Genetic and functional interactions between the regulatory and catalytic domains of Dna2 for DNA metabolism

Abstract

Dna2 is an endonuclease/helicase that plays a critical role in removing secondary-structured 5’ flaps and long single-stranded DNA formed during lagging strand maturation and DNA double-strand break repair. The N-terminal 405 amino-acid domain of Dna2 is associated with two other crucial functions that may collaborate with its enzymatic activities

one is DNA secondary structure binding activity, which is critical to resolve the 5’ flaps containing hairpin structure, and the other is checkpoint activation activity. Here, we show the genetic and functional interactions between the N-terminal domain and the C-terminal catalytic domain using a variety of $dna2$ mutant alleles. We found that the both hairpin binding and checkpoint activation activities become critical when the catalytic activities of Dna2 is malfunctional or when cells are under replication stress. We also found that the activation of replication checkpoint requires hairpin flap binding activity of Dna2. In addition, we investigated the hairpin DNA-binding properties of the N-terminal domain of Dna2 using a variety of structurally distinct substrates and determined that the DNA-protein interaction requires three-way DNA junction with single-stranded DNA end. The N-terminal domain of Dna2 showed the greatest binding affinity to Holliday junction DNA.