DNA Recognition/Processing | DNA Topoisomerases: Type I

ABSTRACT The origin recognition complex (ORC) plays a central role in eukaryotic DNA replication. Here we describe a unique ORC-like complex in Tetrahymena thermophila, TIF4, which bound in an ATP-dependent manner to sequences required for cell cycle-controlled replication and gene amplification (ribosomal DNA [rDNA] type I elements). TIF4's mode of DNA recognition was distinct from that of other characterized ORCs, as it bound exclusively to single-stranded DNA. In contrast to yeast ORCs, TIF4 DNA binding activity was cell cycle regulated and peaked during S phase, coincident with the redistribution of the Orc2-related subunit, p69, from the cytoplasm to the macronucleus. Origin-binding activity and nuclear p69 immunoreactivity were further regulated during development, where they distinguished replicating from nonreplicating nuclei. Both activities were lost from germ line micronuclei following the programmed arrest of micronuclear replication. Replicating macronuclei stained with Orc2 antibodies throughout development in wild-type cells but failed to do so in the amplification-defective rmm11 mutant. Collectively, these findings indicate that the regulation of TIF4 is intimately tied to the cell cycle and developmentally programmed replication cycles. They further implicate TIF4 in rDNA gene amplification. As type I elements interact with other sequence-specific single-strand breaks (in vitro and in vivo), the dynamic interplay of Orc-like (TIF4) and non-ORC-like proteins with this replication determinant may provide a novel mechanism for regulation.

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Improving mobilization of foreign DNA into Zymomonas mobilis ZM4 by removal of multiple restriction systems

Applied and Environmental Microbiology ◽

10.1128/aem.00808-21 ◽

2021 ◽

Author(s):

Piyush Behari Lal ◽

Fritz Wells ◽

Kevin S. Myers ◽

Rajdeep Banerjee ◽

Adam M. Guss ◽

...

Keyword(s):

Zymomonas Mobilis ◽

Plant Biomass ◽

Dna Recognition ◽

Type I ◽

Dna Uptake ◽

Exogenous Dna ◽

Recognition Motif ◽

Restriction System ◽

System A ◽

Foreign Dna

Zymomonas mobilis has emerged as a promising candidate for production of high value bioproducts from plant biomass. However, a major limitation in equipping Z. mobilis with novel pathways to achieve this goal is restriction of heterologous DNA. Here, we characterized the contribution of several defense systems of Z. mobilis strain ZM4 to impeding heterologous gene transfer from an Escherichia coli donor. Bioinformatic analysis revealed that Z. mobilis ZM4 encodes a previously described mrr -like Type IV Restriction Modification (RM) system, a Type I-F CRISPR system, a chromosomal Type I RM ( hsdMS c ) and a previously uncharacterized Type I RM system, located on an endogenous plasmid ( hsdRMS p ). The DNA recognition motif of HsdRMS p was identified by comparing the methylated DNA sequence pattern of mutants lacking one or both of the hsdMS c and hsdRMS p systems to the parent strain. The conjugation efficiency of synthetic plasmids containing single or combinations of the HsdMS c and HsdRMS p recognition sites indicated that both systems are active and decrease uptake of foreign DNA. In contrast, deletions of mrr and cas3 led to no detectable improvement in conjugation efficiency for the exogenous DNA tested. Thus, the suite of markerless restriction - strains that we constructed, and the knowledge of this new restriction system and its DNA recognition motif provide the necessary platform to flexibly engineer the next generation of Z. mobilis strains for synthesis of valuable products. Importance Zymomonas mobilis is equipped with a number of traits that make it a desirable platform organism for metabolic engineering to produce valuable bioproducts. Engineering strains equipped with synthetic pathways for biosynthesis of new molecules requires integration of foreign genes. In this study we have developed an all-purpose strain, devoid of known host restriction systems and free of any antibiotic resistance markers, which dramatically improves the uptake efficiency of heterologous DNA into Z. mobilis ZM4. We also confirmed the role of a previously known restriction system as well as identified a previously unknown Type I RM system on an endogenous plasmid. Elimination of the barriers to DNA uptake as shown here will allow facile genetic engineering of Z. mobilis .

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Synthesis and Use of DNA Containing a 5'-Bridging Phosphorothioate as a Suicide Substrate for Type I DNA Topoisomerases

DNA Topoisomerase Protocols ◽

10.1385/1-59259-057-8:119 ◽

2003 ◽

pp. 119-128

Author(s):

Alex B. Burgin

Keyword(s):

Type I ◽

Dna Topoisomerases ◽

Suicide Substrate

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Cas3 Mediated Target DNA Recognition and Cleavage is Independent of the Composition and Architecture of Cascade Surveillance Complex

10.1101/666776 ◽

2019 ◽

Author(s):

Siddharth Nimkar ◽

B. Anand

Keyword(s):

Rna Processing ◽

Dna Recognition ◽

Nuclease Activity ◽

Helicase Domain ◽

Type I ◽

Conserved Residues ◽

Target Dna ◽

Evolutionarily Conserved ◽

Single Stranded Region

ABSTRACTIn type I CRISPR-Cas system, Cas3 –a nuclease cum helicase– in cooperation with Cascade surveillance complex cleaves the target DNA. Unlike the Cascade/I-E, which is composed of five subunits, the Cascade/I-C is made of only three subunits lacking the CRISPR RNA processing enzyme Cas6, whose role is assumed by Cas5. How these differences in the composition and organisation of Cascade subunits in type I-C influences the Cas3/I-C binding and its target cleavage mechanism is poorly understood. Here, we show that Cas3/I-C is intrinsically a single-strand specific promiscuous nuclease. Apart from the helicase domain, a constellation of highly conserved residues –that are unique to type I-C– located in the uncharacterised C-terminal domain appears to influence the nuclease activity. Recruited by Cascade/I-C, the HD nuclease of Cas3/I-C nicks the single-stranded region of the nontarget strand and positions the helicase motor. Powered by ATP, the helicase motor reels in the target DNA, until it encounters the roadblock en route, which stimulates the HD nuclease. Remarkably, we show that Cas3/I-C supplants Cas3/I-E for CRISPR interference in type I-E in vivo, suggesting that the target cleavage mechanism is evolutionarily conserved between type I-C and type I-E despite the architectural difference exhibited by Cascade/I-C and Cascade/I-E.

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