RPA phosphorylation regulates DNA resection

Soniat, Michael; Lr, Myler; Paull, Tanya T.; Finkelstein, Ilya J.

doi:10.1101/517771

Cited by 4 publications

(8 citation statements)

References 100 publications

(146 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We used a double-stranded DNA (dsDNA) substrate with 3'-ssDNA overhangs that mimic TMEJ resection intermediates. Helicase activity generates ssDNA that can be monitored via a growing RPA-GFP signal (Figure S3A) (41). However, the RPA-GFP intensity did not change when Polθ-h and ATP were added to the flowcell (Figure S3B).…”

Section: Polθ-helicase Strips Rpa From Single-stranded Dnamentioning

confidence: 99%

“…RAD51(K133R) was mutagenized using inverse PCR with primers IF724 and IF725. RPA-GFP (plasmid pIF48), RAD51 (plasmid pIF224), RAD51(K133R) (plasmid pIF582) and PARP-1 (plasmid pIF662) were purified as previously described (41,62,63).…”

Section: Proteins and Nucleic Acidsmentioning

confidence: 99%

“…Polθ-h helicase activity was measured in flowcells containing double-stranded DNA (dsDNA), as used previously for RecQ-family helicases (41,67). The DNA substrate was derived from bacteriophage λ.…”

Section: Polθ-h Helicase Assaysmentioning

confidence: 99%

“…The DNA substrate was derived from bacteriophage λ. The cosL end was ligated with LAB07 and cosR with Lambda Poly-T that produces a 3'-T78 overhang (41). Polθ-h was injected into the flowcell in Imaging Buffer at 0.4 mL min -1 .…”

Section: Polθ-h Helicase Assaysmentioning

confidence: 99%

“…For double-stranded DNA end bridging experiments, we hybridized λ-phage DNA with LAB07 and Lambda Poly-T oligos by thermal melting and subsequent ligation with T4 DNA Ligase (NEB, M0202) as previously described (41). The DNA was fluorescently stained with YOYO-1 to visualize end-tethering.…”

Section: Dna Tethering Assaysmentioning

confidence: 99%

See 4 more Smart Citations

Polymerase theta-helicase promotes end joining by stripping single-stranded DNA-binding proteins and bridging DNA ends

Soniat

Finkelstein

2021

Preprint

Self Cite

View full text Add to dashboard Cite

Homologous recombination-deficient cancers rely on DNA polymerase Theta (Polθ)-Mediated End Joining (TMEJ), an alternative double-strand break repair pathway. Polθ is the only vertebrate polymerase that encodes an N-terminal superfamily 2 (SF2) helicase domain, but the role of this helicase domain in TMEJ remains unclear. Using single-molecule imaging, we demonstrate that Polθ-helicase (Polθ-h) is a highly processive single-stranded DNA (ssDNA) motor protein that can efficiently strip Replication Protein A (RPA) from ssDNA. Polθ-h also has a limited capacity for disassembling RAD51 filaments but is not processive on double- stranded DNA. Polθ-h can bridge two non-complementary DNA strands in trans. PARylation of Polθ-h by PARP-1 resolves these DNA bridges. We conclude that Polθ-h removes RPA and RAD51 filaments and mediates bridging of DNA overhangs to aid in polymerization by the Polθ polymerase domain.

show abstract

Section: Polθ-helicase Strips Rpa From Single-stranded Dnamentioning

confidence: 99%

Section: Proteins and Nucleic Acidsmentioning

confidence: 99%

Section: Polθ-h Helicase Assaysmentioning

confidence: 99%

Section: Polθ-h Helicase Assaysmentioning

confidence: 99%

Section: Dna Tethering Assaysmentioning

confidence: 99%

See 3 more Smart Citations

Polymerase theta-helicase promotes end joining by stripping single-stranded DNA-binding proteins and bridging DNA ends

Soniat

Finkelstein

2021

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

Role of RPA Phosphorylation in the ATR-dependent G2/M Checkpoint and DSB Repair

Liu,

Byrne,

Byrne

et al. 2023

Preprint

View full text Add to dashboard Cite

Cells respond to DNA double-strand breaks by initiating DSB repair and ensuing a cell cycle checkpoint. The primary responder to DSB repair is non-homologous end joining, an error prone repair pathway. However, when DSBs are generated after DNA replication in the G2 phase of the cell cycle, a second DSB repair pathway, homologous recombination, can come into action. Both ATM and ATR are important for DSB-induced DSB repair and checkpoint responses. One method of ATM and ATR working together is through the DNA end-resection of DSBs. As a readout and marker of DNA end-resection, RPA is phosphorylated at Ser4/Ser8 of the N-terminus of RPA32 in response to DSBs. Here, the significance of RPA32 Ser4/Ser8 phosphorylation in response to DNA damage, specifically in S phase to G2 phase of the cell cycle is examined. RPA32 Ser4/Ser8 phosphorylation in G2 synchronized cells is necessary for increases in TopBP1 and Rad9 accumulation on chromatin and full activation of the ATR-dependent G2 checkpoint. In addition, our data suggest RPA Ser4/Ser8 phosphorylation modulates ATM-dependent KAP-1 phosphorylation and Rad51 chromatin loading in G2 cells.

show abstract

The regulation of DNA end resection by chromatin response to DNA double strand breaks

Chen

Sleckman

2022

Front. Cell Dev. Biol.

View full text Add to dashboard Cite

DNA double-strand breaks (DSBs) constantly arise upon exposure to genotoxic agents and during physiological processes. The timely repair of DSBs is important for not only the completion of the cellular functions involving DSBs as intermediates, but also the maintenance of genome stability. There are two major pathways dedicated to DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). The decision of deploying HR or NHEJ to repair DSBs largely depends on the structures of broken DNA ends. DNA ends resected to generate extensive single-strand DNA (ssDNA) overhangs are repaired by HR, while those remaining blunt or minimally processed can be repaired by NHEJ. As the generation and repair of DSB occurs within the context of chromatin, the resection of broken DNA ends is also profoundly affected by the state of chromatin flanking DSBs. Here we review how DNA end resection can be regulated by histone modifications, chromatin remodeling, and the presence of ssDNA structure through altering the accessibility to chromatin and the activity of pro- and anti-resection proteins.

show abstract

RPA phosphorylation regulates DNA resection

Cited by 4 publications

References 100 publications

Polymerase theta-helicase promotes end joining by stripping single-stranded DNA-binding proteins and bridging DNA ends

Polymerase theta-helicase promotes end joining by stripping single-stranded DNA-binding proteins and bridging DNA ends

Role of RPA Phosphorylation in the ATR-dependent G2/M Checkpoint and DSB Repair

The regulation of DNA end resection by chromatin response to DNA double strand breaks

Contact Info

Product

Resources

About