image reconstruction efforts. In this chapter, we outline the current biochemical knowledge about ORC from several eukaryotic systems, with emphasis on the most recent structural and biochemical studies. Despite many species-specific properties, an emerging consensus is that ORC is an ATP-dependent machine that recruits other key proteins to form pre-replicative complexes (pre-RCs) at many origins of DNA replication, enabling the subsequent initiation of DNA replication in S phase
The eukaryotic replisome: a guide to protein structure and function
No full textSuccessful chromosome replication is vital for maintaining the integrity of the genetic material in all forms of cellular life. In humans, there are clear links between chromosome replication defects and genome instability, genetic disease and cancer, making a detailed understanding of the molecular mechanisms of genome duplication vital for future advances in diagnosis and treatment. Inspired by recent exciting breakthroughs in protein structure determination and written by leading experts in the field, The Eukaryotic Replisome: a guide to protein structure and function takes the reader on a guided journey through the intricate molecular machinery of eukaryotic chromosomal DNA replication, from replication origin recognition and the assembly of the pre-replicative complexes in G1 through to the final processing of Okazaki fragments at the end of S-phase. This extensively illustrated book is an invaluable source of information, ideas and inspiration for all those with an interest in chromosome replication, whether from a basic science, translational biology and medical research perspective
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