scholarly journals Nucleotide selection by the Y-family DNA polymerase Dpo4 involves template translocation and misalignment

2013 ◽  
Vol 42 (4) ◽  
pp. 2555-2563 ◽  
Author(s):  
Alfonso Brenlla ◽  
Radoslaw P. Markiewicz ◽  
David Rueda ◽  
Louis J. Romano
Keyword(s):  
Dna Polymerase ◽  
Single Molecule ◽  
Base Pair ◽  
Conformational Dynamics ◽  
Binary Complex ◽  
Translesion Dna Synthesis ◽  
Dna Polymerase Iv ◽  
Y Family ◽  
Dntp Binding ◽  
Base Pair Insertion

Abstract Y-family DNA polymerases play a crucial role in translesion DNA synthesis. Here, we have characterized the binding kinetics and conformational dynamics of the Y-family polymerase Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) using single-molecule fluorescence. We find that in the absence of dNTPs, the binary complex shuttles between two different conformations within ∼1 s. These data are consistent with prior crystal structures in which the nucleotide binding site is either occupied by the terminal base pair (preinsertion conformation) or empty following Dpo4 translocation by 1 base pair (insertion conformation). Most interestingly, on dNTP binding, only the insertion conformation is observed and the correct dNTP stabilizes this complex compared with the binary complex, whereas incorrect dNTPs destabilize it. However, if the n+1 template base is complementary to the incoming dNTP, a structure consistent with a misaligned template conformation is observed, in which the template base at the n position loops out. This structure provides evidence for a Dpo4 mutagenesis pathway involving a transient misalignment mechanism.

scholarly journals Substrate conformational dynamics drive structure-specific recognition of gapped DNA by DNA polymerase

2018 ◽  
Author(s):  
Timothy D. Craggs ◽  
Marko Sustarsic ◽  
Anne Plochowietz ◽  
Majid Mosayebi ◽  
Hendrik Kaju ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Polymerase ◽  
Single Molecule ◽  
Solution Structure ◽  
Binding Proteins ◽  
Conformational Dynamics ◽  
Dna Binding Proteins ◽  
Binary Complex ◽  
Specific Recognition ◽  
Gapped Dna

AbstractDNA-binding proteins utilise different recognition mechanisms to locate their DNA targets. Some proteins recognise specific nucleotide sequences, while many DNA repair proteins interact with specific (often bent) DNA structures. While sequence-specific DNA binding mechanisms have been studied extensively, structure-specific mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I (Pol) substrates both alone and in Pol-DNA complexes. Using a rigid-body docking approach based on a network of 73 distance restraints collected using single-molecule FRET, we determined a novel solution structure of the singlenucleotide-gapped DNA-Pol binary complex. The structure was highly consistent with previous crystal structures with regards to the downstream primer-template DNA substrate; further, our structure showed a previously unobserved sharp bend (~120°) in the DNA substrate; we also showed that this pronounced bending of the substrate is present in living bacteria. All-atom molecular dynamics simulations and single-molecule quenching assays revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Coarsegrained simulations on free gapped substrates reproduced our experimental FRET values with remarkable accuracy (<ΔFRET> = -0.0025 across 34 independent distances) and revealed that the one-nucleotide-gapped DNA frequently adopted highly bent conformations similar to those in the Pol-bound state (ΔG < 4 kT); such conformations were much less accessible to nicked (> 7 kT) or duplex (>> 10 kT) DNA. Our results suggest a mechanism by which Pol and other structure-specific DNA-binding proteins locate their DNA targets through sensing of the conformational dynamics of DNA substrates.Significance StatementMost genetic processes, including DNA replication, repair and transcription, rely on DNA-binding proteins locating specific sites on DNA; some sites contain a specific sequence, whereas others present a specific structure. While sequence-specific recognition has a clear physical basis, structure-specific recognition mechanisms remain obscure. Here, we use single-molecule FRET and computer simulations to show that the conformational dynamics of an important repair intermediate (1nt-gapped DNA) act as central recognition signals for structure-specific binding by DNA polymerase I (Pol). Our conclusion is strongly supported by a novel solution structure of the Pol-DNA complex wherein the gapped-DNA is significantly bent. Our iterative approach combining precise single-molecule measurements with molecular modelling is general and can elucidate the structure and dynamics for many large biomachines.

scholarly journals Substrate conformational dynamics facilitate structure-specific recognition of gapped DNA by DNA polymerase

2019 ◽  
Vol 47 (20) ◽  
pp. 10788-10800 ◽  
Author(s):  
Timothy D Craggs ◽  
Marko Sustarsic ◽  
Anne Plochowietz ◽  
Majid Mosayebi ◽  
Hendrik Kaju ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Polymerase ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Coarse Grained ◽  
General Mechanism ◽  
Binary Complex ◽  
Specific Recognition ◽  
Gapped Dna ◽  
Dna Substrate

Abstract DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets; some proteins recognise specific DNA sequences, while others interact with specific DNA structures. While sequence-specific DNA binding has been studied extensively, structure-specific recognition mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I Klenow Fragment (Pol) substrates both alone and in DNA–Pol complexes. Using a docking approach based on a network of 73 distances collected using single-molecule FRET, we determined a novel solution structure of the single-nucleotide-gapped DNA–Pol binary complex. The structure resembled existing crystal structures with regards to the downstream primer-template DNA substrate, and revealed a previously unobserved sharp bend (∼120°) in the DNA substrate; this pronounced bend was present in living cells. MD simulations and single-molecule assays also revealed that 4–5 nt of downstream gap-proximal DNA are unwound in the binary complex. Further, experiments and coarse-grained modelling showed the substrate alone frequently adopts bent conformations with 1–2 nt fraying around the gap, suggesting a mechanism wherein Pol recognises a pre-bent, partially-melted conformation of gapped DNA. We propose a general mechanism for substrate recognition by structure-specific enzymes driven by protein sensing of the conformational dynamics of their DNA substrates.

scholarly journals Investigating the trade-off between folding and function in a multidomain Y-family DNA polymerase

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Xiakun Chu ◽  
Zucai Suo ◽  
Jin Wang
Keyword(s):  
Dna Binding ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Conformational Dynamics ◽  
Dna Lesions ◽  
Trade Off ◽  
Dna Polymerase Iv ◽  
Functional Dna ◽  
And Function ◽  
Y Family

The way in which multidomain proteins fold has been a puzzling question for decades. Until now, the mechanisms and functions of domain interactions involved in multidomain protein folding have been obscure. Here, we develop structure-based models to investigate the folding and DNA-binding processes of the multidomain Y-family DNA polymerase IV (DPO4). We uncover shifts in the folding mechanism among ordered domain-wise folding, backtracking folding, and cooperative folding, modulated by interdomain interactions. These lead to ‘U-shaped’ DPO4 folding kinetics. We characterize the effects of interdomain flexibility on the promotion of DPO4–DNA (un)binding, which probably contributes to the ability of DPO4 to bypass DNA lesions, which is a known biological role of Y-family polymerases. We suggest that the native topology of DPO4 leads to a trade-off between fast, stable folding and tight functional DNA binding. Our approach provides an effective way to quantitatively correlate the roles of protein interactions in conformational dynamics at the multidomain level.

scholarly journals Human DNA Polymerase ι Utilizes Different Nucleotide Incorporation Mechanisms Dependent upon the Template Base

2004 ◽  
Vol 24 (2) ◽  
pp. 936-943 ◽  
Author(s):  
M. Todd Washington ◽  
Robert E. Johnson ◽  
Louise Prakash ◽  
Satya Prakash
Keyword(s):  
Dna Polymerase ◽  
Base Pair ◽  
Translesion Dna Synthesis ◽  
Human Dna ◽  
Nucleotide Incorporation ◽  
Steady State Kinetic ◽  
Y Family ◽  
State Kinetic ◽  
Dna Polymerase Ι ◽  
Better Than

ABSTRACT Human DNA polymerase ι (Polι) is a member of the Y family of DNA polymerases involved in translesion DNA synthesis. Polι is highly unusual in that it possesses a high fidelity on template A, but has an unprecedented low fidelity on template T, preferring to misincorporate a G instead of an A. To understand the mechanisms of nucleotide incorporation opposite different template bases by Polι, we have carried out pre-steady-state kinetic analyses of nucleotide incorporation opposite templates A and T. These analyses have revealed that opposite template A, the correct nucleotide is preferred because it is bound tighter and is incorporated faster than the incorrect nucleotides. Opposite template T, however, the correct and incorrect nucleotides are incorporated at very similar rates, and interestingly, the greater efficiency of G misincorporation relative to A incorporation opposite T arises predominantly from the tighter binding of G. Based on these results, we propose that the incipient base pair is accommodated differently in the active site of Polι dependent upon the template base and that when T is the templating base, Polι accommodates the wobble base pair better than the Watson-Crick base pair.

scholarly journals Global Conformational Dynamics of a Y-Family DNA Polymerase during Catalysis

PLoS Biology ◽  
2009 ◽  
Vol 7 (10) ◽  
pp. e1000225 ◽  
Author(s):  
Cuiling Xu ◽  
Brian A. Maxwell ◽  
Jessica A. Brown ◽  
Likui Zhang ◽  
Zucai Suo
Keyword(s):  
Dna Polymerase ◽  
Conformational Dynamics ◽  
Y Family

scholarly journals Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair

2020 ◽  
Vol 48 (15) ◽  
pp. 8490-8508 ◽  
Author(s):  
Sarah S Henrikus ◽  
Camille Henry ◽  
Amy E McGrath ◽  
Slobodan Jergic ◽  
John P McDonald ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Single Molecule ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
E Coli ◽  
Pol Iv ◽  
Dna Polymerase Iv

Abstract Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.

scholarly journals Role of Pseudomonas aeruginosa dinB-Encoded DNA Polymerase IV in Mutagenesis

2006 ◽  
Vol 188 (24) ◽  
pp. 8573-8585 ◽  
Author(s):  
Laurie H. Sanders ◽  
Andrea Rockel ◽  
Haiping Lu ◽  
Daniel J. Wozniak ◽  
Mark D. Sutton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dna Polymerase ◽  
Opportunistic Pathogen ◽  
Binding Assays ◽  
Mutator Phenotype ◽  
Cell Extracts ◽  
Dna Polymerase Iv ◽  
Mechanisms Of Mutagenesis ◽  
Y Family

ABSTRACT Pseudomonas aeruginosa is a human opportunistic pathogen that chronically infects the lungs of cystic fibrosis patients and is the leading cause of morbidity and mortality of people afflicted with this disease. A striking correlation between mutagenesis and the persistence of P. aeruginosa has been reported. In other well-studied organisms, error-prone replication by Y family DNA polymerases contributes significantly to mutagenesis. Based on an analysis of the PAO1 genome sequence, P. aeruginosa contains a single Y family DNA polymerase encoded by the dinB gene. As part of an effort to understand the mechanisms of mutagenesis in P. aeruginosa, we have cloned the dinB gene of P. aeruginosa and utilized a combination of genetic and biochemical approaches to characterize the activity and regulation of the P. aeruginosa DinB protein (DinB Pa ). Our results indicate that DinB Pa is a distributive DNA polymerase that lacks intrinsic proofreading activity in vitro. Modest overexpression of DinB Pa from a plasmid conferred a mutator phenotype in both Escherichia coli and P. aeruginosa. An examination of this mutator phenotype indicated that DinB Pa has a propensity to promote C→A transversions and −1 frameshift mutations within poly(dGMP) and poly(dAMP) runs. The characterization of lexA + and ΔlexA::aacC1 P. aeruginosa strains, together with in vitro DNA binding assays utilizing cell extracts or purified P. aeruginosa LexA protein (LexA Pa ), indicated that the transcription of the dinB gene is regulated as part of an SOS-like response. The deletion of the dinB Pa gene sensitized P. aeruginosa to nitrofurazone and 4-nitroquinoline-1-oxide, consistent with a role for DinB Pa in translesion DNA synthesis over N 2 -dG adducts. Finally, P. aeruginosa exhibited a UV-inducible mutator phenotype that was independent of dinB Pa function and instead required polA and polC, which encode DNA polymerase I and the second DNA polymerase III enzyme, respectively. Possible roles of the P. aeruginosa dinB, polA, and polC gene products in mutagenesis are discussed.

How Y-Family DNA polymerase IV is more accurate than Dpo4 at dCTP insertion opposite an N2-dG adduct of benzo[a]pyrene

DNA Repair ◽  
2015 ◽  
Vol 35 ◽  
pp. 144-153 ◽  
Author(s):  
Gabriel Sholder ◽  
Amanda Creech ◽  
Edward L. Loechler
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerase Iv ◽  
Y Family
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