Okazaki fragment processing-independent role for human Dna2 enzyme during DNA replication

TY - JOUR

T1 - Okazaki fragment processing-independent role for human Dna2 enzyme during DNA replication

AU - Duxin, Julien P

AU - Moore, Hayley R

AU - Sidorova, Julia

AU - Karanja, Kenneth K

AU - Honaker, Yuchi

AU - Dao, Benjamin

AU - Piwnica-Worms, Helen

AU - Campbell, Judith L

AU - Monnat, Raymond J

AU - Stewart, Sheila A

PY - 2012/6/22

Y1 - 2012/6/22

N2 - 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.

AB - 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.

KW - Cell Cycle

KW - Cell Line, Tumor

KW - Cell Nucleus

KW - Chromatin

KW - DNA

KW - DNA Damage

KW - DNA Helicases

KW - DNA Repair

KW - DNA Replication

KW - Gene Expression Regulation

KW - HeLa Cells

KW - Humans

KW - Kinetics

KW - Micronucleus Tests

KW - Microscopy, Fluorescence

KW - Journal Article

KW - Research Support, N.I.H., Extramural

KW - Research Support, Non-U.S. Gov't

KW - Research Support, U.S. Gov't, Non-P.H.S.

U2 - 10.1074/jbc.m112.359018

DO - 10.1074/jbc.m112.359018

M3 - Journal article

C2 - 22570476

SN - 0021-9258

VL - 287

SP - 21980

EP - 21991

JO - The Journal of Biological Chemistry

JF - The Journal of Biological Chemistry

IS - 26

ER -