Abstract
Interactions between helicases and the tracking strand of a DNA substrate are well-characterized; however, the role of the displaced strand is a less understood characteristic of DNA unwinding. Dda helicase exhibited greater processivity when unwinding a DNA fork compared to a ss/ds DNA junction substrate. The lag phase in the unwinding progress curve was reduced for the forked DNA compared to the ss/ds junction. Fewer kinetic steps were required to unwind the fork compared to the ss/ds junction, suggesting that binding to the fork leads to disruption of the duplex. DNA footprinting confirmed that interaction of Dda with a fork leads to two base pairs being disrupted whereas no disruption of base pairing was observed with the ss/ds junction. Neutralization of the phosphodiester backbone resulted in a DNA-footprinting pattern similar to that observed with the ss/ds junction, consistent with disruption of the interaction between Dda and the displaced strand. Several basic residues in the 1A domain which were previously proposed to bind to the incoming duplex DNA were replaced with alanines, resulting in apparent loss of interaction with the duplex. Taken together, these results suggest that Dda interaction with the tracking strand, displaced strand and duplex coordinates DNA unwinding.
Highlights
Helicases are ubiquitous molecular motor proteins which participate in various aspects of nucleic acid metabolism such as DNA replication, recombination, repair, transcription, translation and splicing of transcripts by providing ssDNA intermediates [1,2,3,4,5,6]
Dda helicase unwinds forked DNA with enhanced product formation compared to a ss/dsDNA junction under excess enzyme conditions
The X-ray crystallographic structure of Dda bound to ssDNA revealed the interactions with the tracking strand [28]
Summary
Helicases are ubiquitous molecular motor proteins which participate in various aspects of nucleic acid metabolism such as DNA replication, recombination, repair, transcription, translation and splicing of transcripts by providing ssDNA intermediates [1,2,3,4,5,6]. Mutations in helicase genes involved in DNA repair processes have been linked to numerous human diseases [7,8,9,10,11,12] characterized by genomic instability, premature aging and predisposition to cancer [10,13,14,15]. It is essential to understand the mechanisms by which helicases perform different biochemical functions so that the relationship between mutations and specific disease states can be understood at the molecular level. The possibility that the efficacy of chemotherapeutic agents could be increased by administering drugs that target helicases along with the anti-cancer drugs [17] raises the need to study the mechanisms of helicases
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