Characterization of the Caenorhabditis elegans HIM-6/BLM Helicase: Unwinding Recombination Intermediates

Hana Jung,Byungchan Ahn,Jin A Lee,Seoyoon Choi,Hyunwoo Lee,Michael Lichten

doi:10.1371/journal.pone.0102402

Abstract

Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3'-5' direction and needs at least five 3' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates.

Highlights

The RecQ family of proteins belongs to superfamily 2 of DNA helicases, which was named after the Escherichia coli ortholog, RecQ [1]
We found that HIM-6 displaced the 3’tailed substrate in a concentration-dependent manner (Figure 3A), and 40% strand displacement occurred at a protein concentration of approximately 10 nM (Figure 3D)
As expected for a RecQ homolog, our data indicated that HIM-6 has DNA-dependent ATPase and 3’-5’ DNA helicase activities that can unwind displacement loop (D-loop) and Holliday junction (HJ) structures

Summary

Introduction

The RecQ family of proteins belongs to superfamily 2 of DNA helicases, which was named after the Escherichia coli ortholog, RecQ [1]. Most prokaryotes have a single RecQ homologue, whereas eukaryotic organisms often possess more than one RecQ family member The RecQ helicases are implicated in maintaining genome stability across various species. In Caenorhabditis elegans, there are 4 RecQ helicase members including HIM-6, WRN-1, RECQ1, and RECQ5 [2,3]. The C. elegans him-6 gene encodes a 988 amino acid BLM helicase ortholog. Worms with him-6 mutations are radiation sensitive and exhibit genomic instability and decreased frequency of meiotic recombination. Mitotically proliferating germ cells of him-6 mutants show increased frequency of double-strand breaks (DSBs) [4,5,6]

Methods

Results

Conclusion