Xenopus egg extract: A powerful tool to study genome maintenance mechanisms

Hoogenboom, Wouter S.; Douwel, Daisy Klein; Knipscheer, Puck

doi:10.1016/j.ydbio.2017.03.033

Cited by 46 publications

(36 citation statements)

References 110 publications

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“…First, we assessed whether FA-RAD51 mutations alleviate protection of nascent DNA at stalled RF using Xenopus laevis cellfree egg extracts-a well-established system for studying DNA replication and genome maintenance-associated functions of proteins with a high degree of conservation between Xenopus and human proteins (Hoogenboom et al, 2017;Sannino et al, 2016). Xenopus extracts containing replicating chromatin were exposed to a short pulse of biotin-labeled dUTPs, followed by an aphidicolin (APH) treatment, and the remaining biotin in the nascent DNA was then quantified at various time points (Figure 1A) (Kolinjivadi et al, 2017).…”

Section: Fa-rad51 Mutants Are Defective At Protection Of Stalled Rfmentioning

confidence: 99%

Fanconi-Anemia-Associated Mutations Destabilize RAD51 Filaments and Impair Replication Fork Protection

et al. 2017

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Fanconi anemia (FA) is a genetic disorder characterized by a defect in DNA interstrand crosslink (ICL) repair, chromosomal instability, and a predisposition to cancer. Recently, two RAD51 mutations were reported to cause an FA-like phenotype. Despite the tight association of FA/HR proteins with replication fork (RF) stabilization during normal replication, it remains unknown how FA-associated RAD51 mutations affect replication beyond ICL lesions. Here, we report that these mutations fail to protect nascent DNA from MRE11-mediated degradation during RF stalling in Xenopus laevis egg extracts. Reconstitution of DNA protection in vitro revealed that the defect arises directly due to altered RAD51 properties. Both mutations induce pronounced structural changes and RAD51 filament destabilization that is not rescued by prevention of ATP hydrolysis due to aberrant ATP binding. Our results further interconnect the FA pathway with DNA replication and provide mechanistic insight into the role of RAD51 in recombination-independent mechanisms of genome maintenance.

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Section: Fa-rad51 Mutants Are Defective At Protection Of Stalled Rfmentioning

confidence: 99%

Fanconi-Anemia-Associated Mutations Destabilize RAD51 Filaments and Impair Replication Fork Protection

et al. 2017

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“…Xenopus egg extracts have a long track record of dissecting nuclear dynamics and interrogating processes such as DNA replication, chromosome segregation, and DNA repair (Heald et al, 1996; Hoogenboom et al, 2017; Kalab et al, 2006; Knipscheer et al, 2009; Lebofsky et al, 2009). We therefore used high-speed supernatant (HSS) of total Xenopus laevis egg lysate (Lebofsky et al, 2009) to look for cellular factors that promote the dissociation of a pre-formed Cas9 RNP-DNA complex.…”

Section: Resultsmentioning

confidence: 99%

The histone chaperone FACT induces Cas9 multi-turnover behavior and modifies genome manipulation in human cells

Wang

Chen

et al. 2019

Preprint

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is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but 29 turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about 30 the factors responsible for dislodging Cas9 or how they influence genome engineering. Using a 31 proximity labeling system for unbiased detection of transient protein interactions in cell-free 32 Xenopus laevis egg extract, we identified the dimeric histone chaperone FACT as an interactor 33 of substrate-bound Cas9. Immunodepletion of FACT subunits from extract potently inhibits Cas9 34 unloading and converts Cas9's activity from multi-turnover to single-turnover. In human cells, 35 depletion of FACT delays genome editing and alters the balance between indel formation and 36 homology directed repair. Depletion of FACT also increases epigenetic marking by dCas9-37 based transcriptional effectors with concomitant enhancement of transcriptional modulation. 38 FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most 39 likely by determining Cas9 residence times.40 41 42 43 44 45 46 47 48 49 50 51 52acetyltransferases or methyltransferases around a transcription start site (TSS) to manipulate 79 expression of endogenous genes (Gilbert et al., 2013; Hilton et al., 2015). Such transcriptional 80 engineering presumably relies on providing these histone modifiers sufficient time at the TSS to 81 deposit the appropriate epigenetic marks. Conversely, Cas9's utility as a targeted nuclease may 82 be predicated on its removal from the genome because Cas9 itself masks the DSB from cellular 83 repair enzymes (Clarke et al., 2018; Richardson et al., 2016b). Cas9 residence times and 84 unloading might thus play crucial roles in Cas9-based interventions. 85 86 While some Cas9 molecules behave as multi-turnover enzymes (Yourik et al., 2019), the widely 87 used Streptococcus pyogenes Cas9 and dCas9 exhibit extremely stable protein-DNA 88 interactions and possess residence times of over five hours in vitro (Raper et al., 2018; 89 Richardson et al., 2016b; Sternberg et al., 2014). Estimates of residence times in live cells vary 90 (Ma et al., 2016; Shao et al., 2016), but some experiments indicate that S. pyogenes Cas9 stays 91 bound to its target in mammalian cells for as little as five minutes (Knight et al., 2015) and imply 92 that cellular factors promote turnover. The ability to detect resolved genomic edits only a few 93 hours after electroporation of Cas9 RNPs (Kim et al., 2014) further suggests that cells actively 94 remove Cas9 from the genome either purposefully or as a byproduct of normal genome 95 metabolism. 97Prior work has suggested that RNA polymerases can dislodge Cas9 from DNA in vitro when the 98 gRNA anneals to the non-coding DNA strand but not to the coding strand (Clarke et al., 2018). 99Targeting the non-coding strand with a gRNA roughly correlated with increased editing rates in 100 human cells and an increased ability of a bacterially-encoded CRISPR system to fight phage 101 infection. However, t...

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“…Many regulators of ATR accumulate on chromatin in egg extracts upon replication stress [25,[32][33][34]. By mass spectrometry analysis, Haahr et al [21] observed enrichment of ETAA1 on chromatin following treatment with the DNA polymerase inhibitor aphidicolin (APH).…”

Section: Xenopus Etaa1 Associates With Chromatin In a Checkpoint-depementioning

confidence: 99%

RPA-coated single-stranded DNA promotes the ETAA1-dependent activation of ATR

2019

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Besides TopBP1, ETAA1 has been identified more recently as an activator of the ATR-ATRIP complex in human cells. We have examined the role of ETAA1 in the Xenopus egg-extract system, which has been instrumental in the study of ATR-ATRIP. Depletion of ETAA1 from egg extracts did not noticeably reduce the activation of ATR-ATRIP in response to replication stress, as monitored by the ATR-dependent phosphorylation of Chk1 and RPA. Moreover, lack of ETAA1 did not appear to affect DNA replication during an unperturbed S-phase. Significantly, we find that TopBP1 is considerably more abundant than ETAA1 in egg extracts. We proceeded to show that ETAA1 could support the activation of ATR-ATRIP in response to replication stress if we increased its concentration in egg extracts by adding extra full-length recombinant ETAA1. Thus, TopBP1 appears to be the predominant activator of ATR-ATRIP in response to replication stress in this system. We have also explored the biochemical mechanism by which ETAA1 activates ATR-ATRIP. We have developed an in vitro system in which full-length recombinant ETAA1 supports activation of ATR-ATRIP in the presence of defined components. We find that binding of ETAA1 to RPA associated with single-stranded DNA (ssDNA) greatly stimulates its ability to activate ATR-ATRIP. Thus, RPA-coated ssDNA serves as a direct positive effector in the ETAA1-mediated activation of ATR-ATRIP.

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Xenopus egg extract: A powerful tool to study genome maintenance mechanisms

Cited by 46 publications

References 110 publications

Fanconi-Anemia-Associated Mutations Destabilize RAD51 Filaments and Impair Replication Fork Protection

Fanconi-Anemia-Associated Mutations Destabilize RAD51 Filaments and Impair Replication Fork Protection

The histone chaperone FACT induces Cas9 multi-turnover behavior and modifies genome manipulation in human cells

RPA-coated single-stranded DNA promotes the ETAA1-dependent activation of ATR

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