Differential binding kinetics of replication protein A during replication and the pre- and post-incision steps of nucleotide excision repair

Gourdin, Audrey M.; Cuijk, Loes van; Tresini, Maria; Luijsterburg, Martijn S.; Nigg, Alex L.; Giglia-Mari, Giuseppina; Houtsmuller, Adriaan B.; Vermeulen, Wim; Marteijn, Jürgen A.

doi:10.1016/j.dnarep.2014.09.013

Cited by 3 publications

(3 citation statements)

References 56 publications

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“…However, the abundance of Rad6/Rad18 complexes is maintained at a low level (approximately ≤795/cell) and does not change following UVR exposure. , How can Rad6/Rad18 complexes effectively compete with the vast overabundance of competitive PCNA binding proteins in human cells? This study along with previous work from our group and others suggests that the selectivity and efficiency of PCNA monoubiquitination are achieved through nonspecific Rad18·RPA interactions. On native DNA templates, RPA filaments adjacent to progressing P/T junctions are short and transient due to the minimal exposure of native ssDNA templates and their rapid conversion to double-stranded DNA duplexes by the replicative pols ε and δ (Figure , left) . Here, Rad18·RPA interactions on ssDNA are prohibited and Rad6/Rad18 complexes must engage loaded PCNA directly from solution.…”

Section: Discussionmentioning

confidence: 99%

“…37 This study along with previous work from our group and others suggests that the selectivity and efficiency of PCNA monoubiquitination are achieved through nonspecific Rad18• RPA interactions. On native DNA templates, RPA filaments adjacent to progressing P/T junctions are short and transient 38 due to the minimal exposure of native ssDNA templates 4 and their rapid conversion to double-stranded DNA duplexes by the replicative pols ε and δ (Figure 6, left). 39 Here, Rad18•RPA interactions on ssDNA are prohibited and Rad6/Rad18 complexes must engage loaded PCNA directly from solution.…”

Section: ■ Discussionmentioning

confidence: 99%

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PCNA Monoubiquitination Is Regulated by Diffusion of Rad6/Rad18 Complexes along RPA Filaments

Sengupta

Benkovic

et al. 2020

Biochemistry

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Translesion DNA synthesis (TLS) enables DNA replication through damaging modifications to template DNA and requires monoubiquitination of the proliferating cell nuclear antigen (PCNA) sliding clamp by the Rad6/Rad18 complex. This posttranslational modification is critical to cell survival following exposure to DNA-damaging agents and is tightly regulated to restrict TLS to damaged DNA. Replication protein A (RPA), the major single-strand DNA (ssDNA) binding protein complex, forms filaments on ssDNA exposed at TLS sites and plays critical yet undefined roles in regulating PCNA monoubiquitination. Here, we utilize kinetic assays and single-molecule FRET microscopy to monitor PCNA monoubiquitination and Rad6/Rad18 complex dynamics on RPA filaments, respectively. Results reveal that a Rad6/ Rad18 complex is recruited to an RPA filament via Rad18•RPA interactions and randomly translocates along the filament. These translocations promote productive interactions between the Rad6/Rad18 complex and the resident PCNA, significantly enhancing monoubiquitination. These results illuminate critical roles of RPA in the specificity and efficiency of PCNA monoubiquitination and represent, to the best of our knowledge, the first example of ATP-independent translocation of a protein complex along a protein filament.

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Section: Discussionmentioning

confidence: 99%

Section: ■ Discussionmentioning

confidence: 99%

PCNA Monoubiquitination Is Regulated by Diffusion of Rad6/Rad18 Complexes along RPA Filaments

Sengupta

Benkovic

et al. 2020

Biochemistry

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show abstract

“…The present study along with previous work from our group and others suggests that selectivity and efficiency of PCNA monoubiquitination is achieved through non-specific Rad18RPA interactions. On native DNA templates, RPA filaments adjacent to progressing P/T junctions are short and transient 33 due to the minimal exposure of native ssDNA templates 7 and their rapid conversion to doublestranded DNA duplexes by the replicative pols ε and  (Fig. 3, left) 34 .…”

Section: Discussionmentioning

confidence: 99%

PCNA monoubiquitination is regulated by diffusion of Rad6/Rad18 complexes along RPA filaments

Sengupta

Benkovic

et al. 2020

Preprint

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Translesion DNA synthesis (TLS) enables DNA replication through damaging modifications to template DNA and requires monoubiquitination of the PCNA sliding clamp by the Rad6/Rad18 complex. This posttranslational modification is critical to cell survival following exposure to DNA damaging agents and is tightly regulated to restrict TLS to damaged DNA. RPA, the major single strand DNA (ssDNA) binding protein, forms filaments on ssDNA exposed at TLS sites and plays critical yet undefined roles in regulating PCNA monoubiquitination. Here, we utilize kinetic assays and single molecule FRET microscopy to monitor PCNA monoubiquitination and Rad6/Rad18 complex dynamics on RPA filaments, respectively. Results reveal that a Rad6/Rad18 complex is recruited to an RPA filament via Rad18•RPA interactions and randomly translocates along the filament. These translocations promote productive interactions between the Rad6/Rad18 complex and the resident PCNA, significantly enhancing monoubiquitination. These results illuminate critical roles of RPA in the specificity and efficiency of PCNA monoubiquitination.

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Multi-scale cellular imaging of DNA double strand break repair

Heemskerk,

van de Kamp,

Essers

et al. 2023

DNA Repair

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Differential binding kinetics of replication protein A during replication and the pre- and post-incision steps of nucleotide excision repair

Cited by 3 publications

References 56 publications

PCNA Monoubiquitination Is Regulated by Diffusion of Rad6/Rad18 Complexes along RPA Filaments

PCNA Monoubiquitination Is Regulated by Diffusion of Rad6/Rad18 Complexes along RPA Filaments

PCNA monoubiquitination is regulated by diffusion of Rad6/Rad18 complexes along RPA filaments

Multi-scale cellular imaging of DNA double strand break repair

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