Testing water-mediated DNA recognition by the Hin recombinase

Nitrogen-containing heterocycles are one of the most common structural motifs in approximately 80% of the marketed drugs. Of these, benzimidazoles analogues are known to elicit a wide spectrum of pharmaceutical activities such as anticancer, antibacterial, antiparasitic, antiviral, antifungal as well as chemosensor effect. Based on the benzimidazole core fused heterocyclic compounds, crescent-shaped bisbenzimidazoles were developed which provided an early breakthrough in the sequence-specific DNA recognition. Over the years, a number of functional variations in the bisbenzimidazole core have led to the emergence of their unique properties and established them as versatile ligands against several classes of pathogens. The present review provides an overview of diverse pharmacological activities of the bisbenzimidazole analogues in the past decade with a brief account of its development through the years.

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The structure of ORC–Cdc6 on an origin DNA reveals the mechanism of ORC activation by the replication initiator Cdc6

Nature Communications ◽

10.1038/s41467-021-24199-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xiang Feng ◽

Yasunori Noguchi ◽

Marta Barbon ◽

Bruce Stillman ◽

Christian Speck ◽

...

Keyword(s):

Dna Sequences ◽

Origin Recognition Complex ◽

Molecular Level ◽

Dna Recognition ◽

Winged Helix ◽

Replicative Helicase ◽

The Core ◽

Α Helix ◽

Replication Initiator ◽

Winged Helix Domain

AbstractThe Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6. It has been unclear at the molecular level how Cdc6 activates ORC, converting it to an active recruiter of the Mcm2-7 hexamer, the core of the replicative helicase. Here we report the cryo-EM structure at 3.3 Å resolution of the yeast ORC–Cdc6 bound to an 85-bp ARS1 origin DNA. The structure reveals that Cdc6 contributes to origin DNA recognition via its winged helix domain (WHD) and its initiator-specific motif. Cdc6 binding rearranges a short α-helix in the Orc1 AAA+ domain and the Orc2 WHD, leading to the activation of the Cdc6 ATPase and the formation of the three sites for the recruitment of Mcm2-7, none of which are present in ORC alone. The results illuminate the molecular mechanism of a critical biochemical step in the licensing of eukaryotic replication origins.

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In Vivo Identification of Intermediate Stages of the DNA Inversion Reaction Catalyzed by the Salmonella Hin Recombinase

Genetics ◽

10.1093/genetics/149.4.1649 ◽

1998 ◽

Vol 149 (4) ◽

pp. 1649-1663

Author(s):

Oliver Z Nanassy ◽

Kelly T Hughes

Keyword(s):

Amino Acids ◽

Biochemical Analysis ◽

Mutational Analysis ◽

Recombination Reaction ◽

Recombination Site ◽

Dna Inversion ◽

Hin Recombinase ◽

One Step ◽

A Site

Abstract The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.

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