Okazaki Fragment Processing-independent Role for Human Dna2 Enzyme during DNA Replication

Abstract

Background: Dna2 and FEN1 putatively cooperate in Okazaki fragment (OF) processing in yeast. Results: FEN1 depletion leads to OF maturation defects that do not impact replication fork kinetics, whereas hDna2 depletion leads to DNA damage independent of OF maturation. Conclusion: hDna2 participates in DNA replication in a manner distinct from FEN1. Significance: hDna2 participates in replication in a FEN1, OF maturation-independent manner.

Preventing over-resection by DNA2 helicase/nuclease suppresses repair defects in Fanconi anemia cells

“…24 Consistent with this hypothesis, DNA2 knockdown cells accumulate in G 2 and show mitotic defects, such as aneuploidy, increased internuclear chromatin bridges, increased micronuclei, and pulverized chromosomes, 25,26 similar-though not identical-to phenotypes of FA and BLM cell lines. The DNA2 nuclease functions in flap removal during Okazaki fragment processing, in protection against unwanted fork regression at stalled forks, and is one of 2 major nucleases, the other being EXO1, required for the critical step of resection in HDR to produce the 3′ overhang essential for recombination.…”

“…DNA2 is an intrinsic replication fork protein and might be poised to recognize reversed forks. 26,62 In fact, in fission yeast, DNA2 is proposed to prevent deleterious recombinogenic replication fork reversal by cleaving nascent ssDNA at stalled replication forks. 33 In normal cells, FANCD2 may downregulate the resection of reversed forks.…”

“…25,26,67 Briefly, cells were transfected with pLKO.1.shSCR, pLKO.1.shDna2, pLKO.1.shDna2, or pLKO.1.shDna2* and pCMVΔR8.2 and pCMV-VSV-G using BioT (Bioland Scientific). 25,26 Virus was recovered 48 h post-transfection, and infections were performed in cells overnight in the presence of 10 μg/ml of protamine sulfate. Transduced cells were selected with 2 μg/ml of puromycin for 48 h. The following sequences were used for the hDNA2 short hairpins: 5′-CATAGCCAGT AGTATTCGAT G-3′ for shDna2, 5′-CCGGCCAGCT TTGAAGATGG ATTAACTCGA GTTAATCCAT CTTCAAAGCT GGTTTTTG-3′ for shDna2*.…”

“…This accumulation is also seen in cells lacking the DNA2 partner, BLM helicase. 17 A consequence of DNA2 depletion is the accumulation of endogenous DNA replication fork stress 26 that, like nucleotide deprivation, 7 may recruit FANCD2 to stalled or damaged DNA replication forks. We conclude that DNA2 is required downstream of MRE11 but not for FANCD2 ubiquitylation and focus formation.…”

Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity

“…It is of interest that overexpression of nuclease-dead Dna2 proteins had a dominant negative effect in dna2-1 mutants with impaired both helicase and nuclease functions of Dna2 under certain growth conditions (20). Similarly, overexpression of nuclease-dead human DNA2 was more detrimental than overexpression of both nuclease and helicase-dead construct (40). This result, together with our data showing that the nuclease activity limits the helicase function, implies that hyperactive and unregulated Dna2 helicase is toxic, and highlights the importance of restricting the helicase activity of Dna2 in vivo.…”