Phage φ29 protein p56 prevents viral DNA replication impairment caused by uracil excision activity of uracil-DNA glycosylase

Serrano‐Heras, Gemma; Bravo, Alicia; Salas, Margarita

doi:10.1073/pnas.0808797105

Cited by 34 publications

(36 citation statements)

References 45 publications

(45 reference statements)

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“…The first, containing DUF950, was previously characterized as SAUGI, a uracil-DNA glycosylase (UDGase) inhibitor, although its context on a mobile genetic element was not noted 16 . That SAUGI is always present in SCC elements suggests that they can replicate because the only other characterized UDGase inhibitors are encoded by phages to protect their newly replicated DNA from the base excision activity of their host’s UDGase 17 . The roles of the other two ORFs remain unknown.…”

Section: Resultsmentioning

confidence: 99%

Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative

Mir-Sanchis

Roman

Misiura

et al. 2016

Nat Struct Mol Biol

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Methicillin resistant Staphylococcus aureus (MRSA) is a public health threat worldwide. Although the mobile genomic island responsible for this phenotype, called SCC, was labeled non-replicative, we predicted DNA replication-related functions for some of their conserved proteins. We show that one of these, Cch, is homologous to the self-loading initiator helicases of an unrelated family of genomic islands, that it is an active 3’ to 5' helicase, and that the adjacent ORF encodes an ssDNA-binding protein. Our 2.9Å crystal structure of intact Cch shows that it forms a hexameric ring. Cch belongs to the pre-sensor II insert clade of AAA+ ATPases, as do the archaeal and eukaryotic MCM-family replicative helicases. Additionally, we find that SCC elements are part of a broader family of mobile elements that all encode a replication initiator upstream of their recombinases. Replication after excision would enhance the efficiency of horizontal gene transfer.

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Section: Resultsmentioning

confidence: 99%

Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative

Mir-Sanchis

Roman

Misiura

et al. 2016

Nat Struct Mol Biol

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“…If uracil residues appear in ssDNA regions of replicative intermediates, the action of host uracil DNA-glycosylase (UDG) will introduce a nick into the phosphodiester backbone producing the loss of the terminal region. To avoid this process, phage 29 encodes a UDG inhibitor called p56 (57,58).…”

Section: Resultsmentioning

confidence: 99%

Global Transcriptional Analysis of Virus-Host Interactions between Phage ϕ29 and Bacillus subtilis

Mojardín

Salas

2016

J Virol

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The study of phage-host relationships is essential to understanding the dynamic of microbial systems. Here, we analyze genomewide interactions of Bacillus subtilis and its lytic phage 29 during the early stage of infection. Simultaneous high-resolution analysis of virus and host transcriptomes by deep RNA sequencing allowed us to identify differentially expressed bacterial genes. Phage 29 induces significant transcriptional changes in about 0.9% (38/4,242) and 1.8% (76/4,242) of the host protein-coding genes after 8 and 16 min of infection, respectively. Gene ontology enrichment analysis clustered upregulated genes into several functional categories, such as nucleic acid metabolism (including DNA replication) and protein metabolism (including translation). Surprisingly, most of the transcriptional repressed genes were involved in the utilization of specific carbon sources such as ribose and inositol, and many contained promoter binding-sites for the catabolite control protein A (CcpA). Another interesting finding is the presence of previously uncharacterized antisense transcripts complementary to the well-known phage 29 messenger RNAs that adds an additional layer to the viral transcriptome complexity. IMPORTANCEThe specific virus-host interactions that allow phages to redirect cellular machineries and energy resources to support the viral progeny production are poorly understood. This study provides, for the first time, an insight into the genome-wide transcriptional response of the Gram-positive model Bacillus subtilis to phage 29 infection. Due to their small dimension and limited size of genomes, bacteriophages have optimized the exploitation of host resources to increase the production of the viral progeny. A comprehensive understanding of these host-virus interactions requires the analysis of associated transcriptional changes in both organisms. Thus, we used the recently developed RNA sequencing (RNA-Seq) technology to monitor to a high level of accuracy and depth the genome-wide effect of the bacteriophage 29 on Bacillus subtilis transcription. The transcriptome profiles were analyzed at two early infection time points (8 and 16 min postinfection) so that the identification of the bacterial genes corresponding to these stages could allow the identification of potential phage targets.Phage 29 is a well-characterized lytic virus that belongs to the Podoviridae family. Over the years, it has been the subject of many extensive studies that have contributed to the understanding of several molecular mechanisms of biological processes, such as transcription regulation, viral DNA packaging, viral morphogenesis, and DNA replication (1). Phage 29 genome consists of a linear double-stranded DNA (dsDNA) molecule of 19,285 bp, which encodes 28 open reading frames (ORFs) transcribed from four early and one late promoters. The viral genes are expressed in a temporal sequence to ensure that DNA replication, and the production and assembly of viral components occur in an orderly fashion. Thus, bacterial cells inf...

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“…Two of these (UGI and p56) are encoded by different bacteriophages [phages PBS1/PBS2 and phi29 of Bacillus subtilis (Wang and Mosbaugh, 1989) and (Serrano-Heras et al, 2008), respectively]. The UGI function encoded in phages is either required to allow synthesis of uracil-enriched DNA (in the case of phages PBS1/PBS2) or protects against the cleavage of phage genome at uracil positions thereby facilitating viral DNA replication (Serrano-Heras et al, 2008). The third protein with UNG inhibitory activity was recently identified in S. aureus (SaUGI) and interestingly, this is the first such case where a UNG inhibitor is encoded in the cellular genome itself (Wang et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

Life without dUTPase

et al. 2016

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Fine-tuned regulation of the cellular nucleotide pools is indispensable for faithful replication of Deoxyribonucleic Acid (DNA). The genetic information is also safeguarded by DNA damage recognition and repair processes. Uracil is one of the most frequently occurring erroneous bases in DNA; it can arise from cytosine deamination or thymine-replacing incorporation. Two enzyme activities are primarily involved in keeping DNA uracil-free: dUTPase (dUTP pyrophosphatase) activity that prevent thymine-replacing incorporation and uracil-DNA glycosylase activity that excise uracil from DNA and initiate uracil-excision repair. Both dUTPase and the most efficient uracil-DNA glycosylase (UNG) is thought to be ubiquitous in free-living organisms. In the present work, we have systematically investigated the genotype of deposited fully sequenced bacterial and Archaeal genomes. We have performed bioinformatic searches in these genomes using the already well described dUTPase and UNG gene sequences. For dUTPases, we have included the trimeric all-beta and the dimeric all-alpha families and also, the bifunctional dCTP (deoxycytidine triphosphate) deaminase-dUTPase sequences. Surprisingly, we have found that in contrast to the generally held opinion, a wide number of bacterial and Archaeal species lack all of the previously described dUTPase gene(s). The dut– genotype is present in diverse bacterial phyla indicating that loss of this (or these) gene(s) has occurred multiple times during evolution. We discuss potential survival strategies in lack of dUTPases, such as simultaneous lack or inhibition of UNG and possession of exogenous or alternate metabolic enzymes involved in uracil-DNA metabolism. The potential that genes previously not associated with dUTPase activity may still encode enzymes capable of hydrolyzing dUTP is also discussed. Our data indicate that several unicellular microorganisms may efficiently cope with a dut– genotype lacking all of the previously described dUTPase genes, and potentially leading to an unusual uracil-enrichment in their genomic DNA.

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Phage φ29 protein p56 prevents viral DNA replication impairment caused by uracil excision activity of uracil-DNA glycosylase

Cited by 34 publications

References 45 publications

Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative

Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative

Global Transcriptional Analysis of Virus-Host Interactions between Phage ϕ29 and Bacillus subtilis

Life without dUTPase

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