Mapping of Amino Acid Residues in the p34 Subunit of Human Single-stranded DNA-binding Protein Phosphorylated by DNA-dependent Protein Kinase and Cdc2 Kinasein Vitro

Human replication protein A (hRPA) is an essential single-stranded-DNA-binding protein that stimulates the activities of multiple DNA replication and repair proteins through physical interaction. To understand DNA binding and its role in hRPA heterologous interaction, we examined the physical structure of hRPA complexes with single-stranded DNA (ssDNA) by scanning transmission electron microscopy. Recent biochemical studies have shown that hRPA combines with ssDNA in at least two binding modes: by interacting with 8 to 10 nucleotides (hRPA8nt) and with 30 nucleotides (hRPA30nt). We find the relatively unstable hRPA8nt complex to be notably compact with many contacts between hRPA molecules. In contrast, on similar lengths of ssDNA, hRPA30nt complexes align along the DNA and make few intermolecular contacts. Surprisingly, the elongated hRPA30nt complex exists in either a contracted or an extended form that depends on ssDNA length. Therefore, homologous-protein interaction and available ssDNA length both contribute to the physical changes that occur in hRPA when it binds ssDNA. We used activated DNA-dependent protein kinase as a biochemical probe to detect alterations in conformation and demonstrated that formation of the extended hRPA30nt complex correlates with increased phosphorylation of the hRPA 29-kDa subunit. Our results indicate that hRPA binds ssDNA in a multistep pathway, inducing new hRPA alignments and conformations that can modulate the functional interaction of other factors with hRPA.

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Genetic and functional analyses of the øX174 DNA binding protein: the effects of substitutions for amino acid residues that spatially organize the two DNA binding domains

Virology ◽

10.1016/j.virol.2003.09.018 ◽

2004 ◽

Vol 318 (1) ◽

pp. 204-213 ◽

Cited By ~ 14

Author(s):

Susan L. Hafenstein ◽

Min Chen ◽

Bentley A. Fane

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Binding Protein ◽

Dna Binding Protein ◽

Amino Acid Residues ◽

Dna Binding Domains ◽

Binding Domains ◽

Functional Analyses

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Identification of amino acid residues involved in substrate recognition by the catalytic subunit of bovine cyclic AMP dependent protein kinase: peptide-based affinity labels

Biochemistry ◽

10.1021/bi00410a025 ◽

1988 ◽

Vol 27 (10) ◽

pp. 3691-3696 ◽

Cited By ~ 15

Author(s):

Shahriar Mobashery ◽

E. T. Kaiser

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Cyclic Amp ◽

Substrate Recognition ◽

Catalytic Subunit ◽

Amino Acid Residues ◽

Dependent Protein Kinase ◽

Affinity Labels ◽

Dependent Protein

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Cloning and characterization of a novel sequence-specific single-stranded-DNA-binding protein

Biochemical Journal ◽

10.1042/bj3310447 ◽

1998 ◽

Vol 331 (2) ◽

pp. 447-452 ◽

Cited By ~ 25

Author(s):

Dashzeveg BAYARSAIHAN ◽

Ricardo J. SOTO ◽

Lewis N. LUKENS

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Binding Protein ◽

Mammalian Species ◽

Dna Binding Protein ◽

Cdna Expression Library ◽

Collagen Gene ◽

Expression Library ◽

Significant Similarity ◽

Single Stranded Dna

The promoter region of the chicken α2(I) collagen gene contains a pyrimidine-rich element that is well conserved in different mammalian species. This sequence can also form an unusual DNA structure as shown by its sensitivity to SI nuclease in vitro and it lies in a region that is DNase I-hypersensitive only when this promoter is active. We have recently reported that fibroblast nuclear proteins, including chicken Y-box-binding protein 1, bind to this single-stranded pyrimidine-rich sequence. Here we report the isolation, from a chick embryo fibroblast cDNA expression library, of a partial cDNA clone encoding a previously unknown protein, designated SSDP (sequence-specific single-stranded DNA-binding protein), that binds this single-stranded sequence. This clone contains 1199 bp of chicken sequence and has a single long open reading frame that encodes 284 amino acid residues. The affinity-purified recombinant protein encoded by this cDNA binds sequence-specifically to the single-stranded pyrimidine sequence. This cDNA sequence lacks significant similarity to any known gene in the data banks, but it is highly conserved in expressed sequence tags derived from both mouse and human. The corresponding amino acid sequence is remarkably conserved, having 97% identity with mouse and human expressed sequences. The corresponding mRNA is approx. 1800 nt in length and is expressed in both fibroblasts and chondrocytes. The high affinity of this protein for this conserved pyrimidine-rich region suggests that it might be involved in the transcriptional regulation of the α2(I) collagen gene.

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Critical amino acid residues of AIP, a highly specific inhibitory peptide of calmodulin-dependent protein kinase II

FEBS Letters ◽

10.1016/s0014-5793(98)00405-0 ◽

1998 ◽

Vol 427 (1) ◽

pp. 115-118 ◽

Cited By ~ 42

Author(s):

Atsuhiko Ishida ◽

Yasushi Shigeri ◽

Yoshiro Tatsu ◽

Koichi Uegaki ◽

Isamu Kameshita ◽

...

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Amino Acid Residues ◽

Inhibitory Peptide ◽

Dependent Protein Kinase ◽

Critical Amino Acid ◽

Protein Kinase Ii ◽

Dependent Protein

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Phosphorylation of native and truncated isoforms of protein τ by the double-stranded DNA-dependent protein kinase (DNA-PK) shows that the primary phosphorylation sites are localized between amino acid residues 212-231 of the longest τ

IUBMB Life ◽

10.1080/15216549600201492 ◽

1996 ◽

Vol 40 (1) ◽

pp. 21-31

Author(s):

Joseph Wu ◽

Yuping Chen ◽

Tze-chen Hsieh ◽

Roland Brandt ◽

Gloria Lee

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Phosphorylation Sites ◽

Amino Acid Residues ◽

Dependent Protein Kinase ◽

Double Stranded Dna ◽

Dependent Protein ◽

Truncated Isoforms

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The putative single-stranded DNA-binding protein of the filamentous bacteriophage, If1. Amino acid sequence of the protein and structure of the gene

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.1991.0083 ◽

1991 ◽

Vol 245 (1312) ◽

pp. 23-30 ◽

Cited By ~ 5

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Amino Acid Sequence ◽

Binding Protein ◽

Dna Binding Protein ◽

Single Stranded Dna ◽

Filamentous Bacteriophage

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Identification and Characterization of the Single-Stranded DNA-Binding Protein of Bacteriophage P1

Journal of Bacteriology ◽

10.1128/jb.181.20.6463-6468.1999 ◽

1999 ◽

Vol 181 (20) ◽

pp. 6463-6468 ◽

Cited By ~ 11

Author(s):

Hansjörg Lehnherr ◽

Jannick D. Bendtsen ◽

Fabian Preuss ◽

Tatiana V. Ilyina

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Binding Protein ◽

Dna Binding Protein ◽

Temperature Sensitive ◽

Single Stranded Dna ◽

Fusion Construct ◽

Bacteriophage P1 ◽

E Coli ◽

Close Relationship

ABSTRACT The genome of bacteriophage P1 harbors a gene coding for a 162-amino-acid protein which shows 66% amino acid sequence identity to the Escherichia coli single-stranded DNA-binding protein (SSB). The expression of the P1 gene is tightly regulated by P1 immunity proteins. It is completely repressed during lysogenic growth and only weakly expressed during lytic growth, as assayed by anssb-P1/lacZ fusion construct. When cloned on an intermediate-copy-number plasmid, the P1 gene is able to suppress the temperature-sensitive defect of an E. coli ssb mutant, indicating that the two proteins are functionally interchangeable. Many bacteriophages and conjugative plasmids do not rely on the SSB protein provided by their host organism but code for their own SSB proteins. However, the close relationship between SSB-P1 and the SSB protein of the P1 host, E. coli, raises questions about the functional significance of the phage protein.

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