Is a fully wrapped SSB–DNA complex essential for<i>Escherichia coli</i>survival?

Waldman, Vincent M.; Weiland, Elizabeth; Козлов, А. Г.; Lohman, Timothy M.

doi:10.1093/nar/gkw262

Cited by 12 publications

(22 citation statements)

References 77 publications

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“…Both proteins display ionic strength-dependent transitions between ssDNA-binding modes that involve switches in the size of ssDNA occluded 20 . Furthermore, binding of either protein to ssDNA is non-cooperative at physiological ionic strength (200 mM) 18, 19, 21–23 . However, several key features distinguish these functional homologs.…”

Section: Resultsmentioning

confidence: 99%

Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA

2017

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In humans, PCNA sliding clamps encircling DNA coordinate various aspects of DNA metabolism throughout the cell cycle. A critical aspect of this is restricting PCNA to the vicinity of its DNA target site. For example, PCNA must be maintained at/near primer/template (P/T) junctions during DNA synthesis. With a diverse array of cellular factors implicated, many of which interact with PCNA, DNA, or both, it is unknown how this critical feat is achieved. Furthermore, current biochemical assays that examine the retention of PCNA near P/T junctions are inefficient, discontinuous, qualitative, and significantly deviate from physiologically-relevant conditions. To overcome these challenges and limitations, we recently developed a novel and convenient FRET assay that directly and continuously monitors the retention of human PCNA at a P/T junction. Here we describe in detail the design, methodology, interpretation, and limitations of this quantitative FRET assay using the single strand DNA (ssDNA) binding protein, SSB, from Escherichia coli as an example. This powerful tool is broadly applicable to any ssDNA-binding protein and may be utilized and/or expanded upon to dissect DNA metabolic pathways that are dependent upon PCNA.

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Section: Resultsmentioning

confidence: 99%

Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA

2017

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“…A single molecule investigation conducted under near-physiological buffer conditions, and where the generation of ssDNA is coupled to DNA replication on a fork substrate, has shown that the human mitochondrial SSB (HmSSB) binds primarily using a low site size binding mode, likely the (SSB)35 mode [99] . Using linked EcSSB subunits, Waldman et al investigated the effect of preventing the formation of a fully wrapped ssDNA-SSB tetramer on cell viability [72] . By covalently linking two SSB subunits, they generated EcSSB "dimers" in which each subunit contains two OB-domains.…”

Section: Cellular Roles Of Dna Binding Modesmentioning

confidence: 99%

Dynamics of E. coli single stranded DNA binding (SSB) protein-DNA complexes

Antony

Lohman

2019

Seminars in Cell & Developmental Biology

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109

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Single stranded DNA binding proteins (SSB) are essential to the cell as they stabilize transiently open single stranded DNA (ssDNA) intermediates, recruit appropriate DNA metabolism proteins, and coordinate fundamental processes such as replication, repair and recombination. Escherichia coli single stranded DNA binding protein (EcSSB) has long served as the prototype for the study of SSB function. The structure, functions, and DNA binding properties of EcSSB are well established: The protein is a stable homotetramer with each subunit possessing an N-terminal DNA binding core, a C-terminal protein-protein interaction tail, and an intervening intrinsically disordered linker (IDL). EcSSB wraps ssDNA in multiple DNA binding modes and can diffuse along DNA to remove secondary structures and remodel other protein-DNA complexes. This review provides an update on these features based on recent findings, with special emphasis on the functional and mechanistic relevance of the IDL and DNA binding modes.

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“…SSB is a functional homolog of human RPA and binds ssDNA noncooperatively and with very tight affinity (in pM range) at 200 mM ionic strength (28,29,(37)(38)(39). We previously demonstrated that, like RPA, SSB binds tightly to ssDNA adjacent to a P/T junction and restricts PCNA to the upstream duplex region by physically blocking diffusion of the sliding clamp along the adjacent ssDNA (36).…”

Section: Rpa Regulates Monoubiquitination Of Pcna On and Off Dnamentioning

confidence: 99%

Replication protein A dynamically regulates monoubiquitination of proliferating cell nuclear antigen

Hedglin

Aitha

Pedley

et al. 2019

Journal of Biological Chemistry

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Edited by Patrick SungDNA damage tolerance permits bypass of DNA lesions encountered during S-phase and may be carried out by translesion DNA synthesis (TLS). Human TLS requires selective monoubiquitination of proliferating cell nuclear antigen (PCNA) sliding clamps encircling damaged DNA. This posttranslational modification (PTM) is catalyzed by Rad6/Rad18. Recent studies revealed that replication protein A (RPA), the major ssDNA-binding protein, is involved in the regulation of PCNA monoubiquitination and interacts directly with Rad18 on chromatin and in the nucleoplasm. However, it is unclear how RPA regulates this critical PTM and what functional role(s) these interactions serve. Here, we developed an in vitro assay to quantitatively monitor PCNA monoubiquitination under in vivo scenarios. Results from extensive experiments revealed that RPA regulates Rad6/Rad18 activity in an ssDNA-dependent manner. We found that "DNA-free" RPA inhibits monoubiquitination of free PCNA by directly interacting with Rad18. This interaction is promoted under native conditions when there is an overabundance of free RPA in the nucleoplasm where Rad6/ Rad18 and a significant fraction of PCNA reside. During DNA replication stress, RPA binds the ssDNA exposed downstream of stalled primer/template (P/T) junctions, releasing Rad6/ Rad18. RPA restricted the resident PCNAs to the upstream duplex regions by physically blocking diffusion of PCNA along ssDNA, and this activity was required for efficient monoubiquitination of PCNA on DNA. Furthermore, upon binding ssDNA, RPA underwent a conformational change that increased its affinity for Rad18. Rad6/Rad18 complexed with ssDNA-bound RPA was active, and this interaction may selectively promote monoubiquitination of PCNA on long RPAcoated ssDNA.

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Is a fully wrapped SSB–DNA complex essential forEscherichia colisurvival?

Cited by 12 publications

References 77 publications

Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA

Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA

Dynamics of E. coli single stranded DNA binding (SSB) protein-DNA complexes

Replication protein A dynamically regulates monoubiquitination of proliferating cell nuclear antigen

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