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Published June 22, 2012 | Supplemental Material + Published
Journal Article Open

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


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/G2 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.

Additional Information

© 2012 by The American Society for Biochemistry and Molecular Biology, Received March 5, 2012. Revision received April 25, 2012. This work was supported, in whole or in part, by National Institutes of Health Grants GM95924 (to S. A. S.), GM04701 and GM04707 (to H. P.-W.), P01 CA77852 (to R. J. M.), and GM078666 (to J. L. C.). This work was also supported by the Department of Defense Grant CDMRP 09-0-0041, Cancer Biology Pathway, Siteman Cancer Center at Barnes-Jewish Hospital, and Washington University School of Medicine in St Louis (to J. P. D. and H. R. M.). We thank Dr. Soza and Dr. Montecucco for the BrdU-comet assay protocol and technical assistance with the procedure, Dr. Junran Zhang and Dr. Wei Shi for the RPA immunofluorescence protocol and aliquots of the antibody, Megan Ruhland, Avi Silver, and Keffy Kehrli for technical assistance, and members of the Stewart, Campbell, and Monnat laboratories for useful comments. We also thank Ermira Pazolli and Daniel Teasley for critical reading of the manuscript. We thank the The RNAi Consortium, Children's Discovery Institute and Washington University Genome Center for the RNAi constructs.

Attached Files

Published - Duxin2012p19109J_Biol_Chem.pdf

Supplemental Material - jbc.M112.359018-1.pdf


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August 19, 2023
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