Concerted and differential actions of two enzymatic domains underlie Rad5 contributions to DNA damage tolerance

Choi, Kyungoh; Batke, Sabrina; Szakál, Barnabás; Lowther, Jonathan; Hao, Fanfan; Sarangi, Prabha; Branzei, Dana; Ulrich, Helle D.; Zhao, Xiaolan

doi:10.1093/nar/gkv004

Cited by 46 publications

(60 citation statements)

References 44 publications

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“…These findings are consistent with previously reported negative interactions of RAD5 with these genes in MMS and positive interactions when no drug stress is added (St Onge et al , ; Table EV4). The dynamic interactions of RAD5 with these two gene groups may reflect the previously reported multifunctional nature of RAD5 and its ability to coordinate repair events and replication fork progression differently in response to different types of lesions (Choi et al , ).…”

Section: Resultsmentioning

confidence: 89%

Mapping DNA damage‐dependent genetic interactions in yeast via party mating and barcode fusion genetics

Díaz-Mejía

Celaj

Mellor

et al. 2018

Molecular Systems Biology

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Condition‐dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State‐of‐the‐art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double‐mutant strains, does not scale readily to multi‐condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG‐GI), by which double‐mutant strains generated via en masse “party” mating can also be monitored en masse for growth to detect genetic interactions. By using site‐specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG‐GI enables multiplexed quantitative tracking of double mutants via next‐generation sequencing. We applied BFG‐GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4‐nitroquinoline 1‐oxide (4NQO), bleomycin, zeocin, and three other DNA‐damaging environments. BFG‐GI recapitulated known genetic interactions and yielded new condition‐dependent genetic interactions. We validated and further explored a subnetwork of condition‐dependent genetic interactions involving MAG1,SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53.

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Section: Resultsmentioning

confidence: 89%

Mapping DNA damage‐dependent genetic interactions in yeast via party mating and barcode fusion genetics

Díaz-Mejía

Celaj

Mellor

et al. 2018

Molecular Systems Biology

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“…While we observed a nine-fold increase in 4NQO-associated recombination in either a rad5 strain containing pR28 ( RAD5 ) or in a wild type strain, we only observed a four-fold and three-fold increase in 4NQO-associated SCE in rad5 deletion mutants containing pR-19 ( rad5 C914, C917/AA) or pR-30 ( rad5 D681, E682/AA), respectively. Although this would suggest that both ubiquitin ligase and helicase functions are required for 4NQO-associated recombination, Choi et al [45] suggest that the Rad5 helicase motif important for ATP binding is also required for PCNA polyubiquitination.…”

Section: Resultsmentioning

confidence: 99%

Both RAD5-dependent and independent pathways are involved in DNA damage-associated sister chromatid exchange in budding yeast

Fasullo

Sun

2017

AIMS Genetics

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Sister chromatids are preferred substrates for recombinational repair after cells are exposed to DNA damage. While some agents directly cause double-strand breaks (DSBs), others form DNA base adducts which stall or impede the DNA replication fork. We asked which types of DNA damage can stimulate SCE in budding yeast mutants defective in template switch mechanisms and whether PCNA polyubiquitination functions are required for DNA damage-associated SCE after exposure to potent recombinagens. We measured spontaneous and DNA damage-associated unequal sister chromatid exchange (uSCE) in yeast strains containing two fragments of his3 after exposure to MMS, 4-NQO, UV, X rays, and HO endonuclease-induced DSBs. We determined whether other genes in the pathway for template switching, including UBC13, MMS2, SGS1, and SRS2 were required for DNA damage-associated SCE. RAD5 was required for DNA damage-associated SCE after exposure to UV, MMS, and 4-NQO, but not for spontaneous, X-ray-associated, or HO endonuclease-induced SCE. While UBC13, MMS2, and SGS1 were required for MMS and 4NQO-associated SCE, they were not required for UV-associated SCE. DNA damage-associated recombination between his3 recombination substrates on non-homologous recombination was enhanced in rad5 mutants. These results demonstrate that DNA damaging agents that cause DSBs stimulate SCE by RAD5-independent mechanisms, while several potent agents that generate bulky DNA adducts stimulate SCE by multiple RAD5-dependent mechanisms. We suggest that DSB-associated recombination that occurs in G2 is RAD5-independent.

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“…In addition to structure-specific helicase activity, Rad5 also possesses a RING motif associated with ubiquitin ligase activity that plays a role in polyubiquitination of PCNA [34–37]. Two mutations in Rad5, Q1106D and C914A/C917A, were recently described to disrupt its helicase and ubiquitin-ligase activity, respectively [27,38]. Disrupting either of these Rad5 activities in the rrm3Δ mutant significantly increased sensitivity to MMS and to HU, indicating that both activities make important contributions to the repair of DNA lesions that arise in the absence of Rrm3 (Fig 2B).…”

Section: Resultsmentioning

confidence: 99%

“…(B) Ubiquitin ligase and helicase activities of Rad5 contribute to DNA damage tolerance in the absence of Rrm3. Ubiquitin ligase activity of Rad5 was inactivated by C914A/C917A mutations and ATPase/helicase activity by Q1106D [27,38]. (C) Accumulation of rrm3Δ cells in S phase increases upon deletion of RAD5 or RDH54 .…”

Section: Resultsmentioning

confidence: 99%

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

et al. 2016

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In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186–212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.

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Concerted and differential actions of two enzymatic domains underlie Rad5 contributions to DNA damage tolerance

Cited by 46 publications

References 44 publications

Mapping DNA damage‐dependent genetic interactions in yeast via party mating and barcode fusion genetics

Mapping DNA damage‐dependent genetic interactions in yeast via party mating and barcode fusion genetics

Both RAD5-dependent and independent pathways are involved in DNA damage-associated sister chromatid exchange in budding yeast

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

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