Abstract
Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA.
Highlights
DNA replication, repair and recombination require the function of multiple DNA helicases and nucleases (Tsutakawa et al, 2014; Wu and Hickson, 2006)
Without the human Replication Protein A, Human dependent helicase/nuclease 2 (DNA2) (hDNA2) most efficiently degraded ssDNA, while 5’-overhanged, 3’-overhanged and Y-structured DNA were degraded ~7–20-fold less efficiently, based on the hDNA2 concentration required for the degradation of 50% DNA substrate (Figure 1C and Figure 1—figure supplement 1D)
Using a 3’-end labeled ssDNA, we observed that human Replication Protein A (hRPA) directs the nuclease of hDNA2 towards the 5’ terminus; while at the same time inhibits the 3’-5’ nuclease activity (Figure 1D)
Summary
DNA replication, repair and recombination require the function of multiple DNA helicases and nucleases (Tsutakawa et al, 2014; Wu and Hickson, 2006). YDna is one of the nucleases that resect 5’-terminated strands of DNA double-strand breaks (DSBs) (Cejka et al, 2010; Niu et al, 2010; Zhu et al, 2008) This process leads to the formation of 3’-tailed DNA, which becomes a substrate for the strand exchange protein Rad to initiate homology search and accurate DSB repair by the recombination machinery (Cejka, 2015; Heyer et al, 2010; Symington, 2014). The yeast Dna protein contains a large unstructured N-terminal domain, which mediates a physical interaction with yRPA (Bae et al, 2003), is required for Dna2’s checkpoint function (Kumar and Burgers, 2013) and its capacity to melt secondary structures within 5’ DNA flaps (Lee et al, 2013). The N-terminal domain is followed by a RecB-like nuclease domain
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