The enzymatic basis of processivity in lambda exonuclease

Subramanian, Krithika; Rutvisuttinunt, Wiriya; Scott, Walter A.; Myers, Richard S.

doi:10.1093/nar/gkg266

Cited by 113 publications

(141 citation statements)

References 25 publications

Supporting

Mentioning

123

Contrasting

Unclassified

Order By: Relevance

“…Some of these phages encode a homolog of ParB, an enzyme important for DNA binding and segregation (26), as previously pointed out by Aravind's group (7) (e.g., Mycobacteriophage Rosebush). Other phages encode potential helicases and nucleases near the preQ 0 cluster [e.g., Gp11 of 9g and Gp39 of JenKI contain SnfII-like domains (39), Gp11 of Dp-1 is a RecU-like protein (40), Gp40 of JenK1 encodes a putative exonuclease (41), and Gp10 of Dp-1 is a Cas4-like protein (42); SI Appendix, Fig. S12].…”

Section: The Dpd Cluster Is Horizontally Transferred and Found In Genmentioning

confidence: 99%

Novel genomic island modifies DNA with 7-deazaguanine derivatives

Thiaville

Kellner

Yuan

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

161

View full text Add to dashboard Cite

The discovery of ∼20-kb gene clusters containing a family of paralogs of tRNA guanosine transglycosylase genes, called tgtA5, alongside 7-cyano-7-deazaguanine (preQ 0 ) synthesis and DNA metabolism genes, led to the hypothesis that 7-deazaguanine derivatives are inserted in DNA. This was established by detecting 2'-deoxy-preQ 0 and 2'-deoxy-7-amido-7-deazaguanosine in enzymatic hydrolysates of DNA extracted from the pathogenic, Gram-negative bacteria Salmonella enterica serovar Montevideo. These modifications were absent in the closely related S. enterica serovar Typhimurium LT2 and from a mutant of S. Montevideo, each lacking the gene cluster. This led us to rename the genes of the S. Montevideo cluster as dpdA-K for 7-deazapurine in DNA. Similar gene clusters were analyzed in ∼150 phylogenetically diverse bacteria, and the modifications were detected in DNA from other organisms containing these clusters, including Kineococcus radiotolerans, Comamonas testosteroni, and Sphingopyxis alaskensis. Comparative genomic analysis shows that, in Enterobacteriaceae, the cluster is a genomic island integrated at the leuX locus, and the phylogenetic analysis of the TgtA5 family is consistent with widespread horizontal gene transfer. Comparison of transformation efficiencies of modified or unmodified plasmids into isogenic S. Montevideo strains containing or lacking the cluster strongly suggests a restriction-modification role for the cluster in Enterobacteriaceae. Another preQ 0 derivative, 2'-deoxy-7-formamidino-7-deazaguanosine, was found in the Escherichia coli bacteriophage 9g, as predicted from the presence of homologs of genes involved in the synthesis of the archaeosine tRNA modification. These results illustrate a deep and unexpected evolutionary connection between DNA and tRNA metabolism.DNA modification | restriction-modification | 7-deazaguanine | comparative genomics | queuosine H ypermodifications of DNA requiring more than one synthetic enzyme are not as prevalent and chemically diverse as RNA hypermodifications, but around a dozen have been identified in DNA to date (1). The functions of most DNA hypermodifications are still not known, but some have roles in protection against restriction enzymes, whereas others affect thermal stability temperature, DNA packaging, or transcription regulation (2). For example, the hypermodified DNA base β-D-glucosyl-hydroxymethyluracil, or base J, is an epigenetic factor that regulates Pol II transcription initiation in kinetoplastids of trypanosomes (3). The recently discovered phosphorothioate (PT) modification of the DNA backbone in bacteria was found to perform different functions in different organisms (4-6). In Salmonella Cerro 87, PT occurs on each strand of a GAAC/GTTC motif as part of a restriction-modification (R-M) system, whereas in Vibrio cyclitrophicus FF75, which lacks PT restriction enzymes, PT occurs on one strand of C ps CA motifs, and the function remains unclear (6). In 2013, Iyer et al. described the computational prediction of 12 novel DNA hypermodificat...

show abstract

Section: The Dpd Cluster Is Horizontally Transferred and Found In Genmentioning

confidence: 99%

Novel genomic island modifies DNA with 7-deazaguanine derivatives

Thiaville

Kellner

Yuan

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

161

View full text Add to dashboard Cite

show abstract

“…DNA fragments larger than 3 kb also place a limit on Red mediated ssDNA processing, which is most efficient up to 3 kb 30 . Insertion cassettes exceeding 3 kb are dual resected and recombine less efficiently via a beta independent pathway 20 .…”

Section: Discussionmentioning

confidence: 99%

“…A few samples also contained incorrectly gap repaired (lane 2) or mistargeted plasmids (lanes 4 and 5). The failure of concurrent subcloning and targeting in some SPI recombinants highlights the limits of efficient SPI cloning when using large cassettes (> 3 kb), which arise from the constraints on Red processivity of long DNA fragments 30 , or if using short HA. Indeed, increasing the HA of the eYFP cassette to 200 bp increased SPI efficiency and the correct targeting of the cassette (data not shown).…”

Section: Knockin Vectorsmentioning

confidence: 99%

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Reddy

Kelsall

Fevat³

et al. 2015

JoVE

View full text Add to dashboard Cite

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a 'targeting' vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

show abstract

“…We have tested our methodology with a variety other enzymes such as BsmBI and BciVI and the quality and accuracy of the ssDNA molecules generated after the lambda exonuclease treatment was similar to those LPPs used in this study (data not shown). It is likely that this method can be used to produce very large single-stranded molecules, because lambda exonuclease is a very processive enzyme (32,33). Our method of producing singlestranded molecules has a number of applications where variable sizes or large quantities of long ssDNA molecules are needed.…”

Section: Discussionmentioning

confidence: 99%

A comprehensive assay for targeted multiplex amplification of human DNA sequences

Krishnakumar

Zheng

Wilhelmy

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We developed a robust and reproducible methodology to amplify human sequences in parallel for use in downstream multiplexed sequence analyses. We call the methodology SMART (Spacer Multiplex Amplification Reaction), and it is based, in part, on padlock probe technology. As a proof of principle, we used SMART technology to simultaneously amplify 485 human exons ranging from 100 to 500 bp from human genomic DNA. In multiple repetitions, >90% of the targets were successfully amplified with a high degree of uniformity, with 70% of targets falling within a 10-fold range and all products falling within a 100-fold range of each other in abundance. We used long padlock probes (LPPs) >300 bases in length for the assay, and the increased length of these probes allowed for the capture of human sequences up to 500 bp in length, which is optimal for capturing most human exons. To engineer the LPPs, we developed a method that generates ssDNA molecules with precise ends, using an appropriately designed dsDNA template. The template has appropriate restriction sites engineered into it that can be digested to generate nucleotide overhangs that are suitable for lambda exonuclease digestion, producing a singlestranded probe from dsDNA. The SMART technology is flexible and can be easily adapted to multiplex tens of thousands of target sequences in a single reaction.human exons ͉ multiplex PCR ͉ padlock probe ͉ single-strand DNA

show abstract

The enzymatic basis of processivity in lambda exonuclease

Cited by 113 publications

References 25 publications

Novel genomic island modifies DNA with 7-deazaguanine derivatives

Novel genomic island modifies DNA with 7-deazaguanine derivatives

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

A comprehensive assay for targeted multiplex amplification of human DNA sequences

Contact Info

Product

Resources

About