Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases

Datta, Kausiki; Johnson, Neil P.; LiCata, Vince J.; Hippel, Peter H. von

doi:10.1074/jbc.m109.007641

Cited by 26 publications

(36 citation statements)

References 48 publications

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“…Interestingly, Ϸ1% of the polymerase domain binding events were observed to directly transfer the primer from the polymerase domain to the exonuclease domain without prior dissociation from the DNA. Our results yield an intermediary level of exonuclease partitioning relative to previously published data (22)(23)(24) and are consistent with the idea that partitioning to the exonuclease site is increased in the presence of a terminally mismatched primer/ template.…”

Section: Resultssupporting

confidence: 80%

“…Analysis of the binding of KF to a fully paired primer/template showed FRET ratios suggesting Ϸ65% of primers were bound to the polymerase active site and Ϸ35% to the exonuclease site. These results are in agreement with prior studies that used fluorescence to measure the binding position of the primer in KF binary complexes (22)(23)(24). Measurements made during the incorporation of three dNTPs detected the movement of KF on the template in three discrete steps that correlated with the FRET ratios for the binding of the KF to primer/templates in which the primers had these three lengths.…”

Section: Figsupporting

confidence: 80%

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Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution

Christian

Romano

Rueda

2009

Proc. Natl. Acad. Sci. U.S.A.

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The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3 -hydroxyl of the growing DNA chain. Here we report a singlemolecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule Fö rster resonance energy transfer intensity drops in discrete steps to values consistent with single-nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer/template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change after the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the preinsertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process.Klenow Fragment ͉ polymerase and exonuclease site ͉ single molecule fluorescence ͉ single nucleotide resolution ͉ structural dynamics T he catalytic mechanism of Escherichia coli DNA polymerase I has been rigorously studied for more than 40 years (1, 2). The E. coli DNA polymerase I (Klenow fragment [KF]), an active truncated form of polymerase I, is composed of two domains: a polymerase domain that incorporates nucleotides, and a 3Ј-5Ј exonuclease domain that excises misincorporated nucleotides. The polymerase domain consists of three subdomains: the fingers, the palm, and the thumb. The fingers subdomain is primarily involved in interactions with the singlestranded region of the DNA template and the incoming nucleotide; the palm forms the active site of the polymerase upon interaction with the incoming dNTP; and the thumb is responsible for binding double-stranded DNA. The exonuclease domain, located Ϸ30 Å from the polymerase domain, binds to the 3Ј-terminus of the primer when a mismatched base is incorporated (3).Like other high-fidelity polymerases, KF achieves its extraordinary accuracy through a series of steps that discriminate between a correct and incorrect dNTP. A minimal reaction pathway for KF has been proposed with much of the data obtained from chemical quench experiments (4, 5) (Fig. 1A). The rate-limiting step (k 3 ) that precedes the phosphoryl-transfer step (k 4 ) had been tentatively attributed to a conformational change of the fingers domain (6). A comparison of the crystal structures of the binary polymerase-DNA complexes with those of the ternary polymerase-DNA-dNTP complexes reveals a substantial movement upon nucleotide binding, supporting the model that fingers closing was the rate-limiting step (7). However, recent results have shown this step is much too fast to be rate limiting, suggesting additional noncovalent steps th...

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Section: Resultssupporting

confidence: 80%

Section: Figsupporting

confidence: 80%

Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution

Christian

Romano

Rueda

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Using 2-AP dimer probes in the primer strand as a monitor, we have previously established experimental conditions that form "end-state" binary KF-P/T DNA complexes in which the 3′-end of the primer strand is bound entirely in either the pol or the exo site (17). Here 6-MI dimer probes located at positions n;n þ 1 are used to characterize template A B Fig.…”

Section: Distribution Of the P/t Dna Primer Strand Between The Pol Anmentioning

confidence: 99%

“…2B shows spectra in EDTA buffer (containing 2 mM EDTA and no added divalent cations), in which the primer of a fully base-paired P/T DNA construct is totally bound in the pol site (see ref. 17 and SI Text). In contrast, and as shown previously with 2-AP dimer probes positioned at the end of the primer strand, the 3′-ends of fully complementary P/T DNA constructs in Ca 2þ buffer are distributed between the two active sites ( Fig.…”

Section: Distribution Of the P/t Dna Primer Strand Between The Pol Anmentioning

confidence: 99%

“…To investigate the structural origins of this kinetic checkpoint we have monitored local conformational changes within the P/T DNA during dNTP selection by means of fluorescence and near UV circular dichroism (CD) spectra changes for adjacent pairs of 2-aminopurine (2-AP, an adenine analogue) (11) or 6-methyl isoxanthopterin (6-MI, an analogue for guanine) (16) bases at specific positions within the P/T DNA. Previously we used this technique to map local conformations at specific positions within the primer DNA bound at either the pol or the exo active site of KF, and to measure the distribution of the primer terminus between these two enzymatic sites (17). Here we probe the local conformations of both the primer terminus and the template n base bound to KF in the presence of various potential dNTP and rNTP substrates to further define structural aspects of possible checkpoint mechanisms in DNAP fidelity control.…”

mentioning

confidence: 99%

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DNA conformational changes at the primer-template junction regulate the fidelity of replication by DNA polymerase

Datta

Johnson

Hippel

2010

Proc. Natl. Acad. Sci. U.S.A.

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Local conformational changes in primer-template (P/T) DNA are involved in the selective incorporation of dNTP by DNA polymerases (DNAP). Here we use near UV CD and fluorescence spectra of pairs of base analogue probes, substituted either at the primer terminus or in the coding region of the template strand, to monitor and interpret conformational changes at and near the coding base of the template in P/T DNA complexes with Klenow fragment (KF) DNAP as the polymerase moves through the nucleotide addition cycle. Incoming dNTPs and rNTPs encounter binary complexes in which the 3′-end of the primer shuttles between the polymerization (pol) and exonuclease (exo) sites of DNAPs, even for perfectly complementary P/T DNA sequences. We have used spectral changes of probes inserted in both strands to monitor this two-state distribution and determine how it depends on the formation of ternary complexes with both complementary ("correct") and noncomplementary ("incorrect") NTPs and on the local sequence of the P/T DNA. The results show that the relative occupancy of the exo and pol sites is coupled to conformational changes in the P/T DNA of the complex that are partially regulated by the incoming NTP. We find that the coding base on the template strand is unperturbed by the binding of incorrect dNTPs, while binding of complementary rNTPs induces a novel template conformation. We conclude that, in addition to its editing function, primer strand occupancy of the 3′-exo site may also serve as a regulatory checkpoint for accurate dNTP selection in DNA synthesis.DNA editing | Klenow DNA polymerase | primer partitioning in DNAP active sites | low energy circular dichroism | base analogues 6-MI and 2-AP

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Wechselwirkung von Polyelektrolyt‐Architekturen mit Proteinen und Biosystemen

et al. 2020

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Gegenionen, die die Ladungen von Polyelektrolyten wie DNA oder Heparin neutralisieren, können im Wasser dissoziieren und beeinflussen stark deren Wechselwirkung mit Biomolekülen, insbesondere mit Proteinen. In diesem Artikel geben wir einen Überblick über Studien zur Wechselwirkung von Proteinen mit Polyelektrolyten und den Nutzen dieses Wissens für die medizinische Forschung. Hauptantriebskraft für die Bindung von Proteinen an Polyelektrolyte ist die Freisetzung von Gegenionen: Positiv geladene Bereiche des Proteins werden hierbei zu mehrwertigen Gegenionen des Polyelektrolyten, sodass zuvor gebundene Gegenionen des Polyelektrolyten freigesetzt werden und sich zugleich die Entropie erhöht, wie wir anhand von Studien zu Wechselwirkung von Proteinen mit natürlichen und synthetischen Polyelektrolyten zeigen. Ein Schwerpunkt liegt dabei auf sulfatierten dendritischen Polyglycerinen (dPGS). Die Literatur zeigt, dass wir Fortschritte beim Verständnis der Ladungs‐Ladungs‐Wechselwirkung in biologisch relevanten Systemen machen. Die Forschung auf diesem Gebiet wird die Entwicklung synthetischer Polyelektrolyte für die Medizin voranbringen.

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Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases

Cited by 26 publications

References 48 publications

Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution

Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution

DNA conformational changes at the primer-template junction regulate the fidelity of replication by DNA polymerase

Wechselwirkung von Polyelektrolyt‐Architekturen mit Proteinen und Biosystemen

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