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

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.

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Decision letter: Investigating the trade-off between folding and function in a multidomain Y-family DNA polymerase

10.7554/elife.60434.sa1 ◽

2020 ◽

Author(s):

Yong Wang

Keyword(s):

Dna Polymerase ◽

Trade Off ◽

And Function ◽

Y Family

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

Nucleic Acids Research ◽

10.1093/nar/gkt1149 ◽

2013 ◽

Vol 42 (4) ◽

pp. 2555-2563 ◽

Cited By ~ 24

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.

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Effects of electrostatic interactions on global folding and local conformational dynamics of a multidomain Y-family DNA polymerase

Physical Chemistry Chemical Physics ◽

10.1039/d1cp02832d ◽

2021 ◽

Author(s):

Qing-Miao Nie ◽

Li-Zhen Sun ◽

Hai-Bin Li ◽

Xiakun Chu ◽

Jin Wang

Keyword(s):

Dna Binding ◽

Dna Polymerase ◽

Electrostatic Interactions ◽

Conformational Dynamics ◽

Individual Domain ◽

The Individual ◽

Y Family

Electrostatic interactions can facilitate the folding of the multidomain DNA polymerase Dpo4 by refining the folding order of the individual domain and promote the functional conformational dynamics of Dpo4 during the DNA-binding recognition.

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Author response: Investigating the trade-off between folding and function in a multidomain Y-family DNA polymerase

10.7554/elife.60434.sa2 ◽

2020 ◽

Author(s):

Xiakun Chu ◽

Zucai Suo ◽

Jin Wang

Keyword(s):

Dna Polymerase ◽

Author Response ◽

Trade Off ◽

And Function ◽

Y Family

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Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

Journal of Bacteriology ◽

10.1128/jb.00101-15 ◽

2015 ◽

Vol 197 (17) ◽

pp. 2792-2809 ◽

Cited By ~ 20

Author(s):

Sarita Mallik ◽

Ellen M. Popodi ◽

Andrew J. Hanson ◽

Patricia L. Foster

Keyword(s):

Dna Damage ◽

Dna Polymerase ◽

Dna Helicase ◽

Dna Lesions ◽

Replication Forks ◽

Content Type ◽

Pol Iv ◽

Dna Polymerase Iv ◽

Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

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The Trade-off between Thermostability and Function in Designed DNA-Binding Proteins

Biophysical Journal ◽

10.1016/j.bpj.2020.11.379 ◽

2021 ◽

Vol 120 (3) ◽

pp. 19a

Author(s):

Lauren A. Verheyden ◽

Lily A. Schumacher ◽

Andrew Bigler ◽

Natali A. Gonzalez ◽

Emily Hamlin ◽

...

Keyword(s):

Dna Binding ◽

Binding Proteins ◽

Dna Binding Proteins ◽

Trade Off ◽

And Function

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Transactivation Functions of the N-Terminal Domains of Nuclear Hormone Receptors: Protein Folding and Coactivator Interactions

Molecular Endocrinology ◽

10.1210/me.2002-0258 ◽

2003 ◽

Vol 17 (1) ◽

pp. 1-10 ◽

Cited By ~ 125

Author(s):

Raj Kumar ◽

E. Brad Thompson

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Hormone Receptors ◽

Nuclear Hormone Receptor ◽

Binding Domain ◽

Dna Binding Domain ◽

Protein Protein Interactions ◽

Carboxy Terminal ◽

And Function ◽

Terminal Domains

Abstract The N-terminal domains (NTDs) of many members of the nuclear hormone receptor (NHR) family contain potent transcription-activating functions (AFs). Knowledge of the mechanisms of action of the NTD AFs has lagged, compared with that concerning other important domains of the NHRs. In part, this is because the NTD AFs appear to be unfolded when expressed as recombinant proteins. Recent studies have begun to shed light on the structure and function of the NTD AFs. Recombinant NTD AFs can be made to fold by application of certain osmolytes or when expressed in conjunction with a DNA-binding domain by binding that DNA-binding domain to a DNA response element. The sequence of the DNA binding site may affect the functional state of the AFs domain. If properly folded, NTD AFs can bind certain cofactors and primary transcription factors. Through these, and/or by direct interactions, the NTD AFs may interact with the AF2 domain in the ligand binding, carboxy-terminal portion of the NHRs. We propose models for the folding of the NTD AFs and their protein-protein interactions.

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