C-termini are essential and distinct for nucleic acid binding of human NABP1 and NABP2

Vidhyasagar, Venkatasubramanian; He, Yong; Guo, Mingke; Ding, Hao; Talwar, Tanu; Nguyen, Vi; Nwosu, Jessica; Katselis, George; Wu, Yuliang

doi:10.1016/j.bbagen.2015.11.003

Cited by 11 publications

(38 citation statements)

References 60 publications

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“…The nucleic acid-binding ability of NABP1/2 has been well documented [ 6 , 18 ]; however, the characteristics of INTS3 FL have never been reported. Thus, to understand the nucleic acid-binding ability of INTS3 FL , we incubated the protein with ssDNA (dT 30 ) and ssRNA (rU 30 ).…”

Section: Resultsmentioning

confidence: 99%

“…As hNABP1/2 binds effectively with increasing length of ssDNA [ 18 ], next we assessed the binding ability of INTS3 FL with various lengths of ssDNA such as dT 10 , dT 20 , dT 60 , and dT 90 . We found that INTS3 FL requires a minimum of 30 nucleotides and its affinity increases with the length of ssDNA (Supplementary Figure S3B).…”

Section: Resultsmentioning

confidence: 99%

“…The primers used for the amplification of genes are listed in Supplementary Table S1. The vector construction of hNABP1/2 were described recently [ 18 ].…”

Section: Methodsmentioning

confidence: 99%

“…The His-tagged INTS3 C protein was purified similarly, except the gel filtration buffer B has 50 mM of l -arginine and 50 mM of glutamic acid (Sigma). The hNABP1 and hNABP2 proteins were purified as described recently [ 18 ].…”

Section: Methodsmentioning

confidence: 99%

“…Using the previously purified full-length hNABP1 and hNABP2 proteins [ 18 ], rabbit polyclonal antisera were made by the GenScript. The goat polyclonal antibody against INTS3 (sc-138355) and rabbit polyclonal antibody against C9ORF80 (sc-137357) were purchased from Santa Cruz.…”

Section: Methodsmentioning

confidence: 99%

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Biochemical characterization of INTS3 and C9ORF80, two subunits of hNABP1/2 heterotrimeric complex in nucleic acid binding

Vidhyasagar

Guo

et al. 2018

Biochemical Journal

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Human nucleic acid-binding protein 1 and 2 (hNABP1 and hNABP2, also known as hSSB2 and hSSB1 respectively) form two separate and independent complexes with two identical proteins, integrator complex subunit 3 (INTS3) and C9ORF80. We and other groups have demonstrated that hNABP1 and 2 are single-stranded (ss) DNA- and RNA-binding proteins, and function in DNA repair; however, the function of INTS3 and C9OFR80 remains elusive. In the present study, we purified recombinant proteins INTS3 and C9ORF80 to near homogeneity. Both proteins exist as a monomer in solution; however, C9ORF80 exhibits anomalous behavior on SDS–PAGE and gel filtration because of 48% random coil present in the protein. Using electrophoretic mobility shift assay (EMSA), INTS3 displays higher affinity toward ssRNA than ssDNA, and C9ORF80 binds ssDNA but not ssRNA. Neither of them binds dsDNA, dsRNA, or RNA : DNA hybrid. INTS3 requires minimum of 30 nucleotides, whereas C9OFR80 requires 20 nucleotides for its binding, which increased with the increasing length of ssDNA. Interestingly, our GST pulldown results suggest that the N-terminus of INTS3 is involved in protein–protein interaction, while EMSA implies that the C-terminus is required for nucleic acid binding. Furthermore, we purified the INTS3–hNABP1/2–C9ORF80 heterotrimeric complex. It exhibits weaker binding compared with the individual hNABP1/2; interestingly, the hNABP1 complex prefers ssDNA, whereas hNABP2 complex prefers ssRNA. Using reconstituted heterotrimeric complex from individual proteins, EMSA demonstrates that INTS3, but not C9ORF80, affects the nucleic acid-binding ability of hNABP1 and hNABP2, indicating that INTS3 might regulate hNABP1/2's biological function, while the role of C9ORF80 remains unknown.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…The primers used for the amplification of genes are listed in Supplementary Table S1. The vector construction of hNABP1/2 were described recently [ 18 ].…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Biochemical characterization of INTS3 and C9ORF80, two subunits of hNABP1/2 heterotrimeric complex in nucleic acid binding

Vidhyasagar

Guo

et al. 2018

Biochemical Journal

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DNA‐dependent phase separation by human SSB2 (NABP1/OBFC2A) protein points to adaptations to eukaryotic genome repair processes

Kovács,

Harami,

Pálinkás

et al. 2024

Protein Science

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Single‐stranded DNA binding proteins (SSBs) are ubiquitous across all domains of life and play essential roles via stabilizing and protecting single‐stranded (ss) DNA as well as organizing multiprotein complexes during DNA replication, recombination, and repair. Two mammalian SSB paralogs (hSSB1 and hSSB2 in humans) were recently identified and shown to be involved in various genome maintenance processes. Following our recent discovery of the liquid–liquid phase separation (LLPS) propensity of Escherichia coli (Ec) SSB, here we show that hSSB2 also forms LLPS condensates under physiologically relevant ionic conditions. Similar to that seen for EcSSB, we demonstrate the essential contribution of hSSB2's C‐terminal intrinsically disordered region (IDR) to condensate formation, and the selective enrichment of various genome metabolic proteins in hSSB2 condensates. However, in contrast to EcSSB‐driven LLPS that is inhibited by ssDNA binding, hSSB2 phase separation requires single‐stranded nucleic acid binding, and is especially facilitated by ssDNA. Our results reveal an evolutionarily conserved role for SSB‐mediated LLPS in the spatiotemporal organization of genome maintenance complexes. At the same time, differential LLPS features of EcSSB and hSSB2 point to functional adaptations to prokaryotic versus eukaryotic genome metabolic contexts.

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The Essential, Ubiquitous Single-Stranded DNA-Binding Proteins

Oliveira

Ciesielski

2021

Methods in Molecular Biology

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C-termini are essential and distinct for nucleic acid binding of human NABP1 and NABP2

Cited by 11 publications

References 60 publications

Biochemical characterization of INTS3 and C9ORF80, two subunits of hNABP1/2 heterotrimeric complex in nucleic acid binding

Biochemical characterization of INTS3 and C9ORF80, two subunits of hNABP1/2 heterotrimeric complex in nucleic acid binding

DNA‐dependent phase separation by human SSB2 (NABP1/OBFC2A) protein points to adaptations to eukaryotic genome repair processes

The Essential, Ubiquitous Single-Stranded DNA-Binding Proteins

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