Bulk and single-molecule analysis of a bacterial DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor.

Oliver J Wilkinson,Fernando Moreno-Herrero,Carolina Carrasco,Clara Aicart-Ramos,Mark S Dillingham

doi:10.1093/nar/gkaa562

Oliver J Wilkinson, Fernando Moreno-Herrero + Show 3 more

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https://doi.org/10.1093/nar/gkaa562

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Abstract

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase–nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5′ to 3′ ssDNA translocase and 5′ to 3′ helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase–nucleases and DNA looping motor proteins in general.

Highlights

DNA2 is an essential replication and repair factor found widely in eukaryotic proteomes [1]
DNA2 is a DNA helicase–nuclease that is ubiquitous in eukaryotic cells and has been shown to be an essential DNA replication and repair factor [1]
In addition to four amino acid motifs associated with nuclease activity that are shared by all members of the RecB nuclease family, they contain four strictly

Summary

Introduction

DNA2 is an essential replication and repair factor found widely in eukaryotic proteomes [1] It is a multi-functional protein with roles in the processing of Okazaki fragments and stalled replication forks, and in the repair of double-stranded DNA breaks [2,3,4,5,6].The primary structure of DNA2 comprises an N-terminal RecB-family nuclease domain fused to a C-terminal SF1B helicase domain [7]. We hypothesised that the nuclease domain found in DNA2 and closely-related enzymes belonged to a new class of 4Fe–4S cluster associated domains [8], and this was later confirmed experimentally [9]. Given that DNA2 was thought to be restricted to eukaryotic and archaeal organisms, we were surprised to find DNA2-like enzymes sporadically distributed in restricted niches of bacteria including Geobacilli and Mycobacteria (see Supplementary Figure S1 for further information)

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