Surface plasmon resonance measurements reveal stable complex formation between p53 and DNA polymerase α

Kühn, Claudia; Müller, Friedemann; Melle, Christian; Nasheuer, Heinz-Peter; Janus, Friedemann; Deppert, Wolfgang; Grosse, Frank

doi:10.1038/sj.onc.1202327

Cited by 29 publications

(12 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…When the prim 2 was directly immobilized with amino or thiol couplings, binding of primase and Tag was no longer detectable (data not shown). Similar findings were reported earlier when the physical interactions of RPA with p53 were analyzed (74).…”

Section: Figsupporting

confidence: 90%

“…The surface plasmon resonance studies supported our findings that primase directly binds to Tag and that this association does not require either the p180 or the p68 subunits of pol-prim (Table II). The calculated dissociation constants for the interactions of Tag with pol-prim and prim 2 , respectively, were very similar and were 2-fold higher than the calculated constant for the interactions of Tag and the tumor suppressor protein p53 (74). Additionally, the protein overlay experiments allowed us to determine that both p58 and p48 independently contributed to the binding of Tag.…”

Section: Figmentioning

confidence: 66%

See 1 more Smart Citation

Protein-Protein Interactions of the Primase Subunits p58 and p48 with Simian Virus 40 T Antigen Are Required for Efficient Primer Synthesis in a Cell-free System

Weißhart¹,

Förster²,

Kremmer³

et al. 2000

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

DNA polymerase ␣-primase (pol-prim, consisting of p180-p68-p58-p48), and primase p58-p48 (prim 2 ) synthesize short RNA primers on single-stranded DNA. In the SV40 DNA replication system, only pol-prim is able to start leading strand DNA replication that needs unwinding of double-stranded (ds) DNA prior to primer synthesis. At high concentrations, pol-prim and prim 2 indistinguishably reduce the unwinding of dsDNA by SV40 T antigen (Tag). RNA primer synthesis on ssDNA in the presence of replication protein A (RPA) and Tag has served as a model system to study the initiation of Okazaki fragments on the lagging strand in vitro. On ssDNA, Tag stimulates whereas RPA inhibits the initiation reaction of both enzymes. Tag reverses and even overcompensates the inhibition of primase by RPA. Physical binding of Tag to the primase subunits and RPA, respectively, is required for these activities. Each subunit of the primase complex, p58 and p48, performs physical contacts with Tag and RPA independently of p180 and p68. Using surface plasmon resonance, the dissociation constants of the Tag/pol-prim and Tag/primase interactions were 1.2⅐10 ؊8 M and 1.3⅐10 ؊8 M, respectively.In eukaryotic cells the duplication of the genome is a highly accurate and tightly coordinated process (reviewed in Ref. 1). For each reaction a specific set of enzymes and accessory proteins is required to replicate chromosomal DNA (1-4). The first step in leading strand DNA replication is accomplished by the synthesis of oligoribonucleotides, called RNA primers, at the origin of DNA replication. This process is carried out by a special enzyme, DNA primase (5-7). The eukaryotic enzyme consists of two subunits, the catalytic subunit p48 and p58, which serves to stabilize the primase activity (8 -13). In eukaryotic cells these two subunits assemble together with the catalytic DNA polymerase subunit, p180, and the p68 polypeptide into a heterotetrameric DNA polymerase ␣-primase complex (pol-prim) 1 (1-7, 14, 15).The initiation of DNA replication requires the interaction of several proteins in vivo and in vitro (1). The start of DNA synthesis de novo occurs by two independent processes: the singular priming event on the leading strand at the origin and the multiple priming steps for Okazaki fragment synthesis on the lagging strand. Both tasks are carried out by the primase activity (1-3, 14 -17). Two cell-free initiation reactions have served as model systems to investigate these processes, primer synthesis in the cell-free SV40 DNA replication system and primer synthesis on natural single-stranded (ss) DNA templates bound by replication protein A (RPA) (17, 18). Using these cell-free systems, it was shown that the initiation of SV40 DNA replication at the origin is species-specific and requires the activity of the p180 subunit of primate pol-prim (19 -21). In contrast to these results, the initiation of Okazaki fragments during lagging strand DNA synthesis is not species-specific and the human as well as the bovine pol-prim can perform lagging strand initiat...

show abstract

Section: Figsupporting

confidence: 90%

Section: Figmentioning

confidence: 66%

Protein-Protein Interactions of the Primase Subunits p58 and p48 with Simian Virus 40 T Antigen Are Required for Efficient Primer Synthesis in a Cell-free System

Weißhart¹,

Förster²,

Kremmer³

et al. 2000

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Janus et al (1999) proposed that wild type RS-p53 is involved in DNA damage repair, or in the control of homologous recombination through its exonuclease activity. This is supported by observations that RSp53 enhanced the fidelity of DNA replication by DNA polymerase a (Huang, 1998), and the formation of a stable complex between the two proteins (Kuhn et al, 1999). It was also observed that the human recombinase hRad51 stimulates exonucleolytic DNA degradation by p53, mediated by the ternary complex p53/hRad51/ junction DNA, thus suggesting a model for p53-dependent correction of DNA exchange events (Susse et al, 2000).…”

Section: Discussionmentioning

confidence: 80%

DNA binding and 3′–5′ exonuclease activity in the murine alternatively-spliced p53 protein

et al. 2002

View full text Add to dashboard Cite

In this study we show that the naturally occurring Cterminally alternative spliced p53 (referred to as AS-p53) is active as a sequence-specific DNA binding protein as well as a 3' -5'-exonuclease in the presence of Mg 2+ ions. The two activities are positively correlated as the sequence-specific DNA target is more efficiently degraded than a non-specific target. In contrast, a mutated AS-p53 protein that is deficient in DNA binding lacks exonuclease activity. The use of modified p53 binding sites, where the 3'-phosphate is replaced by a phosphorothioate group, enabled the inhibition of DNA degradation under the binding conditions. We demonstrate that AS-p53 interacts with its specific DNA target by two distinct binding modes: a high-affinity mode characterized by a low-mobility protein-DNA complex at the nanomolar range, and a low-affinity mode shown by a high-mobility complex at the micromolar range. Comparison of the data on the natural and the modified p53 binding sites suggests that the high-affinity mode is related to AS-p53 function as a transcription factor and that the low-affinity mode is associated with its exonuclease activity. The implications of these findings to a specific cellular role of AS-p53 are discussed.

show abstract

“…Biomolecular Interaction Analysis-Interaction analysis was performed using the BIAcore 2000 apparatus from BIAcore AB (Freiburg, Germany) as described previously (34,49). Sensor chips CM5, surfactant P20, and the amine coupling kit were purchased from BIAcore AB.…”

Section: Construction Of Baculoviruses Expressing Chimeric P180 Protementioning

confidence: 99%

Species Specificity of Simian Virus 40 DNA Replication in Vitro Requires Multiple Functions of Human DNA Polymerase α

Smith¹,

Steffen²,

Grosse³

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Human cell extracts support the replication of SV40 DNA, whereas mouse cell extracts do not. Species specificity is determined at the level of initiation of DNA replication, and it was previously found that this requires the large subunit, p180, of DNA polymerase ␣-primase to be of human origin. Furthermore, a functional interaction between SV40 large T antigen (TAg) and p180 is essential for viral DNA replication. In this study we determined that the N-terminal regions of human p180, which contain the TAg-binding sites, can be replaced with those of murine origin without losing the ability to support SV40 DNA replication in vitro. The same substitutions do not prevent SV40 TAg from stimulating the activity of DNA polymerase ␣-primase on single-stranded DNA in the presence of replication protein A. Furthermore, biophysical studies show that the interactions of human and murine DNA polymerase ␣-primase with SV40 TAg are of a similar magnitude. These studies strongly suggest that requirement of SV40 DNA replication for human DNA polymerase ␣ depends neither on the TAg-binding site being of human origin nor on the strength of the binary interaction between SV40 TAg and DNA polymerase ␣-primase but rather on sequences in the C-terminal region of human p180.Polyomavirus DNA replication has been studied extensively because of the ease with which viruses of this family, which include SV40 and mouse polyoma virus (PyV), 1 can be grown in cell culture and moreover because of the availability of an in vitro replication system, which allows detailed investigation of the individual factors that play a role in the replication process (1, 2). Polyomavirus DNA replication has been found to be largely dependent upon factors involved in replication of the host DNA but does contribute one essential trans-acting factor known as large T antigen (TAg) to the replication complex. TAg is responsible for recognition of the viral origin of replication, at which it forms a double hexamer (3). In the presence of the single-stranded DNA-binding protein, RPA (replication protein A), and topoisomerase I, TAg proceeds to unwind the origin DNA and recruits the DNA polymerase ␣-primase heterotetramer, which then initiates bidirectional DNA synthesis (4 -11). DNA polymerase ␣-primase initiates DNA replication through the action of its smallest subunit, p48, which synthesizes RNA primers that are subsequently elongated by the large subunit, p180 (12-15). The bulk of DNA synthesis is, however, carried out by a more processive enzyme complex consisting of DNA polymerase ␦ and its processivity factor, PCNA (16 -18). The transition between DNA synthesis by DNA polymerases ␣ and ␦ is mediated by the PCNA loading factor replication factor C (16, 19). Throughout the viral replication cycle, TAg double hexamers are thought to act as the replicative helicase (20). For a more extensive account of the DNA replication process and its participating factors see Refs. 1, 2, and 21-23. SV40 and PyV are very similar with regard to DNA replication; their respectiv...

show abstract

Surface plasmon resonance measurements reveal stable complex formation between p53 and DNA polymerase α

Cited by 29 publications

References 35 publications

Protein-Protein Interactions of the Primase Subunits p58 and p48 with Simian Virus 40 T Antigen Are Required for Efficient Primer Synthesis in a Cell-free System

Protein-Protein Interactions of the Primase Subunits p58 and p48 with Simian Virus 40 T Antigen Are Required for Efficient Primer Synthesis in a Cell-free System

DNA binding and 3′–5′ exonuclease activity in the murine alternatively-spliced p53 protein

Species Specificity of Simian Virus 40 DNA Replication in Vitro Requires Multiple Functions of Human DNA Polymerase α

Contact Info

Product

Resources

About