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

Julien P Duxin,Hayley R Moore,Julia Sidorova,Kenneth Karanja,Yuchi Honaker,Benjamin Dao,Helen Piwnica-Worms,Judith L Campbell,Raymond J Monnat,Sheila A Stewart

doi:10.1074/jbc.m112.359018

Abstract

Dna2 is an essential helicase/nuclease that is postulated to cleave long DNA flaps that escape FEN1 activity during Okazaki fragment (OF) maturation in yeast. We previously demonstrated that the human Dna2 orthologue (hDna2) localizes to the nucleus and contributes to genomic stability. Here we investigated the role hDna2 plays in DNA replication. We show that Dna2 associates with the replisome protein And-1 in a cell cycle-dependent manner. Depletion of hDna2 resulted in S/G(2) phase-specific DNA damage as evidenced by increased γ-H2AX, replication protein A foci, and Chk1 kinase phosphorylation, a readout for activation of the ATR-mediated S phase checkpoint. In addition, we observed reduced origin firing in hDna2-depleted cells consistent with Chk1 activation. We next examined the impact of hDna2 on OF maturation and replication fork progression in human cells. As expected, FEN1 depletion led to a significant reduction in OF maturation. Strikingly, the reduction in OF maturation had no impact on replication fork progression, indicating that fork movement is not tightly coupled to lagging strand maturation. Analysis of hDna2-depleted cells failed to reveal a defect in OF maturation or replication fork progression. Prior work in yeast demonstrated that ectopic expression of FEN1 rescues Dna2 defects. In contrast, we found that FEN1 expression in hDna2-depleted cells failed to rescue genomic instability. These findings suggest that the genomic instability observed in hDna2-depleted cells does not arise from defective OF maturation and that hDna2 plays a role in DNA replication that is distinct from FEN1 and OF maturation.

Highlights

Dna2 and flap endonuclease 1 (FEN1) putatively cooperate in Okazaki fragment (OF) processing in yeast
HDna2 Contributes to Genomic Stability—We previously reported that human Dna2 orthologue (hDna2) depletion in U-2-OS cells leads to genomic instability characterized by the appearance of aneuploid cells, internuclei bridges (ICBs), and an accumulation of cells in the late S/G2 phase of the cell cycle [1]
We further report that hDna2 depletion results in an increase in ␥-H2AX, a well characterized marker of DNA damage including double strand breaks (DSBs), and the appearance of micronuclei indicative of aberrant mitosis

Summary

Background

Dna 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 hDna depletion leads to DNA damage independent of OF maturation. RPAbound OF flaps recruit Dna, which cleaves the RPA-coated DNA and displaces RPA, leaving a short 5– 6-nucleotide RNAfree DNA flap that is further processed by FEN1 to produce a ligatable nick (9, 10, 14 –18) In support of this long flap model, both PIF1 helicase and polymerase ␦ processivity subunit (Pol 32) promote long flap formation in vitro, and their deletion rescues the lethality associated with yeast Dna loss, presumably because long flaps no longer form (12, 19 –22). In vivo and in vitro studies in yeast and humans indicate that Exo can compensate for Dna nuclease activity in this process [24, 29, 31] This suggests that the essential function of Dna is not its resection activity during DSB repair but rather its function in removing long flaps during DNA replication. We provide evidence that hDna ensures genomic stability by virtue of a critical role in DNA replication that is independent of FEN1 and OF processing

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION