Visualization of the physical and functional interaction between hMYH and hRad9 by Dronpa bimolecular fluorescence complementation

Agustina, Lia; Hahm, Soo-Hyun; Han, Se Hee; Tran, An Hue Vy; Chung, Ji Hyung; Park, Jong-Hwa; Park, Jin Woo; Han, Ye

doi:10.1186/1471-2199-15-17

Cited by 4 publications

(4 citation statements)

References 29 publications

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“…Recently, Agustina et al . [45] showed that hRad9 interacts with N-terminal region (residues 1–74) of hMYH. Thus, the physical interactions of MYH with Hus1 and Rad9 appear different.…”

Section: Discussionmentioning

confidence: 99%

“…We have shown previously that deletion of fission yeast S. pombe SpMyh1 strongly abrogated the association of SpHus1 with telomeres [51]. hMYH knockdown in human cells decreases Chk1 and Cdc25C phosphorylation in response to H 2 O 2 treatment [52] and decreases p-Chk1 and p-Cdk2 after hydroxyurea treatment [45]. Thus, we suggest that MYH first recognizes a mismatch then recruits 9-1-1 to sites of DNA damage through MYH-Hus1 interaction.…”

Section: Discussionmentioning

confidence: 99%

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Association of the Rad9–Rad1–Hus1 checkpoint clamp with MYH DNA glycosylase and DNA

et al. 2015

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Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9-1-1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9-1-1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134–155) is a key determinant of MYH binding. Both the N- (residues 1–146) and C-terminal (residues 147–280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1K136A mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad91-266 (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 91-266-1-1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 91-266-1-1 to 5′-recessed DNA substrates is an intrinsic property of this complex and is dependent of complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9-1-1 subunits to MYH-directed BER based on subunit asymmetry in protein-protein interactions and DNA binding events.

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“…Recently, Agustina et al . [45] showed that hRad9 interacts with N-terminal region (residues 1–74) of hMYH. Thus, the physical interactions of MYH with Hus1 and Rad9 appear different.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Association of the Rad9–Rad1–Hus1 checkpoint clamp with MYH DNA glycosylase and DNA

et al. 2015

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“…HU is an inhibitor of ribonucleotide reductase that inhibits DNA replication in cells [ 18 , 21 , 22 ]. HU induces the accumulation of ROS and cell death.…”

Section: Resultsmentioning

confidence: 99%

“…In our previous study, we found that depletion of hMYH disrupts ATR and Chk1 activation following hydroxyurea (HU) and ultraviolet treatment [ 17 ]. In addition, we also observed endogenous interactions between hTopBP1 and hMYH as well as hRad9 and hMYH [ 5 , 18 ]. These observations support the claim that hMYH is an important component of the ATR signaling pathway.…”

Section: Introductionmentioning

confidence: 99%

A physical association between the human mutY homolog (hMYH) and DNA topoisomerase II-binding protein 1 (hTopBP1) regulates Chk1-induced cell cycle arrest in HEK293 cells

et al. 2015

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BackgroundHuman DNA topoisomerase II-binding protein 1 (hTopBP1) plays an important role in DNA replication and the DNA damage checkpoint pathway. The human mutY homolog (hMYH) is a base excision repair DNA glycosylase that excises adenines or 2-hydroxyadenines that are mispaired with guanine or 7,8-dihydro-8-oxoguanine (8-oxoG). hTopBP1 and hMYH were involved in ATR-mediated Chk1 activation, moreover, both of them were associated with ATR and hRad9 which known as checkpoint-involved proteins. Therefore, we investigated whether hTopBP1 interacted with hMYH, and what the function of their interaction is.ResultsWe documented the interaction between hTopBP1 and hMYH and showed that this interaction increased in a hydroxyurea-dependent manner. We also mapped the hMYH-interacting region of hTopBP1 (residues 444–991). In addition, we investigated several cell cycle-related proteins and found that co-knockdown of hTopBP1 and hMYH significantly diminished cell cycle arrest due to compromised checkpoint kinase 1 (Chk1) activation. Moreover, we observed that hMYH was essential for the accumulation of hTopBP1 on damaged DNA, where hTopBP1 interacts with hRad9, a component of the Rad9-Hus1-Rad1 complex. The accumulation of hTopBP1 on chromatin and its subsequent interaction with hRad9 lead to cell cycle arrest, a process mediated by Chk1 phosphorylation and ataxia telangiectasia and Rad3-related protein (ATR) activation.ConclusionsOur results suggested that hMYH is necessary for the accumulation of hTopBP1 to DNA damage lesion to induce the association of hTopBP1 with 9-1-1 and that the interaction between hMYH and hTopBP1 is essential for Chk1 activation. Therefore, we suggest that the interaction between hMYH and hTopBP1 is crucial for activation of the ATR-mediated cell cycle checkpoint.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-015-0042-x) contains supplementary material, which is available to authorized users.

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Functional interaction between hMYH and hTRADD in the TNF-α-mediated survival and death pathways of HeLa cells

Tran

Hahm

Han

et al. 2015

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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Visualization of the physical and functional interaction between hMYH and hRad9 by Dronpa bimolecular fluorescence complementation

Cited by 4 publications

References 29 publications

Association of the Rad9–Rad1–Hus1 checkpoint clamp with MYH DNA glycosylase and DNA

Association of the Rad9–Rad1–Hus1 checkpoint clamp with MYH DNA glycosylase and DNA

A physical association between the human mutY homolog (hMYH) and DNA topoisomerase II-binding protein 1 (hTopBP1) regulates Chk1-induced cell cycle arrest in HEK293 cells

Functional interaction between hMYH and hTRADD in the TNF-α-mediated survival and death pathways of HeLa cells

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