The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA

Abstract

Background:The biochemical function of variable N-terminal regions of eukaryotic Dna2 remains unclear. Results: The N-terminal 45-kDa domain of yeast Dna2 targets the enzyme specifically to a secondary structure flap. Conclusion:The hairpin binding activity of Dna2 contributes to efficient removal of hairpin flaps together with endonuclease and helicase activities during Okazaki fragment processing. Significance: The hairpin binding activity of Dna2 is critical for genome stability.

Rad52/Rad59-dependent Recombination as a Means to Rectify Faulty Okazaki Fragment Processing

“…2B). It was shown that the N-terminal 405-aa deletion is not a loss-of-function mutation in enzymatic activity; the Dna2⌬405N mutant enzyme is catalytically as active or more active than wild type Dna2 under some circumstances (28,31). This observation suggests that the types of DNA damages caused by dna2⌬405N are not critically dependent on the elevated levels of Rad51 or Rad52 for repair.…”

“…It was interpreted that the Rad27-n,p allows Okazaki fragments to be processed by other alternative nucleases such as Dna2 or even by the 3Ј to 5Ј exonuclease activity of Pol ␦ (63). Recently, it was shown that Dna2 is involved in the activation of the Mec1 kinase, a sensor protein for the intra-S-phase checkpoint activation (27,28). Therefore, it is possible that Dna2-K1080E in a complex with the 5Ј-structured flap could cause cell death due to the hyperactivation of the intra-S-phase checkpoint.…”

“…3B) rules out the possibility that the suppression observed by Rad27 is due to the ability of Rad27 to dissociate Dna2 from DNA. It was shown that Dna2⌬405N, mutant Dna2 lacking the N-terminal 405-aa domain, was not able to recognize and bind to secondary structured flaps and that the inability of Dna2⌬405N to target to a secondary structured flap rendered this mutant enzyme severely defective in resolving secondary structure (28). Although Dna2-K1080E and Dna2⌬405N are similar with respect to their inability to process secondary structured flaps, dna2-K1080E cells displayed much more severe growth defects than those of dna2⌬405N; the former are lethal, whereas the latter grow in a temperature-sensitive manner.…”

“…The endonuclease activity of Dna2 is critical for both Okazaki fragment processing and DSB resection (23,24), whereas the N-terminal 405-aa domain of Dna2 is required for the checkpoint function and also for DSB resection to some extent (27,36). This N-terminal domain of Dna2 becomes critical in the processing of secondary structured flaps in Okazaki fragment (28). In contrast to the multiple functions of the endonuclease and N-terminal DNA binding activities of Dna2, the helicase activity of Dna2 seems to be involved exclusively in DNA replication, most likely in the processing of secondary structure or higher order structure flaps present in a subset of Okazaki fragments (23,27,32,36,37).…”

“…Among the enzymes involved in the maturation of Okazaki fragments, Dna2 appears to link diverse DNA metabolisms by playing multiple roles in Okazaki fragment processing (11,17,21,22), long-range resection of double strand break (DSB) repair (23)(24)(25)(26), and S-phase checkpoint activation (27,28). It has been shown that yeast Dna2 consists of three domains, each of which encodes a distinct biochemical activity: helicase, endonuclease, and secondary structure-specific DNA binding activity (28 -32).…”