Replication and recombination in bacteriophage

Zusammenfassung Experimente zur Replikation und Rekombination von Bakteriophagen werden diskutiert unter dem Gesichtspunkt der Frage, ob die infizierende DNA eines Phagen in Fragmente zerlegt und bruchstückweise repliziert wird (partial replica) oder ob bei der Vermehrung stets vollständige Genomstrukturen erhalten bleiben.Ein molekulares Modell wird vorgeschlagen, das auf einigen Annahmen über die Replikationsweise von DNA beruht und das die meisten Befunde der Rekombinationsgenetik von Phagen dutet. Dieses wird für einige neuere Resultate speziell diskutiert.

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With 22 Figures in the Text     

Replication fork arrest and DNA recombination

Replication arrests are associated with genome rearrangements, which result from either homologous or non-homologous recombination. Interestingly, proteins involved in homologous recombination are able to convert an arrested replication fork into a recombination intermediate, which promotes replication restart and thus presumably prevents genome rearrangements.     

Replication and recombination of herpes simplex virus DNA

Replication of herpes simplex virus takes place in the cell nucleus and is carried out by a replisome composed of six viral proteins: the UL30-UL42 DNA polymerase, the UL5-UL8-UL52 helicase-primase, and the UL29 single-stranded DNA-binding protein ICP8. The replisome is loaded on origins of replication by the UL9 initiator origin-binding protein. Virus replication is intimately coupled to recombination and repair, often performed by cellular proteins. Here, we review new significant developments: the three-dimensional structures for the DNA polymerase, the polymerase accessory factor, and the single-stranded DNA-binding protein; the reconstitution of a functional replisome in vitro; the elucidation of the mechanism for activation of origins of DNA replication; the identification of cellular proteins actively involved in or responding to viral DNA replication; and the elucidation of requirements for formation of replication foci in the nucleus and effects on protein localization.     

Replication, recombination, and repair: going for the gold

DNA recombination is now appreciated to be integral to DNA replication and cell survival. Recombination allows replication to successfully maneuver through the roadblocks of damaged or collapsed replication forks. The signals and controls that permit cells to transition between replication and recombination modes are now being identified.     

Replication and recombination in adenovirus-infected cells are temporally and functionally related.

We have studied the temporal and functional relationships between DNA replication and recombination in adenovirus-infected cells by using Southern blot hybridization to detect recombinant products among intracellular viral genomes. The data show that recombination can be detected soon after DNA replication has commenced and that the proportion of recombinant products increases thereafter. To determine the functional relationship between DNA replication and recombination, replication was blocked with the protein synthesis inhibitor anisomycin, the replication inhibitor cytosine arabinoside, and conditionally lethal mutations in either the virus-specified DNA-binding protein or the DNA polymerase. All treatments that directly or indirectly blocked DNA replication caused a delay in the appearance of recombinant products and a marked decline in their abundance relative to products of parental genotype. These data strongly suggest that DNA replication and recombination are interrelated, either because both processes share functions or because DNA structures produced by replication are suitable substrates for recombination. In addition, we have shown that some recombination function(s) is intrinsically thermolabile at 40.9 degrees C, even in wild-type crosses, since the appearance of recombinant products is delayed and their extent is reduced compared with that from crosses performed at 39.9 degrees C.     

Replication blocking lesions present a unique substrate for homologous recombination

Homologous recombination (HR) plays a critical role in the restart of blocked replication forks, but how this is achieved remains poorly understood. We show that mutants in the single Rad51 paralog in Caenorhabditis elegans, rfs-1, permit discrimination between HR substrates generated at DNA double-strand breaks (DSBs), or following replication fork collapse from HR substrates assembled at replication fork barriers (RFBs). Unexpectedly, RFS-1 is dispensable for RAD-51 recruitment to meiotic and ionizing radiation (IR)-induced DSBs and following replication fork collapse, yet, is essential for RAD-51 recruitment to RFBs formed by DNA crosslinking agents and other replication blocking lesions. Deletion of rfs-1 also suppresses the accumulation of toxic HR intermediates in him-6; top-3 mutants and accelerates deletion formation at presumed endogenous RFBs formed by poly G/C tracts in the absence of DOG-1. These data suggest that RFS-1 is not a general mediator of HR-dependent DSB repair, but acts specifically to promote HR at RFBs. HR substrates generated at conventional DSBs or following replication fork collapse are therefore intrinsically different from those produced during normal repair of blocked replication forks.     

Replication fork arrest, recombination and the maintenance of ribosomal DNA stability

There is increasing interest in the role of replication fork arrest and collapse in stimulating genomic instability. Changes in the copy number of the rDNA repeats mediated by homologous recombination has been linked to programmed replication fork barriers (RFBs) and this has been proposed to serve as a paradigm to help understand the links between replication and recombination. Here we review recent advances in our understanding of the initiation and regulation rDNA recombination and discuss historical observations in the context of recently developed models. We contrast the outcome of replication fork arrest at the rDNA RFB with those at an alternative RFB and suggest that, while there are potential similarities in response, there are also important differences which reflect the highly specialised nature of rDNA metabolism.     

Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5' ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis.     

Biomarkers intersect with the exposome

The exposome concept promotes use of omic tools for discovering biomarkers of exposure and biomarkers of disease in studies of diseased and healthy populations. A two-stage scheme is presented for profiling omic features in serum to discover molecular biomarkers and then for applying these biomarkers in follow-up studies. The initial component, referred to as an exposome-wide-association study (EWAS), employs metabolomics and proteomics to interrogate the serum exposome and, ultimately, to identify, validate and differentiate biomarkers of exposure and biomarkers of disease. Follow-up studies employ knowledge-driven designs to explore disease causality, prevention, diagnosis, prognosis and treatment.     

Replication protein A in Pyrococcus furiosus is involved in homologous DNA recombination

Single-stranded DNA-binding protein in Bacteria and replication protein A (RPA) in Eukarya play crucial roles in DNA replication, repair, and recombination processes. We identified an RPA complex from the hyperthermophilic archaeon, Pyrococcus furiosus. Unlike the single-peptide RPAs from the methanogenic archaea, Methanococcus jannaschii and Methanothermobacter thermoautotrophicus, P. furiosus RPA (PfuRPA) exists as a stable hetero-oligomeric complex consisting of three subunits, RPA41, RPA14, and RPA32. The amino acid sequence of RPA41 has some similarity to those of the eukaryotic RPA70 subunit and the M. jannaschii RPA. On the other hand, RPA14 and RPA32 do not share homology with any known open reading frames from Bacteria and Eukarya. However, six of eight archaea, whose total genome sequences have been published, have the open reading frame homologous to RPA32. The PfuRPA complex, but not each subunit alone, specifically bound to a single-stranded DNA and clearly enhanced the efficiency of an in vitro strand-exchange reaction by the P. furiosus RadA protein. Moreover, immunoprecipitation analyses showed that PfuRPA interacts with the recombination proteins, RadA and Hjc, as well as replication proteins, DNA polymerases, primase, proliferating cell nuclear antigen, and replication factor C in P. furiosus cells. These results indicate that PfuRPA plays important roles in the homologous DNA recombination in P. furiosus.     

Replication and recombination factors contributing to recombination-dependent bypass of DNA lesions by template switch

Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication forks as X-shaped structures involving sister chromatid junctions. The homologous recombination factor Rad51 is required for the formation/stabilization of these intermediates, but its mode of action remains to be investigated. By using a combination of genetic and physical approaches, we show that the homologous recombination factors Rad55 and Rad57, but not Rad59, are required for the formation of template switch intermediates. The replication-proficient but recombination-defective rfa1-t11 mutant is normal in triggering a checkpoint response following DNA damage but is impaired in X-structure formation. The Exo1 nuclease also has stimulatory roles in this process. The checkpoint kinase, Rad53, is required for X-molecule formation and phosphorylates Rad55 robustly in response to DNA damage. Although Rad55 phosphorylation is thought to activate recombinational repair under conditions of genotoxic stress, we find that Rad55 phosphomutants do not affect the efficiency of X-molecule formation. We also examined the DNA polymerase implicated in the DNA synthesis step of template switch. Deficiencies in translesion synthesis polymerases do not affect X-molecule formation, whereas DNA polymerase δ, required also for bulk DNA synthesis, plays an important role. Our data indicate that a subset of homologous recombination factors, together with DNA polymerase δ, promote the formation of template switch intermediates that are then preferentially dissolved by the action of the Sgs1 helicase in association with the Top3 topoisomerase rather than resolved by Holliday Junction nucleases. Our results allow us to propose the choreography through which different players contribute to template switch in response to DNA damage and to distinguish this process from other recombination-mediated processes promoting DNA repair.     

Replication mutations differentially enhance RecA-dependent and RecA-independent recombination between tandem repeats in Bacillus subtilis

We have studied DNA recombination between 513 bp tandem direct repeats present in a kanamycin resistance gene inserted in the Bacillus subtilis chromosome. Tandem repeat deletion was not significantly affected by a recA mutation. However, recombination was stimulated by mutations in genes encoding replication proteins, including the primosomal proteins DnaB, DnaD and the DnaG primase, the putative DNA polymerase III subunits PolC, DnaN and DnaX, as well as the DNA polymerase DnaE. Hyper-recombination was found to be dependent on RecA in the dnaE, dnaN and dnaX mutants, whereas the dnaG and dnaD mutants stimulated recombination independently of RecA. Altogether, these data show that both RecA-dependent and RecA-independent mechanisms contribute to recombination between tandem repeats in B. subtilis and that both types of recombination are stimulated by replication mutations.     

Biomarkers intersect with the exposome

The exposome concept promotes use of omic tools for discovering biomarkers of exposure and biomarkers of disease in studies of diseased and healthy populations. A two-stage scheme is presented for profiling omic features in serum to discover molecular biomarkers and then for applying these biomarkers in follow-up studies. The initial component, referred to as an exposome-wide-association study (EWAS), employs metabolomics and proteomics to interrogate the serum exposome and, ultimately, to identify, validate and differentiate biomarkers of exposure and biomarkers of disease. Follow-up studies employ knowledge-driven designs to explore disease causality, prevention, diagnosis, prognosis and treatment.     

Replication factor A is required in vivo for DNA replication, repair, and recombination.

Replication factor A (RF-A) is a heterotrimeric single-stranded-DNA-binding protein which is conserved in all eukaryotes. Since the availability of conditional mutants is an essential step to define functions and interactions of RF-A in vivo, we have produced and characterized mutations in the RFA1 gene, encoding the p70 subunit of the complex in Saccharomyces cerevisiae. This analysis provides the first in vivo evidence that RF-A function is critical not only for DNA replication but also for efficient DNA repair and recombination. Moreover, genetic evidence indicate that p70 interacts both with the DNA polymerase alpha-primase complex and with DNA polymerase delta.     

Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast

Homologous recombination is believed to play important roles in processing stalled/blocked replication forks in eukaryotes. In accordance with this, recombination is induced by replication fork barriers (RFBs) within the rDNA locus. However, the rDNA locus is a specialised region of the genome, and therefore the action of recombinases at its RFBs may be atypical. We show here for the first time that direct repeat recombination, dependent on Rad22 and Rhp51, is induced by replication fork blockage at a site-specific RFB (RTS1) within a 'typical' genomic locus in fission yeast. Importantly, when the RFB is positioned between the direct repeat, conservative gene conversion events predominate over deletion events. This is consistent with recombination occurring without breakage of the blocked fork. In the absence of the RecQ family DNA helicase Rqh1, deletion events increase dramatically, which correlates with the detection of one-sided DNA double-strand breaks at or near RTS1. These data indicate that Rqh1 acts to prevent blocked replication forks from collapsing and thereby inducing deletion events.     

Replication arrest-stimulated recombination: Dependence on the RecA paralog, RadA/Sms and translesion polymerase, DinB

Difficulties in replication can lead to breakage of the fork. Recombinational reactions restore the integrity of the fork through strand-invasion of the broken chromosome with its sister. If this occurs in the context of repeated DNA sequences, genetic rearrangements can result. We have proposed that this process accounts for stimulation of chromosomal rearrangements by mutations in Escherichia coli's replicative DNA helicase, DnaB. At its permissive temperature for growth, a dnaB107 mutant is a 1000-fold more likely to experience a deletion of a 787bp tandem repeated segment inserted in the E. coli chromosome than is a wild-type strain. We have previously shown that enhanced deletion in a dnaB107 strain is reduced in recA, recB and recG102 (formerly known as radC102) derivatives. Here I show that this enhanced recombination is dependent on other factors: the RuvA Holliday junction helicase, the RecJ single-strand DNA exonuclease, the RadA/Sms RecA-paralog protein of unknown function and, surprisingly, the DinB translesion polymerase. The requirement for these factors in DnaB-stimulated rearrangements is much greater than that observed for recombinational events such as P1 transduction. This may be because strand invasion into the repeats limits the extent of heteroduplex DNA that can be formed in the initial stage of recombination. I propose that RadA, RecG and RuvAB are critically required to stabilize the strand-invasion intermediate and that DinB polymerase extends the invading 3' strand to aid in re-initiation. The role of DinB in bacteria may be analogous to translesion DNA polymerase eta in eukaryotes, recently shown to aid recombination.     

Globotriaosyl ceramide receptor function - Where membrane structure and pathology intersect

The glycosphingolipid globotriaosyl ceramide, (Galα1-4Galß1-4 glucosyl ceramide-Gb₃) also known as CD77 and the P^k blood group antigen, is bound by both verotoxins and by the HIV adhesin, gp120. Gb₃ plays an important receptor role in VT induced hemolytic uremic syndrome (HUS) and HIV infection. The organization of glycolipids, including Gb₃, into lipid rafts is central to both pathologies. The fatty acid heterogeneity within the Gb₃ lipid moiety plays a central role in assembly within such ordered domains. Differential binding of verotoxins and gp120 to such Gb₃ isoforms in model and cell membranes indicates a significant role in the eventual pathogenic outcome. HUS may provide the first example whereby membrane Gb₃ organization provides a predictor for tissue selective in vivo pathology.