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PMID:22570476

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Citation

Duxin, JP, Moore, HR, Sidorova, J, Karanja, K, Honaker, Y, Dao, B, Piwnica-Worms, H, Campbell, JL, Monnat, RJ Jr and Stewart, SA (2012) Okazaki fragment processing-independent role for human Dna2 enzyme during DNA replication. J. Biol. Chem. 287:21980-91

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.

Links

PubMed PMC3381158 Online version:10.1074/jbc.M112.359018

Keywords

Cell Cycle; Cell Line, Tumor; Cell Nucleus/metabolism; Chromatin/metabolism; DNA/chemistry; DNA Damage; DNA Helicases/chemistry; DNA Helicases/physiology; DNA Repair; DNA Replication; Gene Expression Regulation; HeLa Cells; Humans; Kinetics; Micronucleus Tests; Microscopy, Fluorescence/methods

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:DNA2

involved_in

GO:0000076: DNA replication checkpoint signaling

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:DNA2

involved_in

GO:0000076: DNA replication checkpoint

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:DNA2

involved_in

GO:0006260: DNA replication

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

Notes

See also

References

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