The N-terminus of RPA large subunit and its spatial position are important for the 5′-&gt;3′ resection of DNA double-strand breaks

Tammaro, Margaret; Liao, Shuren; McCane, Jill E.; Yan, Hong

doi:10.1093/nar/gkv764

Cited by 11 publications

(19 citation statements)

References 32 publications

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“…Expression and purification of wild-type (WT) MRN complex and a nuclease dead-mutant complex containing Mre11(H130N) were described previously (80,88). Expression and purification of WT RPA complex and the RPA (1NΔ) mutant complex were described previously (61). The RPA1 subunit in RPA (1NΔ) lacks 121 amino acids at the N-terminus (61).…”

Section: Methodsmentioning

confidence: 99%

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Dna2 initiates resection at clean DNA double-strand breaks

Paudyal

Yan

et al. 2017

Nucleic Acids Research

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Nucleolytic resection of DNA double-strand breaks (DSBs) is essential for both checkpoint activation and homology-mediated repair; however, the precise mechanism of resection, especially the initiation step, remains incompletely understood. Resection of blocked ends with protein or chemical adducts is believed to be initiated by the MRN complex in conjunction with CtIP through internal cleavage of the 5′ strand DNA. However, it is not clear whether resection of clean DSBs with free ends is also initiated by the same mechanism. Using the Xenopus nuclear extract system, here we show that the Dna2 nuclease directly initiates the resection of clean DSBs by cleaving the 5′ strand DNA ∼10–20 nucleotides away from the ends. In the absence of Dna2, MRN together with CtIP mediate an alternative resection initiation pathway where the nuclease activity of MRN apparently directly cleaves the 5′ strand DNA at more distal sites. MRN also facilitates resection initiation by promoting the recruitment of Dna2 and CtIP to the DNA substrate. The ssDNA-binding protein RPA promotes both Dna2- and CtIP–MRN-dependent resection initiation, but a RPA mutant can distinguish between these pathways. Our results strongly suggest that resection of blocked and clean DSBs is initiated via distinct mechanisms.

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Section: Methodsmentioning

confidence: 99%

“…Expression and purification of WT RPA complex and the RPA (1NΔ) mutant complex were described previously (61). The RPA1 subunit in RPA (1NΔ) lacks 121 amino acids at the N-terminus (61). …”

Section: Methodsmentioning

confidence: 99%

Dna2 initiates resection at clean DNA double-strand breaks

Paudyal

Yan

et al. 2017

Nucleic Acids Research

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“…Studies in Xenopus egg extracts also show that RPA interacts with both WRN and Dna2 to promote 3′→5′ helicase activity of WRN and 5′→3′ nuclease activity of Dna2 [117,118]. In addition to its role in directing 5′→3′ resection, RPA binds promptly to the newly generated 3′ ssDNA and protects the resection product [85][86][87]92,118,120,123,124]. Functional disruption of RPA in yeast not only abrogates resection extension, but also causes formation of hairpin structures on the short 3′ ssDNA generated by MRX-Sae2, which can be further processed, resulting in genomic instability [123].…”

Section: Termination Of Resectionmentioning

confidence: 97%

“…The long stretch of ssDNA generated by Exo1 and Dna2 serves as the substrate for HR, and in the meantime prevents repair by NHEJ or MMEJ [9,92]. The ssDNA-binding protein RPA promotes resection extension by enhancing the nuclease activity of Dna2 on the 5′ strand and by suppressing the inhibitory effects of the 3′ ssDNA resection product on Exo1 [85][86][87]92,117,118,[122][123][124].…”

Section: Extension Of Resection By Exo1 and Dna2mentioning

confidence: 99%

“…A recent structural study of Dna2-ssDNA-RPA complex and in vitro nuclease assays using mouse Dna2 shows that Dna2 physically interacts with RPA bound to both strands but can only displace RPA from the 5′ strand and hence the resection occurs only on the 5′ strand [120]. Studies in Xenopus egg extracts also show that RPA interacts with both WRN and Dna2 to promote 3′→5′ helicase activity of WRN and 5′→3′ nuclease activity of Dna2 [117,118]. In addition to its role in directing 5′→3′ resection, RPA binds promptly to the newly generated 3′ ssDNA and protects the resection product [85][86][87]92,118,120,123,124].…”

Section: Termination Of Resectionmentioning

confidence: 99%

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Sharpening the ends for repair: mechanisms and regulation of DNA resection

Paudyal¹,

You²

2016

ABBS

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DNA end resection is a key process in the cellular response to DNA double-strand break damage that is essential for genome maintenance and cell survival. Resection involves selective processing of 5′ ends of broken DNA to generate ssDNA overhangs, which in turn control both DNA repair and checkpoint signaling. DNA resection is the first step in homologous recombination-mediated repair and a prerequisite for the activation of the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent checkpoint that coordinates repair with cell cycle progression and other cellular processes. Resection occurs in a cell cycle-dependent manner and is regulated by multiple factors to ensure an optimal amount of ssDNA required for proper repair and genome stability. Here, we review the latest findings on the molecular mechanisms and regulation of the DNA end resection process and their implications for cancer formation and treatment.

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CDK1 phosphorylates WRN at collapsed replication forks

et al. 2016

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Regulation of end-processing is critical for accurate repair and to switch between homologous recombination (HR) and non-homologous end joining (NHEJ). End resection is a two-stage process but very little is known about regulation of the long-range resection, especially in humans. WRN participates in one of the two alternative long-range resection pathways mediated by DNA2 or EXO1. Here we demonstrate that phosphorylation of WRN by CDK1 is essential to perform DNA2-dependent end resection at replication-related DSBs, promoting HR, replication recovery and chromosome stability. Mechanistically, S1133 phosphorylation of WRN is dispensable for relocalization in foci but is involved in the interaction with the MRE11 complex. Loss of WRN phosphorylation negatively affects MRE11 foci formation and acts in a dominant negative manner to prevent long-range resection altogether, thereby licensing NHEJ at collapsed forks. Collectively, we unveil a CDK1-dependent regulation of the WRN-DNA2-mediated resection and identify an undescribed function of WRN as a DSB repair pathway switch.

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The N-terminus of RPA large subunit and its spatial position are important for the 5′->3′ resection of DNA double-strand breaks

Cited by 11 publications

References 32 publications

Dna2 initiates resection at clean DNA double-strand breaks

Dna2 initiates resection at clean DNA double-strand breaks

Sharpening the ends for repair: mechanisms and regulation of DNA resection

CDK1 phosphorylates WRN at collapsed replication forks

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