Biochemical Characterization of Interactions between DNA Polymerase and Single-stranded DNA-binding Protein in Bacteriophage RB69

Sun, Siyang; Shamoo, Yousif

doi:10.1074/jbc.m210497200

Cited by 22 publications

(25 citation statements)

References 41 publications

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“…No other chromatographic peaks at 280 or 260 nm were observed in the elution profile, suggesting that the low estimate is the result of an error in the calculation of dn/dc and the UV calibration constant for the protein⅐nucleic acid complex. A similar underestimate was observed for the mass of DNA polymerase⅐primer-template complexes (40). UP1 alone was somewhat polydispersed at the solution conditions used in these experiments (0.1 M KCl, 10 mM potassium cacodylate, pH 6.5).…”

Section: Up1 Interactions With 6-mi-substituted Telomeric Dnamentioning

confidence: 62%

“…The miniDAWN was calibrated according to the manufacturer's instructions (Wyatt Technology), and BSA was used as a standard to evaluate the accuracy of the system. A dn/dc value of 0.184 was used for BSA and single proteins (40). dn/dc values for complexes were determined by using both UV-visible detector (280 nm) and differential refractometer.…”

Section: Methodsmentioning

confidence: 99%

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Structure-based Incorporation of 6-Methyl-8-(2-deoxy-β-ribofuranosyl)isoxanthopteridine into the Human Telomeric Repeat DNA as a Probe for UP1 Binding and Destabilization of G-tetrad Structures

Myers

Moore

Shamoo

2003

Journal of Biological Chemistry

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Heterogeneous ribonucleoprotein A1 (hnRNP A1) is an abundant nuclear protein that participates in RNA processing, alternative splicing, and chromosome maintenance. hnRNP A1 can be proteolyzed to unwinding protein (UP1), a 22.1-kDa protein that retains a high affinity for purine-rich single-stranded nucleic acids, including the human telomeric repeat (hTR) d(T-TAGGG) n . Using the structure of UP1 bound to hTR as a guide, we have incorporated the fluorescent guanine analog 6-MI at one of two positions within the DNA to facilitate binding studies. One is where 6-MI remains stacked with an adjacent purine, and another is where it becomes fully unstacked upon UP1 binding. The structures of both modified oligonucleotides complexed to UP1 were determined by x-ray crystallography to validate the efficacy of our design, and 6-MI has proven to be an excellent reporter molecule for single-stranded nucleic acid interactions in positions where there is a change in stacking environment upon complex formation. We have shown that UP1 affinity for d(TTAGGG) 2 is ϳ5 nM at 100 mM NaCl, pH 6.0, and our binding studies with d(TTAGG(6-MI)TTAGGG) show that binding is only modestly sensitive to salt and pH. UP1 also has a potent G-tetrad destabilizing activity that reduces the T m of the hTR sequence d(TAGGGT) 4 from 67.0°C to 36.1°C at physiological conditions (150 mM KCl, pH 7.0). Consistent with the structures determined by x-ray crystallography, UP1 is able to bind the hTR sequence in solution as a dimer and supports a model for hnRNP A1 binding to nucleic acids in arrays that may make a contiguous set of anti-parallel single-stranded nucleic acid binding clefts. These data suggest that seemingly disparate roles for hnRNP A1 in alternative splice site selection, RNA processing, RNA transport, and chromosome maintenance reflect its ability to bind a purine-rich consensus sequence (nYAGGn) and destabilize potentially deleterious G-tetrad structures.

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Section: Up1 Interactions With 6-mi-substituted Telomeric Dnamentioning

confidence: 62%

Section: Methodsmentioning

confidence: 99%

Structure-based Incorporation of 6-Methyl-8-(2-deoxy-β-ribofuranosyl)isoxanthopteridine into the Human Telomeric Repeat DNA as a Probe for UP1 Binding and Destabilization of G-tetrad Structures

Myers

Moore

Shamoo

2003

Journal of Biological Chemistry

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“…In the same system, DinI, a RecA regulator, has been shown to interact with activated RecA but not free RecA (80). In bacteriophage RB69, it is suggested that ssDNA binding by phage SSB induces a conformational change in SSB that allows it to associate specifically with adjacent SSB molecules and with its DNA polymerase, gp43 (70). These specific interactions are thought to be important for bacteriophage DNA replication.…”

Section: Discussionmentioning

confidence: 99%

Recruitment of Replication Protein A by the Papillomavirus E1 Protein and Modulation by Single-Stranded DNA

Loo

Melendy

2004

J Virol

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With the exception of viral proteins E1 and E2, papillomaviruses depend heavily on host replication machinery for replication of their viral genome. E1 and E2 are known to recruit many of the necessary cellular replication factors to the viral origin of replication. Previously, we reported a physical interaction between E1 and the major human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA). E1 was determined to bind to the 70-kDa subunit of RPA, RPA70. In this study, using E1-affinity coprecipitation and enzyme-linked immunosorbent assay-based interaction assays, we show that E1 interacts with the major ssDNA-binding domain of RPA. Consistent with our previous report, no measurable interaction between E1 and the two smaller subunits of RPA was detected. The interaction of E1 with RPA was substantially inhibited by ssDNA. The extent of this inhibition was dependent on the length of the DNA. A 31-nucleotide (nt) oligonucleotide strongly inhibited the E1-RPA interaction, while a 16-nt oligonucleotide showed an intermediate level of inhibition. In contrast, a 10-nt oligonucleotide showed no observable effect on the E1-RPA interaction. This inhibition was not dependent on the sequence of the DNA. Furthermore, ssDNA also inhibited the interaction of RPA with papillomavirus E2, simian virus 40 T antigen, human polymerase alpha-primase, and p53. Taken together, our results suggest a potential role for ssDNA in modulating RPA-protein interactions, in particular, the RPA-E1 interactions during papillomavirus DNA replication. A model for recruitment of RPA by E1 during papillomavirus DNA replication is proposed.The Papillomavirinae are small, nonenveloped viruses with double-stranded, circular DNA genomes. With the exception of two virally encoded proteins, E1 and E2, papillomavirus (PV) DNA replication is carried out entirely by the host cellular replication machinery (reviewed in references 14, 42, and 68). A partially reconstituted cell-free system of replication has allowed the identification of many of these cellular factors (34,49,55,57). Among them are cellular factors that were previously found to be necessary and sufficient for simian virus 40 (SV40) DNA replication, including DNA polymerase alphaprimase (pol-prim) and replication protein A (RPA) (reviewed in references 13, 51, and 67). However, unlike SV40, additional cellular factors, some of which have yet to be identified, are required for efficient PV DNA replication (44,46,49). Nevertheless, many parallels have been drawn between the two viral systems, particularly regarding the initiation and elongation of DNA replication.The PV E1 protein is an ATP-dependent viral DNA helicase (77); E2 is a multifunctional regulator of viral transcription and replication (18,26,35,48). PV DNA replication is initiated by the assembly of E1 as a double hexamer at the origin of replication, resulting in a localized melting of the DNA template (21, 63). The sequence-specific binding of E1 to the origin is directed by its interactions with E2 (45,54,61)...

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“…Functional and physical interactions have been described for all components of the replisome (2,9,19,20). The polymerase physically interacts with gp32, which has important consequences for the properties of gp43, such as higher affinity for the primer-template junction and increased processivity during DNA synthesis (21). The polymerase is in turn attached to the clamp protein through a well described interaction between the C terminus of the polymerase and the subunit interfaces of the trimeric clamp protein (5,6,22).…”

mentioning

confidence: 99%

Site-directed Mutations of T4 Helicase Loading Protein (gp59) Reveal Multiple Modes of DNA Polymerase Inhibition and the Mechanism of Unlocking by gp41 Helicase

Nelson

Yang

Benkovic

2006

Journal of Biological Chemistry

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The T4 helicase loading protein (gp59) interacts with a multitude of DNA replication proteins. In an effort to determine the functional consequences of these protein-protein interactions, point mutations were introduced into the gp59 protein. Mutations were chosen based on the available crystal structure and focused on hydrophobic residues with a high degree of solvent accessibility. Characterization of the mutant proteins revealed a single mutation, Y122A, which is defective in polymerase binding and has weakened affinity for the helicase. The interaction between single-stranded DNA-binding protein and Y122A is unaffected, as is the affinity of Y122A for DNA substrates. When standard concentrations of helicase are employed, Y122A is unable to productively load the helicase onto forked DNA substrates. As a result of the loss of polymerase binding, Y122A cannot inhibit the polymerase during nucleotide idling or prevent it from removing the primer strand of a D-loop. However, Y122A is capable of inhibiting strand displacement synthesis by polymerase. The retention of strand displacement inhibition by Y122A, even in the absence of a gp59-polymerase interaction, indicates that there are two modes of polymerase inhibition by gp59. Inhibition of the polymerase activity only requires gp59 to bind to the replication fork, whereas inhibition of the exonuclease activity requires an interaction between the polymerase and gp59. The inability of Y122A to interact with both the polymerase and the helicase suggests a mechanism for polymerase unlocking by the helicase based on a direct competition between the helicase and polymerase for an overlapping binding site on gp59.The T4 replisome is considered to be a model for the more complex replication systems (1, 2). The eight protein replisome can be divided into three modules, the leading strand holoenzyme, the lagging strand holoenzyme, and the primosome (3). The leading and lagging strand holoenzymes are formed by the polymerase (gp43) 4 and the polymerase clamp (gp45) with the aid of the clamp loader protein (gp44/62). Contained within gp43 polymerase are two distinct active sites. One is the polymerization site that catalyzes the 5Ј to 3Ј incorporation of deoxynucleotides (dNTPs) into the growing primer strand, and the other is the exonuclease site that is responsible for the removal of dNMPs in the 3Ј to 5Ј direction (4). gp45 is bound to the polymerase and encircles the DNA duplex, thereby increasing its processivity during DNA synthesis (5, 6). gp44/62 aids in the assembly of the polymeraseclamp complex by positioning the clamp at the 3Ј-OH of the primertemplate junction and acting as a chaperone for the interaction between gp45 and gp43 (7,8). The primosome is made up of the helicase (gp41), primase (gp61), single-stranded DNA-binding protein (gp32), and the helicase loading protein (gp59) (9). gp41 is a hexameric protein that travels along the ssDNA in a 5Ј to 3Ј manner using the energy of ATP hydrolysis to unwind duplex DNA (10, 11). gp61 is also hexameric and is respo...

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Biochemical Characterization of Interactions between DNA Polymerase and Single-stranded DNA-binding Protein in Bacteriophage RB69

Cited by 22 publications

References 41 publications

Structure-based Incorporation of 6-Methyl-8-(2-deoxy-β-ribofuranosyl)isoxanthopteridine into the Human Telomeric Repeat DNA as a Probe for UP1 Binding and Destabilization of G-tetrad Structures

Structure-based Incorporation of 6-Methyl-8-(2-deoxy-β-ribofuranosyl)isoxanthopteridine into the Human Telomeric Repeat DNA as a Probe for UP1 Binding and Destabilization of G-tetrad Structures

Recruitment of Replication Protein A by the Papillomavirus E1 Protein and Modulation by Single-Stranded DNA

Site-directed Mutations of T4 Helicase Loading Protein (gp59) Reveal Multiple Modes of DNA Polymerase Inhibition and the Mechanism of Unlocking by gp41 Helicase

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