Understanding DNA replication by the bacteriophage T4 replisome.
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ABSTRACT:
SUBMITTER: Benkovic SJ
PROVIDER: S-EPMC5682956 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The bacteriophage T4 encodes 10 proteins, known collectively as the replisome, that are responsible for the replication of the phage genome. The replisomal proteins can be subdivided into three activities; the replicase, responsible for duplicating DNA, the primosomal proteins, responsible for unwinding and Okazaki fragment initiation, and the Okazaki repair proteins. The replicase includes the gp43 DNA polymerase, the gp45 processivity clamp, the gp44/62 clamp loader complex, and the gp32 single-stranded DNA binding protein. The primosomal proteins include the gp41 hexameric helicase, the gp61 primase, and the gp59 helicase loading protein. The RNaseH, a 5' to 3' exonuclease and T4 DNA ligase comprise the activities necessary for Okazaki repair. The T4 provides a model system for DNA replication. As a consequence, significant effort has been put forth to solve the crystallographic structures of these replisomal proteins. In this review, we discuss the structures that are available and provide comparison to related proteins when the T4 structures are unavailable. Three of the ten full-length T4 replisomal proteins have been determined; the gp59 helicase loading protein, the RNase H, and the gp45 processivity clamp. The core of T4 gp32 and two proteins from the T4 related phage RB69, the gp43 polymerase and the gp45 clamp are also solved. The T4 gp44/62 clamp loader has not been crystallized but a comparison to the E. coli gamma complex is provided. The structures of T4 gp41 helicase, gp61 primase, and T4 DNA ligase are unknown, structures from bacteriophage T7 proteins are discussed instead. To better understand the functionality of T4 DNA replication, in depth structural analysis will require complexes between proteins and DNA substrates. A DNA primer template bound by gp43 polymerase, a fork DNA substrate bound by RNase H, gp43 polymerase bound to gp32 protein, and RNase H bound to gp32 have been crystallographically determined. The preparation and crystallization of complexes is a significant challenge. We discuss alternate approaches, such as small angle X-ray and neutron scattering to generate molecular envelopes for modeling macromolecular assemblies.
| S-EPMC3012046 | biostudies-literature
Project description:The genomes of bacteriophages T4 and RB69 are phylogenetically related but diverge in nucleotide sequence at many loci and are incompatible with each other in vivo. We describe here the biological implications of divergence in a genomic segment that encodes four essential DNA replication proteins: gp45 (sliding clamp), gp44/62 complex (clamp loader), and gp46 (a recombination protein). We have cloned, sequenced, and expressed several overlapping segments of the RB69 gene 46-45.2-(rpbA)-45-44-62 cluster and compared its features to those of the homologous gene cluster from T4. The deduced primary structures of all four RB69 replication proteins and gp45.2 from this cluster are very similar (80 to 95% similarity) to those of their respective T4 homologs. In contrast, the rpbA region (which encodes a nonessential protein in T4) is highly diverged (approximately 49% similarity) between the two phage genomes and does not encode protein in RB69. Expression studies and patterns of high divergence of intercistronic nucleotide sequences of this cluster suggest that T4 and RB69 evolved similar transcriptional and translational control strategies for the cistrons contained therein, but with different specificities. In plasmid-phage complementation assays, we show that posttranslationally, RB69 and T4 homologs of gp45 and the gp44/62 complex can be effectively exchanged between the two phage replicase assemblies; however, we also show results which suggest that mixed clamp loader complexes consisting of T4 gp62 and RB69 gp44 subunits are not active for phage DNA replication. Thus, specificity of the gp44-gp62 interaction in the clamp loader marks a point of departure between the T4 and RB69 replication systems.
| S-EPMC107123 | biostudies-literature
Project description:Double-strand break (DSB) repair and DNA replication are tightly linked in the life cycle of bacteriophage T4. Indeed, the major mode of phage DNA replication depends on recombination proteins and can be stimulated by DSBs. DSB-stimulated DNA replication is dramatically demonstrated when T4 infects cells carrying two plasmids that share homology. A DSB on one plasmid triggered extensive replication of the second plasmid, providing a useful model for T4 recombination-dependent replication (RDR). This system also provides a view of DSB repair in T4-infected cells and revealed that the DSB repair products had been replicated in their entirety by the T4 replication machinery. We analyzed the detailed structure of these products, which do not fit the simple predictions of any of three models for DSB repair. We also present evidence that the T4 RDR system functions to restart stalled or inactivated replication forks. First, we review experiments involving antitumor drug-stabilized topoisomerase cleavage complexes. The results suggest that forks blocked at cleavage complexes are resolved by recombinational repair, likely involving RDR. Second, we show here that the presence of a T4 replication origin on one plasmid substantially stimulated recombination events between it and a homologous second plasmid that did not contain a T4 origin. Furthermore, replication of the second plasmid was increased when the first plasmid contained the T4 origin. Our interpretation is that origin-initiated forks become inactivated at some frequency during replication of the first plasmid and are then restarted via RDR on the second plasmid.
| S-EPMC37434 | biostudies-literature
Project description:After two converging DNA replication forks meet, active replisomes are disassembled and unloaded from chromatin. A key process in replisome disassembly is the unloading of CMG helicases (CDC45-MCM-GINS), which is initiated in Caenorhabditis elegans and Xenopus laevis by the E3 ubiquitin ligase CRL2LRR1. Here, we show that human cells lacking LRR1 fail to unload CMG helicases and accumulate increasing amounts of chromatin-bound replisome components as cells progress through S phase. Markedly, we demonstrate that the failure to disassemble replisomes reduces the rate of DNA replication increasingly throughout S phase by sequestering rate-limiting replisome components on chromatin and blocking their recycling. Continued binding of CMG helicases to chromatin during G2 phase blocks mitosis by activating an ATR-mediated G2/M checkpoint. Finally, we provide evidence that LRR1 is an essential gene for human cell division, suggesting that CRL2LRR1 enzyme activity is required for the proliferation of cancer cells and is thus a potential target for cancer therapy.
| S-EPMC8160578 | biostudies-literature
Project description:DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression as well as bacterial virulence. We report here the crystal structures of the bacteriophage T4Dam DNA adenine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy) and in a ternary complex with a synthetic 12-bp DNA duplex and AdoHcy. T4Dam contains two domains: a seven-stranded catalytic domain that harbors the binding site for AdoHcy and a DNA binding domain consisting of a five-helix bundle and a beta-hairpin that is conserved in the family of GATC-related MTase orthologs. Unexpectedly, the sequence-specific T4Dam bound to DNA in a nonspecific mode that contained two Dam monomers per synthetic duplex, even though the DNA contains a single GATC site. The ternary structure provides a rare snapshot of an enzyme poised for linear diffusion along the DNA.
| S-EPMC4030375 | biostudies-literature
Project description:Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%-25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles.
| S-EPMC3039672 | biostudies-literature
Project description:Nucleosome assembly during DNA replication is tightly coupled to ongoing DNA synthesis. This process, termed DNA replication-coupled (RC) nucleosome assembly, is essential for chromatin replication and has a great impact on both genome stability maintenance and epigenetic inheritance. This review discusses a set of recent findings regarding the role of replisome components contributing to RC nucleosome assembly. Starting with a brief introduction to the factors involved in nucleosome assembly and some aspects of the architecture of the eukaryotic replisome, we discuss studies from yeast to mammalian cells and the interactions of replisome components with histones and histone chaperones. We describe the proposed functions of replisome components during RC nucleosome assembly and discuss their impacts on histone segregation and implications for epigenetic inheritance.
| S-EPMC7066812 | biostudies-literature
Project description:UNLABELLED:The genomic DNAs of tailed bacteriophages are commonly modified by the attachment of chemical groups. Some forms of DNA modification are known to protect phage DNA from cleavage by restriction enzymes, but others are of unknown function. Recently, the CRISPR-Cas nuclease complexes were shown to mediate bacterial adaptive immunity by RNA-guided target recognition, raising the question of whether phage DNA modifications may also block attack by CRISPR-Cas9. We investigated phage T4 as a model system, where cytosine is replaced with glucosyl-hydroxymethylcytosine (glc-HMC). We first quantified the extent and distribution of covalent modifications in T4 DNA by single-molecule DNA sequencing and enzymatic probing. We then designed CRISPR spacer sequences targeting T4 and found that wild-type T4 containing glc-HMC was insensitive to attack by CRISPR-Cas9 but mutants with unmodified cytosine were sensitive. Phage with HMC showed only intermediate sensitivity. While this work was in progress, another group reported examples of heavily engineered CRISRP-Cas9 complexes that could, in fact, overcome the effects of T4 DNA modification, indicating that modifications can inhibit but do not always fully block attack. IMPORTANCE:Bacteria were recently found to have a form of adaptive immunity, the CRISPR-Cas systems, which use nucleic acid pairing to recognize and cleave genomic DNA of invaders such as bacteriophage. Historic work with tailed phages has shown that phage DNA is often modified by covalent attachment of large chemical groups. Here we demonstrate that DNA modification in phage T4 inhibits attack by the CRISPR-Cas9 system. This finding provides insight into mechanisms of host-virus competition and also a new set of tools that may be useful in modulating the activity of CRISPR-Cas9 in genome engineering applications.
| S-EPMC4471564 | biostudies-literature
Project description:Bacteriophage T4 is a large-tailed Escherichia coli virus whose capsid is 120x86 nm. ATP-driven DNA packaging of the T4 capsid results in the loading of a 171-kb genome in less than 5 min during viral infection. We have isolated 50-mg quantities of uniform (15)N- and [epsilon-(15)N]lysine-labeled bacteriophage T4. We have also introduced (15)NH(4)(+) into filled, unlabeled capsids from synthetic medium by exchange. We have examined lyo- and cryoprotected lyophilized T4 using (15)N{(31)P} and (31)P{(15)N} rotational-echo double resonance. The results of these experiments have shown that (i) packaged DNA is in an unperturbed duplex B-form conformation; (ii) the DNA phosphate negative charge is balanced by lysyl amines (3.2%), polyamines (5.8%), and monovalent cations (40%); and (iii) 11% of lysyl amines, 40% of -NH(2) groups of polyamines, and 80% of monovalent cations within the lyophilized T4 capsid are involved in the DNA charge balance. The NMR evidence suggests that DNA enters the T4 capsid in a charge-unbalanced state. We propose that electrostatic interactions may provide free energy to supplement the nanomotor-driven T4 DNA packaging.
| S-EPMC2633174 | biostudies-literature
Project description:T4 phage polynucleotide kinase (PNK) displays 5'-hydroxyl kinase, 3'-phosphatase and 2',3'-cyclic phosphodiesterase activities. The enzyme phosphorylates the 5' hydroxyl termini of a wide variety of nucleic acid substrates, a behavior studied here through the determination of a series of crystal structures with single-stranded (ss)DNA oligonucleotide substrates of various lengths and sequences. In these structures, the 5' ribose hydroxyl is buried in the kinase active site in proper alignment for phosphoryl transfer. Depending on the ssDNA length, the first two or three nucleotide bases are well ordered. Numerous contacts are made both to the phosphoribosyl backbone and to the ordered bases. The position, side chain contacts and internucleotide stacking interactions of the ordered bases are strikingly different for a 5'-GT DNA end than for a 5'-TG end. The base preferences displayed at those positions by PNK are attributable to differences in the enzyme binding interactions and in the DNA conformation for each unique substrate molecule.
| S-EPMC373337 | biostudies-literature